Modulation of Human Eosinophil Polymorphonuclear Leukocyte Migration and Function Edward J. Goetzl, MD
Eosinophil migration toward a concentration gradient of a chemotactic factor is regulated at four levels. Diverse immunologic pathways generate stimuli with eosinophil chemotactic activity, including the complement products C5a and a fragment of C3a and the peptide products of mast cells and basophils activated by IgE-mediated reactions, such as eosinophil chemotactic factor of anaphylaxis (ECF-A) and other oligopeptides. The intrinsic preferential leukocvte activitv of the chemotactic stimuli represents the second level of modulation, with ECF-A and other mast cell-derived peptides exhibiting the most selective action on eosinophils. The third level of control of eosinophil chemotaxis is composed of inactivators and inhibitors of chemotactic stimuli and is exemplified by degradation of C5a by anaphylatoxin inactivator or chemotactic factor inactivator and of ECF-A by carboxvpeptidase-A or aminopeptidases. The activity of ECF-A is uniquely suppressed by equimolar quantities of its NH2terminal tripeptide substituent, presumably by eosinophil membrane receptor competition. Factors comprising the fourth level of regulation, which alter eosinophil responsiveness to chemotactic stimuli, include the chemotactic factors themselves, through deactivation; nonchemotactic inhibitors such as the COOH-terminal tripeptide substituent of ECF-A, the neutrophil-immobilizing factor (NIF), the phagocytosisenhancing factor Thr-Lys-Pro-Arg, and histamine at concentrations greater than 400 ng/ml; and nonchemotactic enhancing principles represented by ascorbate and by histamine at concentrations of 30 ng/ml or less. Local concentrations of eosinophils called to and immobilized at the site of a hypersensitivity reaction may express their regulatory functions by degrading the chemical mediators elaborated including histamine, slow-reacting substance of anaphvlaxis (SRS-A), and platelet-activating factor (PAF) by way of their content of histaminase, arylsulfatase B, and phospholipase D. respectively. Immunologic pathways may thus provide the capability for early and specific host defense reactions with a later influx of eosinophils preventing irreversible local tissue alterations or distant organ effects (Am J Pathol 85:419436, 1976)
EOSINOPHIL POLY\MORPHONNUCLEAR (PMNN) leukocytes originate from myeloid precursors in bone marromv, circulate in peripheral blood for a brief period, and then move to tissue sites localized predominantly in the lungs, skin, intestines, and genitourinary tract.' An elevated level of eosinophils in peripheral blood and in tissues is a characteristic feature of systemic hypersensitivity reactions to a wvide variety of stimuli including drugs, environmental allergens, parasitic and other chronic infectious From the Departments of Mledicine. Harvard \ledical School and the Robert B. Brigham Hospital. Boston. Mlassachusetts. Supported by Grants Al-07722. lX-10:36. and HL-173S2 from the National Institutes of Health: Dr. Goetzl is an Insvestigator of the How-ard Hughes Mledical Institute. Presented at the Sixtieth Xnnual Meeting of the Federation of Xmerican Societies for Experimental Biology. Anaheim. Calif.. April 11. 1976. Address reprint requests to Dr. Edsward J. Goetzl Department of Mledicine. Harxard \ledical School. Boston. MA 0-2115. 419
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agents, and plasma and tissue proteins.3` Although eosinophils arrive at tissue foci of hypersensitivity reactions relatively late in comparison to the time required for full expression of humoral events, they frequently persist in involved tissues for long intervals thereafter without leading to further injurv.7-9 The selective influx of eosinophils to the site of a hypersensitivity reaction is determined by the effective activity of diverse chemotactic factors and the responsiveness of the available eosinophils. The eosinophils are trapped in the involved tissues by the local immobilizing effects of the chemotactic principles themselves and of several inhibitors.10'11 Thus a sufficient number of eosinophils are maintained at the focus of a hypersensitivity reaction to enable the fulfillment of their specific regulatory role. 1012
Generation of Eosiophil Chemotactic Factors by Immune Reactions Cdul Immunity
Eosinophil chemotactic activity in lymphoid tissue has been inferred from histologic studies of animals that received an initial antigen injection and demonstrated a peak of eosinophilia in the perifollicular regions of lymph nodes 4 to 12 hours later.13'14 Mice receiving a booster intraperitoneal injection of Trichinella larvae demonstrated a prominent increase in both eosinophils and mononuclear leukocytes in the peritoneal fluid that was largelv T-lymphocyte dependent.'5 Guinea pigs immunized with thyroid extract in complete Freund's adjuvant developed a thyroiditis consisting of mononuclear leukocyte infiltrates.'16"7 Eosinophils were found in the thyroiditis lesions only in those animals that also had detectable antithyroid antibodies, suggesting a dependence of tissue oesinophilia on both serum antibody and lymphocyte factors. Lymphocyte-related eosinophil chemotactic factors with in vitro activity have been recognized in two settings. Specific antigen stimulation in vitro of lymphocytes from guinea pigs with delayed hypersensitivity generated a precursor macromolecule that exhibited eosinophil chemotactic activity when mixed with homologous immune complexes.'8 Preferential eosinophil chemotactic activity recently was found in supernatants from cultured lymph node cells of patients with Hodgkin's disease and was composed of four peaks of molecular weights ranging from 500 to 30,000 daltons by gel filtration.'9 Humoral Imunity
Both the classic and altemative complement pathways can generate substantial eosinophil chemotactic activity that is attributable to the
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anaphylatoxins (5a and a fragment of the third component of complement (C3).2* The G5a fragment of the fifth component of complement (C5) is a protein of approximate molecular weight of 12,000 that can be generated as well by the cleavage of C5 by a neutrophil lysosomal protease.'2 The C3 fragment is a protein of approximate molecular weight of 9,000 that can also be generated directly by digestion of C3 with plasmin or with a protease in some inflamed tissues.25," Other factors demonstrating eosinophil chemotactic activity include the platelet enzyme products of arachidonic and eicosapentenoic acids 2-29 and those principles whose activities are dependent upon the integrity of an enzyme active site, including kallikrein, plasminogen activator, and the C3 convertase of the alternative complement pathway.3"33 Eosinph Clemotact Factor of A This factor (ECF-A) was discovered in 1971 as the mediator that is responsible for most of the eosinophil chemotactic activitv released during anaphylactic reactions in vitro in guinea pig " and human 35 lung slices. Of the other mediators of immediate hypersensitivity, only histamine stimulated directed migration of eosinophils in vitro; however, its action was transient and it also lacked apparent in vivo chemotactic activity.3"37 The release of ECF-A from human tissues by IgE-dependent mechanisms exhibited a time-course, biochemical requirements, and responsiveness to the intracellular levels of cyclic nucleotides similar to the release of histamine from the same tissues.3' ECF-A was present preformed in purified rat mast cells in association with the granules 3" and was also extractable from human leukemic basophils 40 and mast cell-rich tissues such as human lung and nasal polyps.3"41 Preliminary physicochemical characterization of ECF-A obtained by extraction or immunologic activation of guinea pig and human lung tissues and rat mast cells revealed a molecular weight of approximately 500 by filtration on Sephadex G-25S,"33 inactivation by digestion with subtilisin or pronase but not with trvpsin or chymotrvpsin,42 and two anodal peaks of activitv on high-voltage paper electrophoresis at pH 7.10 The extraction of unchallenged human lung tissue fragments with either butanol: glacial acetic acid (10:1, v/v) or Tyrode's solution buffered at pH 8.6 with 0.1 M Tris-HCI solubilizes eosinophil chemotactic activity in high yield." Gel filtration on Sephadex G-25 of Iyophilized extracts resuspended in 0.1 M acetic acid resolves four peaks of chemotactic activity."'" The largest peak, appearing in the exclusion volume, is preferentially chemotactic for neutrophils and has been termed neutrophil chemotactic factor of anaphylaxis (NCF-A).4'" An intermediate peak of
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approximate molecular weight of 2500 to 3500 is preferentially active for eosinophils but, unlike NCF-A and ECF-A, is present only in anaphylactic diffusates in amounts representing a small percent of the activity in tissue stores. The two smaller peaks, which exhibit molecular weights of 300 to 500 and 150 to 200, are selectively chemotactic for eosinophils and correspond, respectively, to ECF-A and to a region between ECF-A and histamine that may represent a synergistic action of the two factors."', The 300 to 500 molecular weight peak of preferential eosinophil chemotactic activity from extracts of human lung, corresponding to the Sephadex G-25 peak of ECF-A in anaphylactic diffusates, was isolated by sequential purification using ion-exchange chromatography, gel filtration on Sephadex G-10, and paper chromatography.' The ECF-A activitv from IgE-mediated reactions in human lung tissues appeared along with that in extracts of human lung during parallel purification procedures. Fractions of the highly purified extracts with eosinophilotactic activitv contained two acidic tetrapeptides of amino acid sequence Ala-Gly-Ser-Glu and Val-Gly-Ser-Glu. Both the synthetic tetrapeptides and purified ECF-A were maximally active in amounts from 0.1 to 1.0 nmole/ chemotactic chamber, representing concentrations of 10 7 to 10' M.'4 The dose-response relationships of highly purified ECF-A from human lung extracts and the synthetic tetrapeptides were established in the modified Boyden assav (Text-figure 1). Purified ECF-A was quantitated bv acid hydrolysis and analysis of constituent amino acids, and the target leukocytes were from a patient with 88% peripheral blood eosinophilia. For all three stimuli, eosinophilotactic activity was detectable at 10 M and reached a plateau of 10 to 14 eosinophils/high-power field (eos/hpf) bv 107 M. Portions of the initial Sephadex G-25 peak of ECF-A extracted from lung attracted 12 eos/hpf for 25 Al, 28 eos/hpf for 50 Al, and 32 eos/hpf for 100 Ml. Thus, while not achieving a maximum level of eosinophil chemotaxis comparable to that for a crude preparation of ECFA, equivalent doses of purified native ECF-A and svnthetic tetrapeptides were equally and preferentially active on eosinophils.4" The intraperitoneal administration of 0.1 to 1.0 nmole of synthetic valyl-tetrapeptide to guinea pigs elicited an early selective influx of eosinophils followed bv neutrophils." The in vivo potency of the tetrapeptides paralleled the in vitro dose-response relationship, and their effect in guinea pigs correlated with the preferential attraction of eosinophils in vitro, although an ability to induce directed migration of neutrophils was also recognized. A 3(0 to 500 molecular weight chemotactic factor that preferentially attracts eosinophils and, like ECF-A, is inactivated by subtilisin digestion has been recognized in extracts of tumor tissue and supernatants
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TEXT-FIGURE 1-Dose-response of eosinophil chemotactic actisits of ECF-X purified from human lung (open squares) and sy nthetic tetrapeptides-alany ltetrapeptide (open circles) and saly l-tetrapeptide .solid circles). Human lung ECF-A swas purified by using Sephadex G-25. Dowex1. Sephadex G-10. and deseending paper chromatographn- equal quantities of the alan% l- and valyl-tetrapeptide pools were recombined for the chemotactic stimulus. Background eosinophil migration was four eosinophils per high-power field.
423
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of cell cultures of patients with bronchogenic carcinoma and peripheral blood eosinophilia.47 This factor is less acidic than ECF-A and elutes from Dowex-1 at a pH of 4.5 to 3.8. The intermediate molecular weight peak of eosinophil chemotactic activity, released in only small quantities by IgE-dependent reactions, is contained in large amounts along with ECF-A in preformed stores in rat mast cells, in association with the granules,46 and in mast cell-rich tissues." This oligopeptide ECF is inactivated bv digestion with carboxypeptidase-A and is preferentiallv chemotactic for eosinophils; thus, it may be either a precursor of ECF-A or a distinct but functionallv related eosinophil chemotactic factor. A comparable peak of eosinophilotactic activity has been recognized in tumor extracts, urine and cell culture supernatants of patients with bronchogenic carcinoma and eosinophilia,47 and in supernatants of cell cultures from lymph nodes of patients with Hodgkin's disease."9 The availabilitv in mast cell stores of eosinophilotactic peptides of vary'ing sizes provides for rapid establishment of a chemotactic gradient by the smaller factors and maintenance of the gradient bv larger factors, thus ensuring an earlv and continuing movement of eosinophils to a focus of allergic reaction.
Regulation of the Eosinophil Chernotactic Response The generation of chemotactic factors during immunologic and other inflammatory reactions represents the initial pathway in the regulation of the eosinophil chemotactic response (Text-figure 2, Table 1). Immediate hypersensitivityT reactions liberate ECF-A and small quantities of larger ECF peptides.",451' Activation of the classic or alternative (properdin) complement pathways generates the anaphylatoxins C3a and C-a.`0 Other factors with substantial eosinophil chemotactic activity are elabo-
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rated by- exposure of PM N leukocytes to a calcium ionophore or phagocytosable particles,49 antigen activation of lymphocytes,18 and oxidative conversion of arachidonic acid.27The second lev-el of modulation of eosinophil chemotaxis consists of the concentration, potency, and leukocyte specificity of each chemotactic factor wvhich may- be determined by specific structural features. Highlxpurified ECF-A. synthetic ECF-A tetrapeptides, and partially purified larger peptides from human lung tissue or rat mast cells preferentiallxattract human eosinophils in vitro -hen the eosinophils represent 10% or more of the target leukocyte pool.10 43 50 Both the low,-- and intermediatemolecular-w-eight ECF peptides derived from bronchogenic carcinoma are also preferentially chemotactic for eosinophils.47 The complementderixved factors C5a and a fragment of C3 attract eosinophils, neutrophils. and monocvtes equally.l10245051 In contrast, the plasma -globulin enzy-mes, kallikrein and plasminogen activator, and the alternative complement path way C3 convertase are selectiv-ely chemotactic for neutrophils.32.33 Inhibition and Inactivation of Eosiophil Chemotactic Factor Activity
The third level of regulation of eosinophil chemotaxis encompasses the inhibitors or inactivators of chemotactic factors (Table 1. Text-figure 2). The anaphylatoxin inactiv-ator suppresses C5a-mediated leukotaxis.52 The chemotactic factor inactiv-ator (CFI), w hich is composed of tv-o or more exoproteolvtic enzymes present in serum and cells, irreversibly inactivates numerous chemotactic factors.53 While the in vivo pathw-ays of degradation of ECF-A hav-e not vet been elucidated, limited digestion of the valyl- and alanyl-tetrapeptides -with either aminopeptidase- M or carboxv-peptidase-A reduces their eosinophil chemotactic acti-ity in proportion to the extent of liberation of NH2- and COOH-terminal amino acids, respectiv ely. 3 Some hydrophobic amino acid deriv-atives and peptide analogs or sub-
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stituents of ECF-A tetrapeptides can inhibit the chemotactic activity of the intact tetrapeptide as exemplified by valine-amide (val-amide) and the NH2-terminal tripeptides Ala,Aal-Gl%-Ser, which possess neither chemotactic nor deactivating capacity.4554 Both val-amide and Val-Clv-Ser suppress the eosinophilotactic activity of 107M valvl-tetrapeptide wvhen present concomitantly in the stimulus compartment of the Boyden chamber producing 30%c mean inhibition at 10-6M and 10-7WM respectively.54 Val-amide and Val-GIl-Ser presumably inhibit the eosinophil chemotactic response by competing wvith ECF-A for a hydrophobic site on the eosinophil surface and thus do not affect eosinophil random migration. The capacity of Val-Gly-Ser to inhibit eosinophil chemotaxis to equimolar quantities of valvl-tetrapeptide has been observed in vivo as wvell." Cell-Directed Modulation of the Eosinophil Chemotactic Response
The fourth level of regulation of eosinophil chemotactic migration includes factors that alter chemotaxis by influencing the responsiveness of the cells (Table 1 Text-figure 2). The effects of these factors on eosinophils are independent of the specific chemotactic stimulus, result in enhancement or suppression of chemotaxis and generally extend to similar actions on spontaneous migration. This group of noncytotoxic factors consists of the chemotactic factors themselves that induce an eosinophil chemotactic unresponsiveness termed deactivation,55' and a variety of cell-directed inhibitors and enhancers that lack chemotactic activity-.57-62 Cell-Directed Inhibitors
Chemotactic deactivation of leukocytes by prior exposure to an active chemotactic stimulus exhibits a leukocv-te specificity \-hich parallels the chemotactic selectivity of the stimuli.'032 Human eosinophils exposed to chemotactic concentrations of C3a or partially purified ECF-A rapidly become unresponsive to both the homologous and heterologous stimuli.' Concentrations of either stimulus as lowv as one-twentieth the minimal chemotactic dose fully deactivated eosinophils in a time-dependent fashion. Human eosinophils preincubated wvith synthetic alanvl- or valvltetrapeptide at levels of 108 to 10-10 NM for a minutes at 37 C wvere rendered -5 to 80%'- unresponsive to a standard 10_'NI stimulus of either peptide preparation.45.54 With a 10-12 NM concentration of synthetic tetrapeptide deactivation wvas 40% after 10 minutes and reached 75 to 8Occ after 90 minutes. Thus, one-one hundredths of the minimal chemotactic dose of either tetrapeptide comprising ECF-A gave brisk and complete deactivation of human eosinophils. This uniquely lo,- threshold of eosinophils deactivation may account for their local immobilization at involved sites
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without tissue damage, since lysosomal enzyme release and metabolic activation of the eosinophils apparently requires higher concentrations of chemotactic factors than deactivation. Several factors have been identified that are themselves devoid of chemotactic activity but inhibit chemotaxis and spontaneous migration of eosinophils by an action on the cells. The neutrophil-immobilizing factor (NIF) is an inhibitor of polymorphonuclear leukocyte random and directed migration that is generated by incubating human peripheral blood mononuclear or polymorphonuclear leukocytes in acid medium (ANIF), with endotoxin (ENIF), or with phagocytosable particles (PhNIF).s7 Neutrophil-immobilizing factor is separable from coexistent chemotactic activity in the leukocyte supematants by heating and gel filtration on Sephadex G-25, and exhibits an apparent molecular weight of approximately 4000 to 5000 daltons. Partially purified NIF irreversibly suppresses the random migration and chemotactic response to diverse stimuli of eosinophils and neutrophils but not of mononuclear leukocytes.5 Inhibition of migration by NIF is not associated with any effect on leukocyte viability, phagocytosis, adherence to surfaces, or baseline or stimulated HMPS activity." Neutrophil-immobilizing factor also inhibits the stimulation of random migration and chemotaxis that results when neutrophils are preincubated with high concentrations of ascorbate." Human polymorphonuclear and mononuclear leukocytes also contain extractable preformed stores of higher molecular weight chemotactic inhibitors which are released during phagocytosis, but not by acid or endotoxin treatment.57 These larger inhibitors are functionally similar to NIF with an action apparently limited to inhibition of random and directed migration of eosinophil and neutrophil PMN leukocytes. Filtration on Sephadex G100 of the inhibitors in the excluded volume from Sephadex G-25 revealed two factors of molecular weights 30,000 to 40,000 and >120,000. Gly-Ser-Glu, the ECF-A COOH-terminal tripeptide, inhibits chemotaxis by a suppressive action on the cells."'," Preincubation of human eosinophils with Gly-Ser-Glu, which lacks substantial chemotactic activity, resulted in a dose- and time-dependent suppression of their subsequent response to natural or synthetic ECF-A that reached 80% inhibition after a 20-minute exposure to 10-8 M tripeptide." The action of the COOH-terminal tripeptide appears to be on the cells themselves as evidenced by the persistence of its effect after cell washing and the failure of 10 M tripeptide to inhibit significantly when present on the stimulus side of a Boyden chamber. A leukocyte phagocytosis-stimulating tetrapeptide of amino acid sequence L-threonyl- L-lysyl- L-prolyl- L-arginine (Thr-Lys-Pro-Arg), desig-
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nated "Tuftsin", has been purified from digestion mixtures of a phagocvtosis-enhancing cytophilic fraction of human -r -globulin and a proteol tic enzvme contained in human polymorphonuclear leukocytes.596 Nanogram amounts of the synthetic or purified natural peptide enhances by 2 to 2.3-fold the phagocytosis of latex particles or Staphylococcus aureus by human, dog, and guinea pig polvmorphonuclear leukocytes and bv mouse peritoneal and rabbit lung macrophages.59 The synthetic tetrapeptide lacks chemotactic activity, but preincubation of purified populations of human eosinophils, neutrophils, and mononuclear leukocytes with concentrations ranging from 0.1 to 100 ng/ml resulted in a dose-related inhibition of eosinophil and neutrophil random migration and chemotaxis to a preferential stimulus (Text-figure 3). In contrast, mononuclear leukocy-te random and chemotactic migration w,ere uninfluenced by the tetrapeptide. Thus, the inhibitory action of Thr-Lvs-Pro-Arg resembles that of the larger basic peptide NIF in suppressing migration of eosinophils and neutrophils wvithout affecting their viability or hexose monophosphate shunt activity and without decreasing mononuclear leukocyte migration.5 Thr-Lv-s-Pro-Arg but not NIF stimulates leukocyte phagocytosis in the same concentration range that is optimal for inhibition of migration. Cell-Directed Enhancing Factors
A variety of pathways vield factors which directly augment eosinophil random migration and enhance their chemotactic responsiveness. When lymphocytes of mice previously sensitized with schistosomes were incubated wvith specific schistosomal egg antigen or phytohemagglutinin, RANDOM MIGRATION
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TEXT-FIGIRE 3-Thr-Lv s-ProXrg tetrapeptide inhibition of leukoc'te migration. Eosinophils open squaresl from a patient w-ith a peripheral blcxd concentration of S4 '5 'cvere emplosed. Neutrophils solid circles and mononuclear leukoc-tes (open triangles) wsere standard preparations from normal donors." Control random migration was eosinophils. 13 hpf. neutrophils. 14 hpf. mononuclear leukocytes. 12 hpf. Chemotaxis %vas eosinophils to ECF-A. 34 hpf. neutrophils to kallilkrein 48 hpf and mononuclear leukoc'-tes to C.3a. 26 hpf.
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they elaborated a 25,000 to 35,000 molecular weight principle, termed eosinophil stimulation promoter." This factor increased mouse eosinophil random migration from agarose droplets but has not been assessed for its activity on chemotaxis. Ascorbic acid is a nonchemotactic principle that enhances the random migration and chemotactic responsiveness of neutrophils, eosinophils, and mononuclear leukocytes by 100 to 300% of baseline values with concomitant and equivalent stimulation of leukocyte HMPS activity.' Ascorbate, at concentrations of 2 to 10 X 10 3 M, results in dose-related enhancement of both migration and HMPS activity of eosinophils and other leukocytes, but has no effect on phagocytosis. Histamine Effects on Es
M i
Histamine exhibits a triphasic concentration-dependent modulation of eosinophil migration,'," with enhancement of random and directed migration at levels of 1 to 3 x 10' M, inhibition of random and directed migration at levels of 4 X 10' M or higher, and induction of directed migration in the intermediate range. In the midconcentration range of 3 X 10-7 M to 1.25 X 10 M (33 to 139 ng/ml), histamine induces directed migration of eosinophils in some in vitro chambers that employ thin polycarbonate filters or brief incubation periods with standard cellulose nitrate filters." However, its eosinophilotactic activity in a conventional chemotactic assay differs from that of eosinophil chemotactic factors such as ECF-A and CSa. Unlike purified or synthetic ECF-A, histamine failed to induce a true wave of cell movement as assessed by a shift of the maximum density of eosinophils into the filters.'" Further, 105 M histamine added to both sides of the filter often resulted in enhanced random migration of a small fraction of the eosinophils." Finally, histamine has no documented in vivo eosinophil chemotactic activity. Early in vivo studies revealed an influx of eosinophils into pulmonary tissue, in association with a peripheral blood eosinophilia, in response to the intraperitoneal or intravenous injection of large doses of histamine in guinea pigs.'-' However, neither local intraperitoneal nor intradermal eosinophilia followed histamine injection in animals.',' The major actions of histamine on in vitro eosinophil migration involve modulation of their responsiveness to chemotactic factors. The addition of histamine to the cell compartment of Boyden chambers at a dose of 30 ng/ml, which stimulated only minimal eosinophil migration, enhanced the chemotactic response of eosinophils to a low concentration of valyltetrapeptide in a more than additive fashion. An identical protocol utilizing a dose of 600 ng/ml histamine in the cell compartment inhibited the spontaneous migration of eosinophils and suppressed the eosinophil re-
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sponse to valyl-tetrapeptide.5 Thus, the effects of histamine on eosinophil movement include brief directed migration at intermediate doses, and cell-directed modulation of random and directed migration that is stimulatory at low levels and suppressive at high levels. High-dose histamine suppression of both chemotaxis and histamine-induced directed migration has been attributed to elevation of intracellular cyclic AMP levels since it is prevented by the simultaneous addition of H2-blockers, while histamine enhancement of eosinophil chemotactic responsiveness was reduced by H-1 blockers."'" Eosinopil Fuct in Immediate Hypersensitiity After arriving at a site of an immediate hypersensitivity reaction, the eosinophil may manifest regulatory functions that could limit or eventually terminate the allergic reaction. Although under special circumstances eosinophil-derived factors may influence the release or activity of chemical mediators of immediate hypersensitivity,7172 their predominant regulatory role is the degradation of chemical mediators by way of specific constituent enzymes. 1112 " Eosinophil granules contain a histaminase capable of oxidative deamination of histamine,73 an arylsulfatase B which can degrade SRS-A,74 and a phospholipase D with the capacity to inactivate a PAF.75 The arylsulfatase B preferentially present in eosinophils as compared to other leukocytes has been purified by sequential gel filtration on Sephadex G-100 and chromatography on carboxymethyl cellulose.74 It exhibits properties of a Type II arylsulfatase by virtue of preferential cleavage of pnitrocatechol sulfate (PNCS) over p-acetylphenyl sulfate or p-nitrophenyl sulfate and inhibition by sulfate and phosphate but not cyanide.76 Physicochemical characterization identified the eosinophil arylsulfatase as Type B since it is soluble, has a molecular weight of approximately 60,000, and possesses a basic isoelectric point; further it is inhibited by chloride ions in the presence of pyrophosphate.""w Purified eosinophil arylsulfatase, as well as arylsulfatase B from human lung,74'81 inactivates human SRS-A in a time-dependent reaction with a pH optimum of 5.5 to 6.0 that is- comparable to that for cleavage of PNCS.10'74 That the arylsulfatase B active site is responsible for SRS-A inactivation was indicated by the association of both PNCS-cleaving and SRS-A-inactivating activities during purification of the enzyme, and the ability of SRS-A to inhibit cleavage of the preferred synthetic substrate.74 A phospholipase D is also preferentially contained in human eosinophils, and has been purified by sequential anion exchange chromatographv and gel filtration on Sephadex G-100 where it has an apparent
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molecular wveight of 60,000.75 The purified phospholipase D exhibited a pl of 3.8 to 6.2 on polyacrvlamide gel isoelectric focusing. The ability of eosinophil-derived phospholipase D to cleave choline from lecithin and lvsolecithin, and its capacitv to inactivate a rat platelet-activating factor (PAF) were associated during purification of the enzvme, and there was apparent reciprocal inhibition of choline-generating activitv by PAF and of PAF-inactivating activity by phosphatidvl-choline.75 Mediator degradation by intact eosinophils is more efficient and possibly more relevant to in uivo pathwavs since the eosinophils rapidly inactivate SRS-A with an optimal pH of 7.0 to 7.4, as compared to that of 5.5 to 6.0 for eosinophil extracts. In addition, intact eosinophil inactivation of a given quantity of SRS-A occurs more rapidly than with an extract prepared from an identical number of cells.81 The activity of arvlsulfatase B per eosinophil may be high in the active phase of some host reactions, typified by filarial infestations, and drops to the normal range with recovery.82 The precise mechanisms of such enzyme adaptations or of mediator degradation bv eosinophils are unknown, but the expression of SRSA-inactivating capacity of intact eosinophils is dependent on their full metabolic activity.8 References 1.
2.
:3. 4.
a. 6.
8. 9.
10. 11. 12.
Hudson G: Quantitative study of the eosinophil granulocytes. Semin Hematol 5:166-186. 1968 Spry CJF: Mechanism of eosinophilia. \. Kinetics of normal and accelerated eosinopoiesis.Cell Tissue Kinet 4:351 -364, 1971 Zucker-Franklin D: Eosinophil function and disorders. Adv Intern Med 19:1-26, 1974 Gelfand M. Bernberg H: Tropical eosinophilic syndrome: A clinical description of the disorder as seen in S. Rhodesia. Cent Afr J Med 5:405-411, 1939 Lukens JN: Eosinophilia in children. Pediatr Clin N Am 19:969-981, 1972 Chusid MJ. Dale DC, W'est BC, Wolff SM: The hypereosinophilic syndrome: Analysis of fourteen cases with review of the literature. Medicine 34:1-28, 1973 Kav AB: Studies on eosinophil leucocyte migration. I. Eosinophil and neutrophil accumulation following antigen-antiboddy reactions in guinea pig skin. Clin Exp Immunol 6:75-86. 1970 Klein NC, Hargrove RL, Sleisenger NIH, Jeffries GH: Eosinophilic gastroenteritis. Medicine 49:299-319. 1970 Eidinger D, Raff NI, Rose B: Tissue eosinophilia in hypersensitivity reactions as revealed b- the human skin window. Nature 196:6&3-4, 1962 Goetzl EJ, Wasserman SI, Austen KP: Nodulation of the eosinophil chemotactic response in immediate hypersensitivity. Progress in Immunology II, \Vol. 4. Edited by L Brent, J Holborow. Amsterdam, North-Holland Publishing Co, 1974, pp 41-50 Goetzl EJ, %asserman SI, Austen KF: Eosinophil polymorphonuclear leukocvte function in immediate hypersensitivity. Arch Pathol 99:1-4, 1975 Goetzl EJ, W asserman SI. Gigli I, Austen KF: Modulation of eosinophil function in
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immediate hypersensitivitv. New Directions in Asthma. Edited bv Ni Stein. Park Ridge, Ill., American College of Physicians, 1975, pp 173-186 Cohen SG, Sapp TM: Experimental eosinophilia. IV. Eosinophilotactic influences of polysaccharides. Exp Mol Pathol 2:74-2, 1963 Litt M: Studies in experimental eosinophilia. VII. Eosinophils in lymph nodes during the first 24 hours following primarv antigenic stimulation. J Immunol 93:807-813, 1964 Walls RS, Hersey P, Quie PG: Macrophage-eosinophil interactions in the inflammatorv response to Trichinella spirais. Blood 44:131-136, 1974 Rose NR, Kite JH, Jr: Cell damage in autoimmune disease. International Convocation on Immunology. Edited by NR Rose, F Milgram. Basel, S. Karger, 1969, pp 247-259
17. Flax MH: Cell mediated hypersensitivity in the pathogenesis of experimental allergic thyroiditis, and its role in extralymphoid plasmacytosis. Cellular Interactions in the Immune Response. Edited by S Cohen, G Cudkowicz, RT McCluskey. Basel, S. Karger, 1971, pp 282-289 18. Torisu M, Yoshida T, Ward PA, Cohen S: Lymphocvte-derived eosinophil chemotactic factors. II. Studies on the mechanism of activation of the precursor substance bv immune complexes. J Immunol 111:145-1458, 1973 19. Kay AB, NMcVie JG, Stuart AE, Krajewski A, Tumbull LW: Eosinophil chemotaxis of supernatants from cultured Hodgkin's lymph node cells. J Clin Pathol 28:502-505, 1975 20. Mulller-Eberhard HJ: Chemistrv and reaction mechanisms of complement. Adv Immunol 8:1-80, 1968 21. Ruddy S, Gigli I, Austen KF: The complement system of man. N Engl j Med 287:489-495, 545-549, 592-596, 642-646, 1972 22. Vallota EH, Mu1ller-Eberhard HJ: Formation of C3a and C5a anaphvlatoxins in whole human serum after inhibition of the anaphvlatoxin inactivator. j Exp Med 137:1109-1123, 1973 23. Ward PA, Newman LJ: A neutrophil chemotactic factor from human C'5. J Immunol 102:93-99, 1969 24. Ward PA, Hill JH: CS chemotactic fragments produced by an enzyme in lysosomal granules of neutrophils. J Immunol 104:535-543, 1970 25. Ward PA: A plasmin-split fragment of C3 as a new chemotactic factor. J Exp Med 126:189-206, 1967 26. Ward PA: Complement-derived leukotactic factors in pathological fluids. J Exp Med 134 (Suppl):109S-113S, 1971 27. Tumer SR, Campbell JA, Lynn WS: Polymorphonuclear leukocvte chemotaxis toward oxidized lipid components of cell membranes. J Exp Med 141:1437-1441, 1975 28. Turner SR, Tainer JA, Lynn WS: Biogenesis of chemotactic molecules by the arachidonate lipoxygenase system of platelets. Nature 257:680-681, 1975 29. Goetzl EJ, Woods JM, Gorman RR: Preferential stimulation of human eosinophil polymorphonuclear leukocyte chemotaxis and random migration by 12-L-hydroxy5, 8, 10, 14-eicosatetraenoic acid (HETE). Unpublished data 30. Kaplan AP, Kay AB, Austen KF: A prealbumin activator of prekallikrein. III. Appearance of chemotactic activity for human neutrophils bv the conversion of human prekallikrein to kallikrein. J Exp Med 135:81-97, 1972 31. Kaplan AP, Goetzl EJ, Austen KF: The fibrinolvtic pathway of human plasma. II. The generation of chemotactic activity by activation of plasminogen proactivator. J Clin Invest 52:2591-2595, 1973 32. Goetzl EJ, Schreiber AD, Austen KF: Chemotactic activity of components of the kallikrein-kinin and fibrinolytic sequences. Chemistry and Biology of the Kallikrein-
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Kinini S-stem in Health and Disease. Edited b- J Pisano. Bethesda. \Md.. National Institutes of Health (In press) Rudd%- S. Austen KF. Goetzl EJ: Chemotactic activity derived from interaction of factors D and B of the properdin pathwvay wvith cobra venom factor or C:3b. J Clin Invest 55:587-5.392. 1975 Kay AB. Stechschulte DJ. Austen KF: An eosinophil leukocv-te chemotactic factor of anaphylaxis. J Exp NIed 1:33:602-619. 1971 Kay AB. Austen KF: The IgE-mediated release of an eosinophil leukocv-te chemotactic factor from human lung. j Immunol 107:899-902. 1971 Archer RK: The eosinophilic response in the horse to intramedullary and intradermal injections of histamine. ACTH. and cortisone. J Pathol Bacteriol 72:87-94. 1956 Vegad JL. Lancaster MC: Eosinophil leucocvte-attracting effect of histamine in the sheep skin. Ind J Exp Biol 10:147-148. 1972 Wasserman SI. Goetzl EJ. Kaliner MA. Austen KF: Modulation of the immunologic release of the eosinophil chemotactic factor of anaphylaxis from human lung. Immunology 26:677-684. 1974 Wasserman SI. Goetzl EJ. Austen KF: Preformed eosinophil chemotactic factor of anaphylaxis (ECF-A). J Immunol 1 12::351-3:.8. 1974 Lewis RA. Goetzl EJ. Wasserman SI. Valone FH. Rubin RH. Austen KF: The release of four mediators of immediate hypersensitivity from human leukemic basophils. J Immunol 114:87-92. 1975 Kaliner \M. Wasserman SI. Austen KF: Immunologic release of chemical mediators from human nasal polvps. N EngI J Med 289:277-281. 1973 Wasserman SI. Goetzl EJ. Ellman L. Austen KF: Tumor-associated eosinophilotactic factor. N Engl J Med 290:420-424. 1974 Goetzl EJ. Austen KF: Purification and synthesis of eosinophilotactic tetrapeptides of human lung tissue: Identification as eosinophil chemotactic factor of anaphylaxis. Proc Natl Acad Sci U SA 72:4123-4127. 1975 Austen KF. Wasserman SI. Goetz] EJ: Mast cell-derived mediators: Structural and functional diversity and regulation of expression. Proceedings of Nobel Symposium :3;3. Molecular and Biological Aspects of the Accute Allergic Reactions. Edited b\ SGO Johansson. K Strandberg. B Uvnas. )ndcon. Plenum Publishing Co. In press (oetzl EJ. Austen KF: Specificity and modulation of the eosinophil polymorphonuclear leukocyte response to the eosinophil chemotactic factor of anaphVlaxis (ECF-A)." Wasserman SI. Boswell RN. Drazen J\I. Goetzl EJ. Austen KF: Functional specificitv of the human lung acidic tetrapeptides constituting eosinophil chemotactic factor of anaphvlaxis (ECF-A). J Allerg Clin Immunol 57:190. 1976 (Cetzl EJ. Rubin RH. \McDonough J. Tashjian AH Jr. Austen KF: Production of eosinophilotactic peptides by bronchogenic carcinoma in situ and in vitro. Clin Res In press) Abstr Bosw ell NB. Austen KF. Goetzl EJ: Structural and functional characterization of ECF oligopeptides from rat mast cells and leutkemic basophils. U L-npublished data) Czarnetski B\t. K5nig W. Lichtenstein L\I: Antigen-induced eosinophil chem(tactic factor ECF release by human leukocvtes. Inflammation 1:201-213. 1976 Kay AB. Shin JS. Austen KF: Selective attraction of eosinophils and synergism between eosinophil chemotactic factor of anaphylaxis (ECF-A) and a fragment cleaved from the fifth component of complement (C5a). Immunology 24:969-976. 197:3 Snvderman R. Shin HS. Hausman \IH: A chemotactic factor for mononuclear leukocvtes. Proc Soc Exp Biol Med 1:38::387-390. 1971
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52. Bokisch VA, Muller-Eberhard HJ: Anaphylatoxin inactivator of human plasma: Its isolation and characterization as a carboxypeptidase. J Clin Invest 49:2427-2436, 1970 53. Berenberg JL, Ward PA: Chemotactic factor inactivator in normal human serum. J CGn Invest 52:1200-1206, 1973 54. Goetzl EJ, Boswell RN, Austen KF: The tetrapeptides comprising eosinophil chemotactic factor of anaphylaxis (ECF-A): Structural determinants of chemotactic activity. Fed Proc 35:515, 1976 (Abstr) 55. Ward PA, Becker EL: The deactivation of rabbit neutrophils by chemotactic factor and the nature of the activatable esterase. J Exp Med 127:693-709, 1968 56. Ward PA, Becker EL: Biochemical demonstration of the activatable esterase of the rabbit neutrophil involved in the chemotactic response. J Immunol 105:1057-1067, 1970
57. Goetzl EJ, Austen KF: A neutrophil immobilizing factor derived from human leukocytes. I. Generation and partial characterization. J Exp Med 136:1564-1580, 1972
58. Goetzl EJ, Gigli I, Wasserman S, Austen KF: A neutrophil immobilizing factor derived from human leukocvtes. II. Specificity of action on polymorphonuclear leukocyte mobility. J Immunol 111:938-945, 1973 59. Nishioka K, Constantopoulos A, Satoh PS, Najjar VA: The characteristics, isolation and synthesis of the phagocytosis stimulating peptide tuftsin. Biochem Biophys Res Commun 47:172-179, 1972 60. Goetzl EJ: Plasma and cell-derived inhibitors of human neutrophil chemotaxis. Ann NY Acad Sci 256:210-221, 1975 61. Goetzl EJ, Austen KF: Stimulation of human neutrophil leukocyte aerobic glucose metabolism by purified chemotactic factors. J Clin Invest 53:591-599, 1974 62. Goetzl EJ, Wasserman SI, Gigli I, Austen KF: Enhancement of random migration and chemotactic response of human leukocytes by ascorbic acid. J Clin Invest 53:813-818, 1974 63. Wasserman SI, Whitmer D, Goetzl EJ, Austen KF: Chemotactic deactivation of human eosinophils by the eosinophil chemotactic factor of anaphylaxis. Proc Soc Exp Biol Med 148:301-306, 1975 64. Najjar VA: The physiological role of -t -globulin. Adv Enzymol 41:129-178, 1974 65. Colley DG: Eosinophils and immune mechanisms. I. Eosinophil stimulation promoter (ESP): A lymphokine induced by specific antigen or phytohemagglutinin. J Immunol 110:1419-1423, 1973 66. Clark RAF, Galin JI, Kaplan AP: The selective eosinophil chemotactic activity of histamine. J Exp Med 142:1462-1476, 1975 67. Vaughn J: The function of the eosinophile leukocyte. Blood 8:1-15, 1953 68. Parish WE: Investigations on eosinophilia. The influence of histamine, antigen-antibody complexes containing Y 1 or Y 2 globulins, foreign bodies (phagocytosis) and disrupted mast cells. Br J Dermatol 82:42-64, 1970 69. Clark RAF, Galin JI, Kaplan AP: Histamine-dependent inhibition and enhancement of eosinophil migration to chemotactic agents. J Allerg Clin Immunol (In press) 70. Kovacs BA: Antihistamine effect of eosinophil leukocytes. Experientia 6:349-50,
1950 71. Archer RK, Feldberg W, Kovacs BA: Antihistamine activity in extracts of horse eosinophils. Br J Pharmacol 18:101-108, 1962 72. Hubscher T, Eisen AH: A possible immunopharmacological role of human eosinophils in allergic reactions. Mechanisms in Allergy: Reagin-Mediated Hypersensitivity. Edited by L Goodfriend, A Sehon, RP Orange. New York, Marcel Dekker Inc., 1973, pp 413-437 73. Zeiger RS, Yurdin DL, Colten HR: Histamine metabolism. II. Cellular and sub-
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cellular localization of the catabolic enzymes, histaminase and histamine methyl transferase in human leukocvtes. J Allerg Clin Immunol (In press) Wasserman SI, Goetzl EJ, Austen KF: Inactivation of slow reacting substance of anaphvlaxis by human eosinophil arvlsulfatase. J Immunol 114:645-649, 1975 Kater LA, Goetzl EJ, Austen KF: Isolation of human eosinophil phospholipase D. J Clin Invest 57:1173-1180, 1976 Roy AB: The sulfatase of ox liver. I. The complex nature of the enzvme. Biochem J 53:12-15, 1953 Dodgson KS, Spencer B: Assay of sulfatases. Meth Biochem Anal 4:211-255, 1957 Allen E, Rov AB: The sulfatase of ox liver. XI. The isoelectric focussing of a purified preparation of sulfatase B. Biochim Biophys Acta 168:243-251, 1968 Bleszvnski W, Lemicki A, Lewosz J: Kinetic properties of three soluble arvlsulfatases from ox brain, homogeneous in polyacrylamide gel electrophoresis. Enzymologia 37:314-324, 1969 Baum H, Dodgson KS, Spencer B: The assay of arylsulphatases A and B in human urine. Clin Chim Acta 4:453-455, 1959 Wrasserman SI, Goetzl EJ, Austen KF: Inactivation of human SRS-A by intact human eosinophils and by eosinophil arvlsulfatase. J Allerg Clin Immunol 55:72, 1975 (Abstr) Rubin RH, Austen KF, Goetzl EJ: Studies of immediate hypersensitivity in a patient with Acanthocheionema perstans filarial infection. J Infect Dis 131(Suppl):S98-103, 1975
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[End of Article]
Eosinophil migration toward a concentration gradient of a chemotactic factor is regulated at four levels. Diverse immunologic pathways generate stimuli with eosinophil chemotactic activity, including the complement products C5a and a fragment of C3a and the peptide products of mast cells and basophils activated by IgE-mediated reactions, such as eosinophil chemotactic factor of anaphylaxis (ECF-A) and other oligopeptides. The intrinsic preferential leukocvte activitv of the chemotactic stimuli represents the second level of modulation, with ECF-A and other mast cell-derived peptides exhibiting the most selective action on eosinophils. The third level of control of eosinophil chemotaxis is composed of inactivators and inhibitors of chemotactic stimuli and is exemplified by degradation of C5a by anaphylatoxin inactivator or chemotactic factor inactivator and of ECF-A by carboxvpeptidase-A or aminopeptidases. The activity of ECF-A is uniquely suppressed by equimolar quantities of its NH2terminal tripeptide substituent, presumably by eosinophil membrane receptor competition. Factors comprising the fourth level of regulation, which alter eosinophil responsiveness to chemotactic stimuli, include the chemotactic factors themselves, through deactivation; nonchemotactic inhibitors such as the COOH-terminal tripeptide substituent of ECF-A, the neutrophil-immobilizing factor (NIF), the phagocytosisenhancing factor Thr-Lys-Pro-Arg, and histamine at concentrations greater than 400 ng/ml; and nonchemotactic enhancing principles represented by ascorbate and by histamine at concentrations of 30 ng/ml or less. Local concentrations of eosinophils called to and immobilized at the site of a hypersensitivity reaction may express their regulatory functions by degrading the chemical mediators elaborated including histamine, slow-reacting substance of anaphvlaxis (SRS-A), and platelet-activating factor (PAF) by way of their content of histaminase, arylsulfatase B, and phospholipase D. respectively. Immunologic pathways may thus provide the capability for early and specific host defense reactions with a later influx of eosinophils preventing irreversible local tissue alterations or distant organ effects (Am J Pathol 85:419436, 1976)
EOSINOPHIL POLY\MORPHONNUCLEAR (PMNN) leukocytes originate from myeloid precursors in bone marromv, circulate in peripheral blood for a brief period, and then move to tissue sites localized predominantly in the lungs, skin, intestines, and genitourinary tract.' An elevated level of eosinophils in peripheral blood and in tissues is a characteristic feature of systemic hypersensitivity reactions to a wvide variety of stimuli including drugs, environmental allergens, parasitic and other chronic infectious From the Departments of Mledicine. Harvard \ledical School and the Robert B. Brigham Hospital. Boston. Mlassachusetts. Supported by Grants Al-07722. lX-10:36. and HL-173S2 from the National Institutes of Health: Dr. Goetzl is an Insvestigator of the How-ard Hughes Mledical Institute. Presented at the Sixtieth Xnnual Meeting of the Federation of Xmerican Societies for Experimental Biology. Anaheim. Calif.. April 11. 1976. Address reprint requests to Dr. Edsward J. Goetzl Department of Mledicine. Harxard \ledical School. Boston. MA 0-2115. 419
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agents, and plasma and tissue proteins.3` Although eosinophils arrive at tissue foci of hypersensitivity reactions relatively late in comparison to the time required for full expression of humoral events, they frequently persist in involved tissues for long intervals thereafter without leading to further injurv.7-9 The selective influx of eosinophils to the site of a hypersensitivity reaction is determined by the effective activity of diverse chemotactic factors and the responsiveness of the available eosinophils. The eosinophils are trapped in the involved tissues by the local immobilizing effects of the chemotactic principles themselves and of several inhibitors.10'11 Thus a sufficient number of eosinophils are maintained at the focus of a hypersensitivity reaction to enable the fulfillment of their specific regulatory role. 1012
Generation of Eosiophil Chemotactic Factors by Immune Reactions Cdul Immunity
Eosinophil chemotactic activity in lymphoid tissue has been inferred from histologic studies of animals that received an initial antigen injection and demonstrated a peak of eosinophilia in the perifollicular regions of lymph nodes 4 to 12 hours later.13'14 Mice receiving a booster intraperitoneal injection of Trichinella larvae demonstrated a prominent increase in both eosinophils and mononuclear leukocytes in the peritoneal fluid that was largelv T-lymphocyte dependent.'5 Guinea pigs immunized with thyroid extract in complete Freund's adjuvant developed a thyroiditis consisting of mononuclear leukocyte infiltrates.'16"7 Eosinophils were found in the thyroiditis lesions only in those animals that also had detectable antithyroid antibodies, suggesting a dependence of tissue oesinophilia on both serum antibody and lymphocyte factors. Lymphocyte-related eosinophil chemotactic factors with in vitro activity have been recognized in two settings. Specific antigen stimulation in vitro of lymphocytes from guinea pigs with delayed hypersensitivity generated a precursor macromolecule that exhibited eosinophil chemotactic activity when mixed with homologous immune complexes.'8 Preferential eosinophil chemotactic activity recently was found in supernatants from cultured lymph node cells of patients with Hodgkin's disease and was composed of four peaks of molecular weights ranging from 500 to 30,000 daltons by gel filtration.'9 Humoral Imunity
Both the classic and altemative complement pathways can generate substantial eosinophil chemotactic activity that is attributable to the
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anaphylatoxins (5a and a fragment of the third component of complement (C3).2* The G5a fragment of the fifth component of complement (C5) is a protein of approximate molecular weight of 12,000 that can be generated as well by the cleavage of C5 by a neutrophil lysosomal protease.'2 The C3 fragment is a protein of approximate molecular weight of 9,000 that can also be generated directly by digestion of C3 with plasmin or with a protease in some inflamed tissues.25," Other factors demonstrating eosinophil chemotactic activity include the platelet enzyme products of arachidonic and eicosapentenoic acids 2-29 and those principles whose activities are dependent upon the integrity of an enzyme active site, including kallikrein, plasminogen activator, and the C3 convertase of the alternative complement pathway.3"33 Eosinph Clemotact Factor of A This factor (ECF-A) was discovered in 1971 as the mediator that is responsible for most of the eosinophil chemotactic activitv released during anaphylactic reactions in vitro in guinea pig " and human 35 lung slices. Of the other mediators of immediate hypersensitivity, only histamine stimulated directed migration of eosinophils in vitro; however, its action was transient and it also lacked apparent in vivo chemotactic activity.3"37 The release of ECF-A from human tissues by IgE-dependent mechanisms exhibited a time-course, biochemical requirements, and responsiveness to the intracellular levels of cyclic nucleotides similar to the release of histamine from the same tissues.3' ECF-A was present preformed in purified rat mast cells in association with the granules 3" and was also extractable from human leukemic basophils 40 and mast cell-rich tissues such as human lung and nasal polyps.3"41 Preliminary physicochemical characterization of ECF-A obtained by extraction or immunologic activation of guinea pig and human lung tissues and rat mast cells revealed a molecular weight of approximately 500 by filtration on Sephadex G-25S,"33 inactivation by digestion with subtilisin or pronase but not with trvpsin or chymotrvpsin,42 and two anodal peaks of activitv on high-voltage paper electrophoresis at pH 7.10 The extraction of unchallenged human lung tissue fragments with either butanol: glacial acetic acid (10:1, v/v) or Tyrode's solution buffered at pH 8.6 with 0.1 M Tris-HCI solubilizes eosinophil chemotactic activity in high yield." Gel filtration on Sephadex G-25 of Iyophilized extracts resuspended in 0.1 M acetic acid resolves four peaks of chemotactic activity."'" The largest peak, appearing in the exclusion volume, is preferentially chemotactic for neutrophils and has been termed neutrophil chemotactic factor of anaphylaxis (NCF-A).4'" An intermediate peak of
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approximate molecular weight of 2500 to 3500 is preferentially active for eosinophils but, unlike NCF-A and ECF-A, is present only in anaphylactic diffusates in amounts representing a small percent of the activity in tissue stores. The two smaller peaks, which exhibit molecular weights of 300 to 500 and 150 to 200, are selectively chemotactic for eosinophils and correspond, respectively, to ECF-A and to a region between ECF-A and histamine that may represent a synergistic action of the two factors."', The 300 to 500 molecular weight peak of preferential eosinophil chemotactic activity from extracts of human lung, corresponding to the Sephadex G-25 peak of ECF-A in anaphylactic diffusates, was isolated by sequential purification using ion-exchange chromatography, gel filtration on Sephadex G-10, and paper chromatography.' The ECF-A activitv from IgE-mediated reactions in human lung tissues appeared along with that in extracts of human lung during parallel purification procedures. Fractions of the highly purified extracts with eosinophilotactic activitv contained two acidic tetrapeptides of amino acid sequence Ala-Gly-Ser-Glu and Val-Gly-Ser-Glu. Both the synthetic tetrapeptides and purified ECF-A were maximally active in amounts from 0.1 to 1.0 nmole/ chemotactic chamber, representing concentrations of 10 7 to 10' M.'4 The dose-response relationships of highly purified ECF-A from human lung extracts and the synthetic tetrapeptides were established in the modified Boyden assav (Text-figure 1). Purified ECF-A was quantitated bv acid hydrolysis and analysis of constituent amino acids, and the target leukocytes were from a patient with 88% peripheral blood eosinophilia. For all three stimuli, eosinophilotactic activity was detectable at 10 M and reached a plateau of 10 to 14 eosinophils/high-power field (eos/hpf) bv 107 M. Portions of the initial Sephadex G-25 peak of ECF-A extracted from lung attracted 12 eos/hpf for 25 Al, 28 eos/hpf for 50 Al, and 32 eos/hpf for 100 Ml. Thus, while not achieving a maximum level of eosinophil chemotaxis comparable to that for a crude preparation of ECFA, equivalent doses of purified native ECF-A and svnthetic tetrapeptides were equally and preferentially active on eosinophils.4" The intraperitoneal administration of 0.1 to 1.0 nmole of synthetic valyl-tetrapeptide to guinea pigs elicited an early selective influx of eosinophils followed bv neutrophils." The in vivo potency of the tetrapeptides paralleled the in vitro dose-response relationship, and their effect in guinea pigs correlated with the preferential attraction of eosinophils in vitro, although an ability to induce directed migration of neutrophils was also recognized. A 3(0 to 500 molecular weight chemotactic factor that preferentially attracts eosinophils and, like ECF-A, is inactivated by subtilisin digestion has been recognized in extracts of tumor tissue and supernatants
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TEXT-FIGURE 1-Dose-response of eosinophil chemotactic actisits of ECF-X purified from human lung (open squares) and sy nthetic tetrapeptides-alany ltetrapeptide (open circles) and saly l-tetrapeptide .solid circles). Human lung ECF-A swas purified by using Sephadex G-25. Dowex1. Sephadex G-10. and deseending paper chromatographn- equal quantities of the alan% l- and valyl-tetrapeptide pools were recombined for the chemotactic stimulus. Background eosinophil migration was four eosinophils per high-power field.
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of cell cultures of patients with bronchogenic carcinoma and peripheral blood eosinophilia.47 This factor is less acidic than ECF-A and elutes from Dowex-1 at a pH of 4.5 to 3.8. The intermediate molecular weight peak of eosinophil chemotactic activity, released in only small quantities by IgE-dependent reactions, is contained in large amounts along with ECF-A in preformed stores in rat mast cells, in association with the granules,46 and in mast cell-rich tissues." This oligopeptide ECF is inactivated bv digestion with carboxypeptidase-A and is preferentiallv chemotactic for eosinophils; thus, it may be either a precursor of ECF-A or a distinct but functionallv related eosinophil chemotactic factor. A comparable peak of eosinophilotactic activity has been recognized in tumor extracts, urine and cell culture supernatants of patients with bronchogenic carcinoma and eosinophilia,47 and in supernatants of cell cultures from lymph nodes of patients with Hodgkin's disease."9 The availabilitv in mast cell stores of eosinophilotactic peptides of vary'ing sizes provides for rapid establishment of a chemotactic gradient by the smaller factors and maintenance of the gradient bv larger factors, thus ensuring an earlv and continuing movement of eosinophils to a focus of allergic reaction.
Regulation of the Eosinophil Chernotactic Response The generation of chemotactic factors during immunologic and other inflammatory reactions represents the initial pathway in the regulation of the eosinophil chemotactic response (Text-figure 2, Table 1). Immediate hypersensitivityT reactions liberate ECF-A and small quantities of larger ECF peptides.",451' Activation of the classic or alternative (properdin) complement pathways generates the anaphylatoxins C3a and C-a.`0 Other factors with substantial eosinophil chemotactic activity are elabo-
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rated by- exposure of PM N leukocytes to a calcium ionophore or phagocytosable particles,49 antigen activation of lymphocytes,18 and oxidative conversion of arachidonic acid.27The second lev-el of modulation of eosinophil chemotaxis consists of the concentration, potency, and leukocyte specificity of each chemotactic factor wvhich may- be determined by specific structural features. Highlxpurified ECF-A. synthetic ECF-A tetrapeptides, and partially purified larger peptides from human lung tissue or rat mast cells preferentiallxattract human eosinophils in vitro -hen the eosinophils represent 10% or more of the target leukocyte pool.10 43 50 Both the low,-- and intermediatemolecular-w-eight ECF peptides derived from bronchogenic carcinoma are also preferentially chemotactic for eosinophils.47 The complementderixved factors C5a and a fragment of C3 attract eosinophils, neutrophils. and monocvtes equally.l10245051 In contrast, the plasma -globulin enzy-mes, kallikrein and plasminogen activator, and the alternative complement path way C3 convertase are selectiv-ely chemotactic for neutrophils.32.33 Inhibition and Inactivation of Eosiophil Chemotactic Factor Activity
The third level of regulation of eosinophil chemotaxis encompasses the inhibitors or inactivators of chemotactic factors (Table 1. Text-figure 2). The anaphylatoxin inactiv-ator suppresses C5a-mediated leukotaxis.52 The chemotactic factor inactiv-ator (CFI), w hich is composed of tv-o or more exoproteolvtic enzymes present in serum and cells, irreversibly inactivates numerous chemotactic factors.53 While the in vivo pathw-ays of degradation of ECF-A hav-e not vet been elucidated, limited digestion of the valyl- and alanyl-tetrapeptides -with either aminopeptidase- M or carboxv-peptidase-A reduces their eosinophil chemotactic acti-ity in proportion to the extent of liberation of NH2- and COOH-terminal amino acids, respectiv ely. 3 Some hydrophobic amino acid deriv-atives and peptide analogs or sub-
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stituents of ECF-A tetrapeptides can inhibit the chemotactic activity of the intact tetrapeptide as exemplified by valine-amide (val-amide) and the NH2-terminal tripeptides Ala,Aal-Gl%-Ser, which possess neither chemotactic nor deactivating capacity.4554 Both val-amide and Val-Clv-Ser suppress the eosinophilotactic activity of 107M valvl-tetrapeptide wvhen present concomitantly in the stimulus compartment of the Boyden chamber producing 30%c mean inhibition at 10-6M and 10-7WM respectively.54 Val-amide and Val-GIl-Ser presumably inhibit the eosinophil chemotactic response by competing wvith ECF-A for a hydrophobic site on the eosinophil surface and thus do not affect eosinophil random migration. The capacity of Val-Gly-Ser to inhibit eosinophil chemotaxis to equimolar quantities of valvl-tetrapeptide has been observed in vivo as wvell." Cell-Directed Modulation of the Eosinophil Chemotactic Response
The fourth level of regulation of eosinophil chemotactic migration includes factors that alter chemotaxis by influencing the responsiveness of the cells (Table 1 Text-figure 2). The effects of these factors on eosinophils are independent of the specific chemotactic stimulus, result in enhancement or suppression of chemotaxis and generally extend to similar actions on spontaneous migration. This group of noncytotoxic factors consists of the chemotactic factors themselves that induce an eosinophil chemotactic unresponsiveness termed deactivation,55' and a variety of cell-directed inhibitors and enhancers that lack chemotactic activity-.57-62 Cell-Directed Inhibitors
Chemotactic deactivation of leukocytes by prior exposure to an active chemotactic stimulus exhibits a leukocv-te specificity \-hich parallels the chemotactic selectivity of the stimuli.'032 Human eosinophils exposed to chemotactic concentrations of C3a or partially purified ECF-A rapidly become unresponsive to both the homologous and heterologous stimuli.' Concentrations of either stimulus as lowv as one-twentieth the minimal chemotactic dose fully deactivated eosinophils in a time-dependent fashion. Human eosinophils preincubated wvith synthetic alanvl- or valvltetrapeptide at levels of 108 to 10-10 NM for a minutes at 37 C wvere rendered -5 to 80%'- unresponsive to a standard 10_'NI stimulus of either peptide preparation.45.54 With a 10-12 NM concentration of synthetic tetrapeptide deactivation wvas 40% after 10 minutes and reached 75 to 8Occ after 90 minutes. Thus, one-one hundredths of the minimal chemotactic dose of either tetrapeptide comprising ECF-A gave brisk and complete deactivation of human eosinophils. This uniquely lo,- threshold of eosinophils deactivation may account for their local immobilization at involved sites
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without tissue damage, since lysosomal enzyme release and metabolic activation of the eosinophils apparently requires higher concentrations of chemotactic factors than deactivation. Several factors have been identified that are themselves devoid of chemotactic activity but inhibit chemotaxis and spontaneous migration of eosinophils by an action on the cells. The neutrophil-immobilizing factor (NIF) is an inhibitor of polymorphonuclear leukocyte random and directed migration that is generated by incubating human peripheral blood mononuclear or polymorphonuclear leukocytes in acid medium (ANIF), with endotoxin (ENIF), or with phagocytosable particles (PhNIF).s7 Neutrophil-immobilizing factor is separable from coexistent chemotactic activity in the leukocyte supematants by heating and gel filtration on Sephadex G-25, and exhibits an apparent molecular weight of approximately 4000 to 5000 daltons. Partially purified NIF irreversibly suppresses the random migration and chemotactic response to diverse stimuli of eosinophils and neutrophils but not of mononuclear leukocytes.5 Inhibition of migration by NIF is not associated with any effect on leukocyte viability, phagocytosis, adherence to surfaces, or baseline or stimulated HMPS activity." Neutrophil-immobilizing factor also inhibits the stimulation of random migration and chemotaxis that results when neutrophils are preincubated with high concentrations of ascorbate." Human polymorphonuclear and mononuclear leukocytes also contain extractable preformed stores of higher molecular weight chemotactic inhibitors which are released during phagocytosis, but not by acid or endotoxin treatment.57 These larger inhibitors are functionally similar to NIF with an action apparently limited to inhibition of random and directed migration of eosinophil and neutrophil PMN leukocytes. Filtration on Sephadex G100 of the inhibitors in the excluded volume from Sephadex G-25 revealed two factors of molecular weights 30,000 to 40,000 and >120,000. Gly-Ser-Glu, the ECF-A COOH-terminal tripeptide, inhibits chemotaxis by a suppressive action on the cells."'," Preincubation of human eosinophils with Gly-Ser-Glu, which lacks substantial chemotactic activity, resulted in a dose- and time-dependent suppression of their subsequent response to natural or synthetic ECF-A that reached 80% inhibition after a 20-minute exposure to 10-8 M tripeptide." The action of the COOH-terminal tripeptide appears to be on the cells themselves as evidenced by the persistence of its effect after cell washing and the failure of 10 M tripeptide to inhibit significantly when present on the stimulus side of a Boyden chamber. A leukocyte phagocytosis-stimulating tetrapeptide of amino acid sequence L-threonyl- L-lysyl- L-prolyl- L-arginine (Thr-Lys-Pro-Arg), desig-
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nated "Tuftsin", has been purified from digestion mixtures of a phagocvtosis-enhancing cytophilic fraction of human -r -globulin and a proteol tic enzvme contained in human polymorphonuclear leukocytes.596 Nanogram amounts of the synthetic or purified natural peptide enhances by 2 to 2.3-fold the phagocytosis of latex particles or Staphylococcus aureus by human, dog, and guinea pig polvmorphonuclear leukocytes and bv mouse peritoneal and rabbit lung macrophages.59 The synthetic tetrapeptide lacks chemotactic activity, but preincubation of purified populations of human eosinophils, neutrophils, and mononuclear leukocytes with concentrations ranging from 0.1 to 100 ng/ml resulted in a dose-related inhibition of eosinophil and neutrophil random migration and chemotaxis to a preferential stimulus (Text-figure 3). In contrast, mononuclear leukocy-te random and chemotactic migration w,ere uninfluenced by the tetrapeptide. Thus, the inhibitory action of Thr-Lvs-Pro-Arg resembles that of the larger basic peptide NIF in suppressing migration of eosinophils and neutrophils wvithout affecting their viability or hexose monophosphate shunt activity and without decreasing mononuclear leukocyte migration.5 Thr-Lv-s-Pro-Arg but not NIF stimulates leukocyte phagocytosis in the same concentration range that is optimal for inhibition of migration. Cell-Directed Enhancing Factors
A variety of pathways vield factors which directly augment eosinophil random migration and enhance their chemotactic responsiveness. When lymphocytes of mice previously sensitized with schistosomes were incubated wvith specific schistosomal egg antigen or phytohemagglutinin, RANDOM MIGRATION
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TEXT-FIGIRE 3-Thr-Lv s-ProXrg tetrapeptide inhibition of leukoc'te migration. Eosinophils open squaresl from a patient w-ith a peripheral blcxd concentration of S4 '5 'cvere emplosed. Neutrophils solid circles and mononuclear leukoc-tes (open triangles) wsere standard preparations from normal donors." Control random migration was eosinophils. 13 hpf. neutrophils. 14 hpf. mononuclear leukocytes. 12 hpf. Chemotaxis %vas eosinophils to ECF-A. 34 hpf. neutrophils to kallilkrein 48 hpf and mononuclear leukoc'-tes to C.3a. 26 hpf.
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they elaborated a 25,000 to 35,000 molecular weight principle, termed eosinophil stimulation promoter." This factor increased mouse eosinophil random migration from agarose droplets but has not been assessed for its activity on chemotaxis. Ascorbic acid is a nonchemotactic principle that enhances the random migration and chemotactic responsiveness of neutrophils, eosinophils, and mononuclear leukocytes by 100 to 300% of baseline values with concomitant and equivalent stimulation of leukocyte HMPS activity.' Ascorbate, at concentrations of 2 to 10 X 10 3 M, results in dose-related enhancement of both migration and HMPS activity of eosinophils and other leukocytes, but has no effect on phagocytosis. Histamine Effects on Es
M i
Histamine exhibits a triphasic concentration-dependent modulation of eosinophil migration,'," with enhancement of random and directed migration at levels of 1 to 3 x 10' M, inhibition of random and directed migration at levels of 4 X 10' M or higher, and induction of directed migration in the intermediate range. In the midconcentration range of 3 X 10-7 M to 1.25 X 10 M (33 to 139 ng/ml), histamine induces directed migration of eosinophils in some in vitro chambers that employ thin polycarbonate filters or brief incubation periods with standard cellulose nitrate filters." However, its eosinophilotactic activity in a conventional chemotactic assay differs from that of eosinophil chemotactic factors such as ECF-A and CSa. Unlike purified or synthetic ECF-A, histamine failed to induce a true wave of cell movement as assessed by a shift of the maximum density of eosinophils into the filters.'" Further, 105 M histamine added to both sides of the filter often resulted in enhanced random migration of a small fraction of the eosinophils." Finally, histamine has no documented in vivo eosinophil chemotactic activity. Early in vivo studies revealed an influx of eosinophils into pulmonary tissue, in association with a peripheral blood eosinophilia, in response to the intraperitoneal or intravenous injection of large doses of histamine in guinea pigs.'-' However, neither local intraperitoneal nor intradermal eosinophilia followed histamine injection in animals.',' The major actions of histamine on in vitro eosinophil migration involve modulation of their responsiveness to chemotactic factors. The addition of histamine to the cell compartment of Boyden chambers at a dose of 30 ng/ml, which stimulated only minimal eosinophil migration, enhanced the chemotactic response of eosinophils to a low concentration of valyltetrapeptide in a more than additive fashion. An identical protocol utilizing a dose of 600 ng/ml histamine in the cell compartment inhibited the spontaneous migration of eosinophils and suppressed the eosinophil re-
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sponse to valyl-tetrapeptide.5 Thus, the effects of histamine on eosinophil movement include brief directed migration at intermediate doses, and cell-directed modulation of random and directed migration that is stimulatory at low levels and suppressive at high levels. High-dose histamine suppression of both chemotaxis and histamine-induced directed migration has been attributed to elevation of intracellular cyclic AMP levels since it is prevented by the simultaneous addition of H2-blockers, while histamine enhancement of eosinophil chemotactic responsiveness was reduced by H-1 blockers."'" Eosinopil Fuct in Immediate Hypersensitiity After arriving at a site of an immediate hypersensitivity reaction, the eosinophil may manifest regulatory functions that could limit or eventually terminate the allergic reaction. Although under special circumstances eosinophil-derived factors may influence the release or activity of chemical mediators of immediate hypersensitivity,7172 their predominant regulatory role is the degradation of chemical mediators by way of specific constituent enzymes. 1112 " Eosinophil granules contain a histaminase capable of oxidative deamination of histamine,73 an arylsulfatase B which can degrade SRS-A,74 and a phospholipase D with the capacity to inactivate a PAF.75 The arylsulfatase B preferentially present in eosinophils as compared to other leukocytes has been purified by sequential gel filtration on Sephadex G-100 and chromatography on carboxymethyl cellulose.74 It exhibits properties of a Type II arylsulfatase by virtue of preferential cleavage of pnitrocatechol sulfate (PNCS) over p-acetylphenyl sulfate or p-nitrophenyl sulfate and inhibition by sulfate and phosphate but not cyanide.76 Physicochemical characterization identified the eosinophil arylsulfatase as Type B since it is soluble, has a molecular weight of approximately 60,000, and possesses a basic isoelectric point; further it is inhibited by chloride ions in the presence of pyrophosphate.""w Purified eosinophil arylsulfatase, as well as arylsulfatase B from human lung,74'81 inactivates human SRS-A in a time-dependent reaction with a pH optimum of 5.5 to 6.0 that is- comparable to that for cleavage of PNCS.10'74 That the arylsulfatase B active site is responsible for SRS-A inactivation was indicated by the association of both PNCS-cleaving and SRS-A-inactivating activities during purification of the enzyme, and the ability of SRS-A to inhibit cleavage of the preferred synthetic substrate.74 A phospholipase D is also preferentially contained in human eosinophils, and has been purified by sequential anion exchange chromatographv and gel filtration on Sephadex G-100 where it has an apparent
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molecular wveight of 60,000.75 The purified phospholipase D exhibited a pl of 3.8 to 6.2 on polyacrvlamide gel isoelectric focusing. The ability of eosinophil-derived phospholipase D to cleave choline from lecithin and lvsolecithin, and its capacitv to inactivate a rat platelet-activating factor (PAF) were associated during purification of the enzvme, and there was apparent reciprocal inhibition of choline-generating activitv by PAF and of PAF-inactivating activity by phosphatidvl-choline.75 Mediator degradation by intact eosinophils is more efficient and possibly more relevant to in uivo pathwavs since the eosinophils rapidly inactivate SRS-A with an optimal pH of 7.0 to 7.4, as compared to that of 5.5 to 6.0 for eosinophil extracts. In addition, intact eosinophil inactivation of a given quantity of SRS-A occurs more rapidly than with an extract prepared from an identical number of cells.81 The activity of arvlsulfatase B per eosinophil may be high in the active phase of some host reactions, typified by filarial infestations, and drops to the normal range with recovery.82 The precise mechanisms of such enzyme adaptations or of mediator degradation bv eosinophils are unknown, but the expression of SRSA-inactivating capacity of intact eosinophils is dependent on their full metabolic activity.8 References 1.
2.
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Hudson G: Quantitative study of the eosinophil granulocytes. Semin Hematol 5:166-186. 1968 Spry CJF: Mechanism of eosinophilia. \. Kinetics of normal and accelerated eosinopoiesis.Cell Tissue Kinet 4:351 -364, 1971 Zucker-Franklin D: Eosinophil function and disorders. Adv Intern Med 19:1-26, 1974 Gelfand M. Bernberg H: Tropical eosinophilic syndrome: A clinical description of the disorder as seen in S. Rhodesia. Cent Afr J Med 5:405-411, 1939 Lukens JN: Eosinophilia in children. Pediatr Clin N Am 19:969-981, 1972 Chusid MJ. Dale DC, W'est BC, Wolff SM: The hypereosinophilic syndrome: Analysis of fourteen cases with review of the literature. Medicine 34:1-28, 1973 Kav AB: Studies on eosinophil leucocyte migration. I. Eosinophil and neutrophil accumulation following antigen-antiboddy reactions in guinea pig skin. Clin Exp Immunol 6:75-86. 1970 Klein NC, Hargrove RL, Sleisenger NIH, Jeffries GH: Eosinophilic gastroenteritis. Medicine 49:299-319. 1970 Eidinger D, Raff NI, Rose B: Tissue eosinophilia in hypersensitivity reactions as revealed b- the human skin window. Nature 196:6&3-4, 1962 Goetzl EJ, Wasserman SI, Austen KP: Nodulation of the eosinophil chemotactic response in immediate hypersensitivity. Progress in Immunology II, \Vol. 4. Edited by L Brent, J Holborow. Amsterdam, North-Holland Publishing Co, 1974, pp 41-50 Goetzl EJ, %asserman SI, Austen KF: Eosinophil polymorphonuclear leukocvte function in immediate hypersensitivity. Arch Pathol 99:1-4, 1975 Goetzl EJ, W asserman SI. Gigli I, Austen KF: Modulation of eosinophil function in
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immediate hypersensitivitv. New Directions in Asthma. Edited bv Ni Stein. Park Ridge, Ill., American College of Physicians, 1975, pp 173-186 Cohen SG, Sapp TM: Experimental eosinophilia. IV. Eosinophilotactic influences of polysaccharides. Exp Mol Pathol 2:74-2, 1963 Litt M: Studies in experimental eosinophilia. VII. Eosinophils in lymph nodes during the first 24 hours following primarv antigenic stimulation. J Immunol 93:807-813, 1964 Walls RS, Hersey P, Quie PG: Macrophage-eosinophil interactions in the inflammatorv response to Trichinella spirais. Blood 44:131-136, 1974 Rose NR, Kite JH, Jr: Cell damage in autoimmune disease. International Convocation on Immunology. Edited by NR Rose, F Milgram. Basel, S. Karger, 1969, pp 247-259
17. Flax MH: Cell mediated hypersensitivity in the pathogenesis of experimental allergic thyroiditis, and its role in extralymphoid plasmacytosis. Cellular Interactions in the Immune Response. Edited by S Cohen, G Cudkowicz, RT McCluskey. Basel, S. Karger, 1971, pp 282-289 18. Torisu M, Yoshida T, Ward PA, Cohen S: Lymphocvte-derived eosinophil chemotactic factors. II. Studies on the mechanism of activation of the precursor substance bv immune complexes. J Immunol 111:145-1458, 1973 19. Kay AB, NMcVie JG, Stuart AE, Krajewski A, Tumbull LW: Eosinophil chemotaxis of supernatants from cultured Hodgkin's lymph node cells. J Clin Pathol 28:502-505, 1975 20. Mulller-Eberhard HJ: Chemistrv and reaction mechanisms of complement. Adv Immunol 8:1-80, 1968 21. Ruddy S, Gigli I, Austen KF: The complement system of man. N Engl j Med 287:489-495, 545-549, 592-596, 642-646, 1972 22. Vallota EH, Mu1ller-Eberhard HJ: Formation of C3a and C5a anaphvlatoxins in whole human serum after inhibition of the anaphvlatoxin inactivator. j Exp Med 137:1109-1123, 1973 23. Ward PA, Newman LJ: A neutrophil chemotactic factor from human C'5. J Immunol 102:93-99, 1969 24. Ward PA, Hill JH: CS chemotactic fragments produced by an enzyme in lysosomal granules of neutrophils. J Immunol 104:535-543, 1970 25. Ward PA: A plasmin-split fragment of C3 as a new chemotactic factor. J Exp Med 126:189-206, 1967 26. Ward PA: Complement-derived leukotactic factors in pathological fluids. J Exp Med 134 (Suppl):109S-113S, 1971 27. Tumer SR, Campbell JA, Lynn WS: Polymorphonuclear leukocvte chemotaxis toward oxidized lipid components of cell membranes. J Exp Med 141:1437-1441, 1975 28. Turner SR, Tainer JA, Lynn WS: Biogenesis of chemotactic molecules by the arachidonate lipoxygenase system of platelets. Nature 257:680-681, 1975 29. Goetzl EJ, Woods JM, Gorman RR: Preferential stimulation of human eosinophil polymorphonuclear leukocyte chemotaxis and random migration by 12-L-hydroxy5, 8, 10, 14-eicosatetraenoic acid (HETE). Unpublished data 30. Kaplan AP, Kay AB, Austen KF: A prealbumin activator of prekallikrein. III. Appearance of chemotactic activity for human neutrophils bv the conversion of human prekallikrein to kallikrein. J Exp Med 135:81-97, 1972 31. Kaplan AP, Goetzl EJ, Austen KF: The fibrinolvtic pathway of human plasma. II. The generation of chemotactic activity by activation of plasminogen proactivator. J Clin Invest 52:2591-2595, 1973 32. Goetzl EJ, Schreiber AD, Austen KF: Chemotactic activity of components of the kallikrein-kinin and fibrinolytic sequences. Chemistry and Biology of the Kallikrein-
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Kinini S-stem in Health and Disease. Edited b- J Pisano. Bethesda. \Md.. National Institutes of Health (In press) Rudd%- S. Austen KF. Goetzl EJ: Chemotactic activity derived from interaction of factors D and B of the properdin pathwvay wvith cobra venom factor or C:3b. J Clin Invest 55:587-5.392. 1975 Kay AB. Stechschulte DJ. Austen KF: An eosinophil leukocv-te chemotactic factor of anaphylaxis. J Exp NIed 1:33:602-619. 1971 Kay AB. Austen KF: The IgE-mediated release of an eosinophil leukocv-te chemotactic factor from human lung. j Immunol 107:899-902. 1971 Archer RK: The eosinophilic response in the horse to intramedullary and intradermal injections of histamine. ACTH. and cortisone. J Pathol Bacteriol 72:87-94. 1956 Vegad JL. Lancaster MC: Eosinophil leucocvte-attracting effect of histamine in the sheep skin. Ind J Exp Biol 10:147-148. 1972 Wasserman SI. Goetzl EJ. Kaliner MA. Austen KF: Modulation of the immunologic release of the eosinophil chemotactic factor of anaphylaxis from human lung. Immunology 26:677-684. 1974 Wasserman SI. Goetzl EJ. Austen KF: Preformed eosinophil chemotactic factor of anaphylaxis (ECF-A). J Immunol 1 12::351-3:.8. 1974 Lewis RA. Goetzl EJ. Wasserman SI. Valone FH. Rubin RH. Austen KF: The release of four mediators of immediate hypersensitivity from human leukemic basophils. J Immunol 114:87-92. 1975 Kaliner \M. Wasserman SI. Austen KF: Immunologic release of chemical mediators from human nasal polvps. N EngI J Med 289:277-281. 1973 Wasserman SI. Goetzl EJ. Ellman L. Austen KF: Tumor-associated eosinophilotactic factor. N Engl J Med 290:420-424. 1974 Goetzl EJ. Austen KF: Purification and synthesis of eosinophilotactic tetrapeptides of human lung tissue: Identification as eosinophil chemotactic factor of anaphylaxis. Proc Natl Acad Sci U SA 72:4123-4127. 1975 Austen KF. Wasserman SI. Goetz] EJ: Mast cell-derived mediators: Structural and functional diversity and regulation of expression. Proceedings of Nobel Symposium :3;3. Molecular and Biological Aspects of the Accute Allergic Reactions. Edited b\ SGO Johansson. K Strandberg. B Uvnas. )ndcon. Plenum Publishing Co. In press (oetzl EJ. Austen KF: Specificity and modulation of the eosinophil polymorphonuclear leukocyte response to the eosinophil chemotactic factor of anaphVlaxis (ECF-A)." Wasserman SI. Boswell RN. Drazen J\I. Goetzl EJ. Austen KF: Functional specificitv of the human lung acidic tetrapeptides constituting eosinophil chemotactic factor of anaphvlaxis (ECF-A). J Allerg Clin Immunol 57:190. 1976 (Cetzl EJ. Rubin RH. \McDonough J. Tashjian AH Jr. Austen KF: Production of eosinophilotactic peptides by bronchogenic carcinoma in situ and in vitro. Clin Res In press) Abstr Bosw ell NB. Austen KF. Goetzl EJ: Structural and functional characterization of ECF oligopeptides from rat mast cells and leutkemic basophils. U L-npublished data) Czarnetski B\t. K5nig W. Lichtenstein L\I: Antigen-induced eosinophil chem(tactic factor ECF release by human leukocvtes. Inflammation 1:201-213. 1976 Kay AB. Shin JS. Austen KF: Selective attraction of eosinophils and synergism between eosinophil chemotactic factor of anaphylaxis (ECF-A) and a fragment cleaved from the fifth component of complement (C5a). Immunology 24:969-976. 197:3 Snvderman R. Shin HS. Hausman \IH: A chemotactic factor for mononuclear leukocvtes. Proc Soc Exp Biol Med 1:38::387-390. 1971
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52. Bokisch VA, Muller-Eberhard HJ: Anaphylatoxin inactivator of human plasma: Its isolation and characterization as a carboxypeptidase. J Clin Invest 49:2427-2436, 1970 53. Berenberg JL, Ward PA: Chemotactic factor inactivator in normal human serum. J CGn Invest 52:1200-1206, 1973 54. Goetzl EJ, Boswell RN, Austen KF: The tetrapeptides comprising eosinophil chemotactic factor of anaphylaxis (ECF-A): Structural determinants of chemotactic activity. Fed Proc 35:515, 1976 (Abstr) 55. Ward PA, Becker EL: The deactivation of rabbit neutrophils by chemotactic factor and the nature of the activatable esterase. J Exp Med 127:693-709, 1968 56. Ward PA, Becker EL: Biochemical demonstration of the activatable esterase of the rabbit neutrophil involved in the chemotactic response. J Immunol 105:1057-1067, 1970
57. Goetzl EJ, Austen KF: A neutrophil immobilizing factor derived from human leukocytes. I. Generation and partial characterization. J Exp Med 136:1564-1580, 1972
58. Goetzl EJ, Gigli I, Wasserman S, Austen KF: A neutrophil immobilizing factor derived from human leukocvtes. II. Specificity of action on polymorphonuclear leukocyte mobility. J Immunol 111:938-945, 1973 59. Nishioka K, Constantopoulos A, Satoh PS, Najjar VA: The characteristics, isolation and synthesis of the phagocytosis stimulating peptide tuftsin. Biochem Biophys Res Commun 47:172-179, 1972 60. Goetzl EJ: Plasma and cell-derived inhibitors of human neutrophil chemotaxis. Ann NY Acad Sci 256:210-221, 1975 61. Goetzl EJ, Austen KF: Stimulation of human neutrophil leukocyte aerobic glucose metabolism by purified chemotactic factors. J Clin Invest 53:591-599, 1974 62. Goetzl EJ, Wasserman SI, Gigli I, Austen KF: Enhancement of random migration and chemotactic response of human leukocytes by ascorbic acid. J Clin Invest 53:813-818, 1974 63. Wasserman SI, Whitmer D, Goetzl EJ, Austen KF: Chemotactic deactivation of human eosinophils by the eosinophil chemotactic factor of anaphylaxis. Proc Soc Exp Biol Med 148:301-306, 1975 64. Najjar VA: The physiological role of -t -globulin. Adv Enzymol 41:129-178, 1974 65. Colley DG: Eosinophils and immune mechanisms. I. Eosinophil stimulation promoter (ESP): A lymphokine induced by specific antigen or phytohemagglutinin. J Immunol 110:1419-1423, 1973 66. Clark RAF, Galin JI, Kaplan AP: The selective eosinophil chemotactic activity of histamine. J Exp Med 142:1462-1476, 1975 67. Vaughn J: The function of the eosinophile leukocyte. Blood 8:1-15, 1953 68. Parish WE: Investigations on eosinophilia. The influence of histamine, antigen-antibody complexes containing Y 1 or Y 2 globulins, foreign bodies (phagocytosis) and disrupted mast cells. Br J Dermatol 82:42-64, 1970 69. Clark RAF, Galin JI, Kaplan AP: Histamine-dependent inhibition and enhancement of eosinophil migration to chemotactic agents. J Allerg Clin Immunol (In press) 70. Kovacs BA: Antihistamine effect of eosinophil leukocytes. Experientia 6:349-50,
1950 71. Archer RK, Feldberg W, Kovacs BA: Antihistamine activity in extracts of horse eosinophils. Br J Pharmacol 18:101-108, 1962 72. Hubscher T, Eisen AH: A possible immunopharmacological role of human eosinophils in allergic reactions. Mechanisms in Allergy: Reagin-Mediated Hypersensitivity. Edited by L Goodfriend, A Sehon, RP Orange. New York, Marcel Dekker Inc., 1973, pp 413-437 73. Zeiger RS, Yurdin DL, Colten HR: Histamine metabolism. II. Cellular and sub-
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cellular localization of the catabolic enzymes, histaminase and histamine methyl transferase in human leukocvtes. J Allerg Clin Immunol (In press) Wasserman SI, Goetzl EJ, Austen KF: Inactivation of slow reacting substance of anaphvlaxis by human eosinophil arvlsulfatase. J Immunol 114:645-649, 1975 Kater LA, Goetzl EJ, Austen KF: Isolation of human eosinophil phospholipase D. J Clin Invest 57:1173-1180, 1976 Roy AB: The sulfatase of ox liver. I. The complex nature of the enzvme. Biochem J 53:12-15, 1953 Dodgson KS, Spencer B: Assay of sulfatases. Meth Biochem Anal 4:211-255, 1957 Allen E, Rov AB: The sulfatase of ox liver. XI. The isoelectric focussing of a purified preparation of sulfatase B. Biochim Biophys Acta 168:243-251, 1968 Bleszvnski W, Lemicki A, Lewosz J: Kinetic properties of three soluble arvlsulfatases from ox brain, homogeneous in polyacrylamide gel electrophoresis. Enzymologia 37:314-324, 1969 Baum H, Dodgson KS, Spencer B: The assay of arylsulphatases A and B in human urine. Clin Chim Acta 4:453-455, 1959 Wrasserman SI, Goetzl EJ, Austen KF: Inactivation of human SRS-A by intact human eosinophils and by eosinophil arvlsulfatase. J Allerg Clin Immunol 55:72, 1975 (Abstr) Rubin RH, Austen KF, Goetzl EJ: Studies of immediate hypersensitivity in a patient with Acanthocheionema perstans filarial infection. J Infect Dis 131(Suppl):S98-103, 1975
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[End of Article]
