Some Interrelations of Neutrophil Chemotaxis, Lysosomal Enzyme Secretion, and Phagocytosis as Revealed by Synthetic Peptides Elmer L. Becker, MD, PhD
Sy nthetic oligopeptides of appropriate structure stimulate neutrophil random locomotion, chemotaxis, lysosomal enzyme secretion, and phagocytosis. The structure-activity relationships found for enhanced migration and ly-sosomal enzyme secretion strongly suggest that the peptides bind to a structurally specific receptor on the neutrophil surface. It is further suggested that the binding of a peptide to the same receptor initiates all of these neutrophil functions. It is postulated that this is accomplished by the receptor-peptide combination initiating a series of parallel but coordinated and interdependent biochemical sequences leading to either microfilament or microtubule activation in addition to other processes. The various functions of the neutrophil differentially utilize the microfilament and microtubule systems. (Am J Pathol
85:385-394, 1976)
C HE\IOTAXIS.
PH XGOCY TOSIS. AN D LYSOSONIAL ENZY\M E degranula-
tion or secretion are all involved in the important roles the neutrophil play s in inflammation. Thus it is not amiss that wve consider the neutrophil functions in this Symposium. The bases of the w-ork that I shall discuss are sev-eral. We and others have show-n that a variety of chemotactic factors can induce l-sosomal enz-me secretion.'2 There is evidence that under the proper circumstances chemotactic factors can inhibit phagocytosis.3 Schiffmann and his co-wvorkers have demonstrated that simple formylmethiony l peptides stimulated neutrophil movement and mvere presumablv chemotactic.4 Based on this latter finding. %ve have synthesized some 24 peptides. most of them formyl-methionyl peptides and mostly di- and tripeptides, with a few- tetrapeptides. W7e are using these peptides to study their activity as l-sosomal enzyme releasers and enhancers of neutrophil movement 5 and their effect on phagocytosis. In w-hat follows, I shall first described the results of our systematic study of the relation of the structure of these peptides to their ability to enhance both chemotactic and random mo-ement of neutrophils and to induce secretion of their lvsosomal enzymes.5 I shall also showv that w-hen in solution these peptides From the Department of Patholog%-. Universitv of Connecticut Health Center. Farmington. Connecticut Presented at the Sixtieth Annual MIeeting of the Federation of American Societies for Experimental Bioloyv. Xnaheim. Calif. Xpril 11. 1976 Supported bx- Grants AI-06945 and Contract NIDR-N-01-DE-52477 from the US Public Health Serx ice. Xddress reprint requests to Dr Elmer Becker. Department of Pathology. Unixersity of ConnecticuJt Health Center. Farmington CT 06032 385
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inhibit phagocvtosis, and when placed on latex particles they enhance phagocvtosis. I shall then suggest some inferences about the primary interaction of these peptides with the neutrophil surface in relation to the initiation of these various functions. Finally, I shall speculate on the nature of the interrelations of the final pathways in these neutrophil functions. In the work itself, rabbit peritoneal neutrophils were employed throughout and the Boyden chamber technique was utilized to study the ability of the peptides to stimulate their movement. In this technique, neutrophils are placed above a Millipore filter and peptide placed below; the stimulated movement is measured by counting the number of neutrophils induced to penetrate into the pores of the filter after a suitable incubation time. Various substances increase penetration of cells into the filter either by enhancing the cells' random locomotion or their directedthat is, chemotactic-motion or by both means. Zigmond and Hirsch have described a technique for determining if the movement of a cell into the filter under the influence of different gradients is greater than can be expected on the basis of increased random locomotion and to that extent is due to a true chemotactic response.6 We used their technique to test two representative peptides, F-Met-Met-Phe and F-Met-Met-Met. Both peptides were found to stimulate random movement and also to induce a directed, that is, a chemotactic response.5 Thus, the initial conclusion is that these peptides stimulate both random and chemotactic movement of neutrophils. The migration-enhancing activity of the various peptides is assayed by determining dose-response curves for each peptide. For this purpose, the measured stimulated activity is plotted against the logarithm of the concentration. Within a given experiment (and for most experiments), the dose-response curves are sigmoid, reasonably parallel in their linear portions, and have the same maxima. We, therefore, describe the activities of the different peptides in terms of their EDs, i.e., the concentration of peptide giving 50% of the maximum activity.5 The peptides in the presence of 5 Mg/ml of cytochalasin B induce the release of the lysosomal enzvmes, W-glucuronidase and lysozyme, but cause no release of the cytoplasmic marker enzyme, lactic dehydrogenase (LDH). Parallel, sigmoid log dose-response curves are obtained for the release of both enzvmes, allowing the enzyme-secreting activity of the peptides also to be expressed as an ED. for each enzyme. The peptides are highly specific in their activity, small changes in structure being capable of leading to large changes in activity, whether the activity is measured as migration enhancement or the induction of
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Table 1-Effect of Formyl (F) Group on the Activity of Formyl-Methionyl Peptides in Stimulating Migration and Releasing Lysosomal Enzymes*
F-Met-Leu-Phe Met-Leu-Phe F-Met-Phe-Leu Met-Phe-Leu F-Met-Met-Met Met-Met-Met *
EDO
±
Migration-stimulating activity
Lysozyme
3-Glucuronidase
7.0 ± 1.7 x 10 " 6.7 1.9 x 10-7 5.4 1.9 x 108 2.4 ± 1 x 10' 5.1 e0.62 x 10 9 1.0 ± 0.39 x 10'
2.4 ± 0.31 x 10 10 8.9 ± 1.4 x 10-7 3.3 0.7 x 10-7 >1 X 103 4.3 1.4 x 10 3 8.8 4.4 x 10'4
2.6 ± 0.32 x 10 10 9.7 ± 0.12 x 10 7 3.5 1.6 x 10 >1 X 103 3.1 ±0.63 x 10 9 7.2 ± 1.3 x 10 4
SE, molar concentration.
Iysosomal enzyme secretion. This illustrated in Table 1 -here wve see, in confirmation of the findings of Schiffmann et al.,j that the presence of a formyl group on the methionine leads to a 3,000 to 30,000 increase in activity. The approximately 500-fold greater activity of the F-Met-LeuPhe compared to the F-Met-Phe-Leu, -here the position of the last t wo amino acids is simply inverted, is one illustration of the specificity of the peptides. Also apparent in Table 1 is the high activity of some of the peptides: the ED,, for migration enhancement of F-Met-Leu-Phe is 7 X 10-" NI. This peptide is easily detected at 1 x 10- ' NM and wvith sufficiently active cells at 10-12 NI. Yet, we believe that this peptide is not the most active obtainable. The specificity depends not only on the nature of the amino acid but also on the position of the amino acid in the peptide chain. This is illustrated by the structural requirements for increased activity of amino acids in the second position from the NH2-terminal end compared to those in the third position. Table 2 shows that in the second position from the NH2-terminal end, leucine, methionine, and phenylalanine are essentially equivalent; methionine possibly contributing slightly less to the activity than the other tw-o.5 Other work has showvn that F-Met-His, with its positively charged histidine, and F-Met-Glu, wvith its negatively charged glutamic acid, are distinctly less active than the dipeptides in Table 2. Table 2-Structural Requirements for Migration-Stimulating and Enzyme-Releasing Activity of Amino Acids in the Second Position in N-Formyl-Methionyl Peptides*
Migration-stimulating F-Met F-Met-Leu F-Met-Phe F-Met-Met 0
2.1 4.0 4.1 8.8
activity 0.6 x ± 0.45 x ± 0.95 X ± 2.3 x
ED50 ± SE, molar concentration.
10' 10' 10-7 10-7
Enzyme-releasing activity 3-Gkucuronidase Lysozyme >1 X 102 >1.0 X 102 1.7 = 0.17 x 10' 1.9 t 0.2 x 10' 1.5 ± 0.39 x 10 ' 2.0 ± 0.4 x 10'6 2.9 ± 0.47 x 10 ' 7.5 ± 3.5 x 10'6
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Table 3-Structural Requirements for Migration-Stimulating and Enzyme-Releasing Activity of Amino Acids in the Third Position in N-Formyl-Methionyl Peptides*
Migration-stimulating activity F-Met-Leu F-Met-Leu-Phe F-Met-Leu-Leu F-Met-Leu-Glu F-Met-Leu-Arg F-Met-Phe-Leu F-Met-Met F-Met-Met-Ala F-Met-Met-Met F-Met-Met-Phe F-Met-Phe-Met *
4.0 ± 0.45 x 10'7 7.0 ± 0.17 x 10 4.8 0.13 x 106 1.3 0.38 x 10' 3.6 1.0 x 107 5.4 ± 1.9 x 106 8.8 ± 2.3 x 10' 5.4 ± 1.8 x 10 7 5.1 0.62 x 109 2.1 0.4 x 1010 1.5 + 0.33 x 109
Enzyme-releasing activity Lysozyme 1.7 0.17 2.4 ± 0.31 2.8 0.41 5.0 ± 1.3 2.2 0.45 3.3 0.7 2.9 ± 0.47 1.5 ±0.5 4.3 + 1.4 1.9 0.4 2.6 ±0.5
x 10-6 x 10 1"
x 10' x 10' x 10'
x 107 x 10-6 x 10'
x 108 x 109 x 109
3-Glucuronidase 1.9 ± 0.21 x 106 2.6 0.32 x 10"'° 4.5 2.1 x 10' 7.2 ± 0.29 x 10 6 2.6 0.7 x 10' 3.5 ± 1.6 X 107 7.5 ± 3.5 x 10 ' 1.5 ± 0.45 x 10' 3.1 0.63 x 106 1.8 0.24 x 109 3.5 + 0.6 x 106
ED,O ± SE, molar concentration.
This suggests that in the second position, any neutral, sufficiently hydrophobic amino acid is sufficient for maximal or nearly maximal activity. Table 3 show-s that this is not true for amino acids in the third position of the peptide. F-Met-Leu-Phe is approximately 300 times more active than F-Met-Leu-Leu (Table 3). In other words, adding phenvlalanine to F-Met-Leu increased activity much more than adding leucine. This is in contrast to the dipeptides where the contribution of the two amino acids to activitv was the same. Similarly, F-Met-Met-Phe is approximately 25 times more active than F-Met-Met-Met: that is, adding phenylalanine to F-MIet-Mlet increases activity much more than the addition of methionine, -whereas. the addition of phenvlalanine or methionine to F-Met leads to compounds -ith essentially the same activity. Thus, when phenylalanine is in the third position of a tripeptide it contributes much more to the activity than when it is in the second position of either a dipeptide or tripeptide. Turning to consider the ly-sosomal enzyme-releasing activity of the peptides. -ve see from Tables 1-3 that the ability of the various peptides to release either ly sozyme or 0-glucuronidase correlates very well with their ability to stimulate neutrophil movement. In fact, -vhen the logarithm of the ED50 for migration stimulation -vas plotted against the logarithm of the EDsos for either lysozvme or ,B-glucuronidase release, an essentially perfect correlation was found.5 The correlation coefficient for both enzvrmes was 0.98. In this test all 19 peptides which possessed sufficient activitv to act as enzyme releasers as well as stimulators of movement were employed. As is also evident from the data in Tables 1-3, a higher concentration of peptide is required to induce lysosomal enzyme release than the corresponding degree of stimulation of movement. On the aver-
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age, the ED50 for lysozyme release is 7.5 times and that for 0-glucuronidase release is nine times more than the ED50 for movement. In addition to their stimulation of random locomotion, chemotaxis, and ly-sosomal enzyme secretion, these peptides also affect phagocvtosis in two w-ays. Mr. Robert Musson in my laboratory has shown that various chemotactic factors added to a suspension of human neutrophils and complement containing sheep erythrocytes (EAC1423) inhibit the initial rate of erythrophagocytosis but not the final extent of ingestion.3 There is some evidence compatible with the inhibition being competitive, but not enough work has been done to be sure of the mechanism. Table 4, made from data furnished by M r. M usson, showvs that the chemotactically active F-M.et-Mlet and F-Met-Leu inhibit the initial rate of erythrophagocytosis to essentially the same extent, but the chemotacticallv much less active, unformvlated Met-Met has no effect on erythrophagocytosis at a 100-fold higher concentration. More directly, in very preliminary wvork we have demonstrated that the synthetic peptide F-NMet-Leu-Phe, w-hen adsorbed to latex particles, greatly enhances their phagocytosis. The latex spherules were placed in 1 x 106 NI F-Met-Leu-Phe for 1 hour at 37 C, washed thoroughly, and resuspended in buffer. The presence of F-Met-Leu-Phe on the latex spherule increased the initial rate of ingestion approximately 5.5-fold and the final extent of ingestion approximately 3.5 times. These results are similar to the unpublished results of Henson (referred to in Henson 7) that C5a adsorbed to latex beads enhances their ability to be phagocytosed. Summary of Experimental Results 1. Synthetic peptides of appropriate structure can stimulate random locomotion, chemotaxis, I-sosomal enzyme secretion, and phagocvtosis. 2. There is an exact correlation between the ability of the peptides to enhance neutrophil moveement and their ability to induce lvsosomal secretion; how-ever, greater amounts are required for the latter. Table 4-Effect of Synthetic Peptides on the Initial Rate of Phagocytosis of EAC1423 by Human Neutrophils
Rate of ingestion* z SE 1.9 ± 0.1 1.4 ± 0.04t 1.3 ± 0.03t 1.9 ± 0.1 1.9 + 0.1 Rate of ingestion (10k EAC1423/10' PMN/min). t Significantly different from control (P < 0.05). Peptide
Percent inhibition
None 10 5M F-Met-Leu 10-5M F-Met-Met 105M Met-Met 10-3M Met-Met
27 31 0 0
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3. The relation of structure to these activities is extremely specific; verv small changes in structure can make large changes in activity. Moreover, this specificity exhibits a definite regularity and pattern; the activity of a given peptide depends not only on the constituent amino acids but also on their precise position in the peptide chain. Some Interpetive H eses
An attempt at an explanation of these experimental findings will be done in terms of the following series of hypotheses. 1. The primary interaction of neutrophil and synthetic chemotactic peptide is a binding of the peptide with a structurally specific receptor on the neutrophil surface. This is the simplest hypothesis explaining the nature of the relationships between structure and activity just described, both the great specificity of the structural requirements and the manner in which the activity depends upon the position of the amino acid in the peptide chain. The further work necessary to establish this hypothesis, the binding studies, the isolation of the putative receptor, etc., has yet to be done. In fact, the incentive to do such work is one of the virtues of this hypothesis. A wide variety of substances have been reported to be chemotactic.5 Whether some or all of them act through different receptors or, as suggested, bind through nonstructurally specific receptors can not be answered at this time. 2. All the neutrophil functions just described-random locomotion, chemotaxis, lysosomal enzyme secretion and phagocytosis, and probably others-can arise through stimulation of the same receptor. In this view, interaction at a single type of receptor is sufficient to trigger the various neutrophil functions, the conditions under which the triggering occurs, determining the nature of the reaction which ensues. Chemotactic factors stimulate random locomotion at the same concentrations they induce chemotaxis in the few instances where this has been looked at specifically. '6 This provides at least some evidence that random and directed locomotion may be stimulated by the same peptide-receptor interaction. The essentially exact correlation between migration-enhancing and exzyme-secreting activities5 makes plausible the suggestion that enzvme secretion from both primary and specific granules can be triggered bv the same receptor supposedlv responsible for chemotaxis and random locomotion. The demonstration that a chemotactic peptide can enhance phagocytosis is compatible with the single receptor hypothesis. We are in the process of extending this work to see if the structure-activity relations for phagocytosis enhancement are the same as for the other functions of the neutrophil we have studied.
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The fact that the chemotactically inactive Met-Met did not inhibit ervthrophagocytosis, whereas the chemotactically active F-Met-Met did, suggests that in phagocytosis the same structure-activity relationships might hold; here also, however, much more evidence is required. The relationship of the findings cited here to the recent report of Hook et al. that certain of the F-methionyl peptides induce noncytotoxic histamine release from human basophils, another type of granulocyte is unknown.9 3. The various neutrophil responses to the chemotactic peptides result from the triggering of parallel biochemical sequences. The various neutrophil functions can be disassociated from each other in a number of different ways (reviewed in Becker and Henson 1). This suggests that the interaction of the peptide with the putative neutrophil receptor initiates a series of parallel, interdependent, and coordinated biochemical sequences which lead to one or another neutrophil response. 4. The various neutrophil responses are the result of microtubule and /or microfilament involvement. All of the neutrophil functions are forms of cell movement.' Microfilaments and microtubules are generally considered to be involved in cell movement.1'11"2 There are greater or lesser accumulations of evidence suggesting that these two systems are differentially involved in the various neutrophil functions. The evidence is all indirect consisting of either observations of the behavior of neutrophils considered to have a defect of microfilament or microtubule activity or the effects on the various cell functions of inhibitors of one or the other system. Boxer et al. found that the neutrophils from a patient subject to recurrent infections were deficient in their random locomotion and chemotactic and phagocvtic responses but did show enhanced degranulation."3 The number of microfilaments in these neutrophils were fewer and the actin isolated from these neutrophils polymerized to a lesser extent than actin isolated from normal neutrophils. As Dr. Oliver will describe later in this Symposium, a defect in microtubule assembly is thought to be the basic defect in the neutrophils of animals or humans with the Chediak-Higashi svndrome.'4 The cells are defective in their chemotactic responsiveness and ability to degranulate although their random locomotion and phagocytosis is reportedly unaffected or even increased. Cvtochalasin B, a drug reported to inhibit microfilament action as well as other cell responses, inhibits chemotaxis and random locomotion 17-20 and phagocytosis,19'20 but enhances Iysosomal enzyme secretion.21'2 C5a, a chemotactic factor, induces assembly of neutrophil microtubules. "'2' Based on the use of colchicine and other antimicrotubule agents, a number of workers have suggested that microtubules are re-
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quired in directional movement (chemotaxis) but not in random locomotion 25,27
Although there is general agreement that random locomotion is unaffected and even enhanced by colchicine and similar agents, 2.28 wide variations have been reported in the concentrations required to inhibit chemotaxis.27.9-31 Colchicine is reported to inhibit lsosomal enzyme secretion, but frequently, concentrations wvere employed much above those reported to inhibit microtubules.32 In some instances, high concentrations of colchicine Xvere found to have no effect on lvsosomal enzyme release.33 ' A major defect in all of these and similar investigations is that no concomitant ultrastructural studies were done to correlate the inhibitory or lack of inhibitory effect of colchicine on the microtubule system with the effect of the drug on lysosomal enzyme secretion. How-ever, Hoffstein et al.. although not using colchicine, related microtubule assembly to l-sosomal enzyme release under a variety of conditions and concluded that microtubule assembly w as a necessarv but not sufficient condition for secretion.35 Except for the report of Stossel et al., colchicine has been found either not to affect phagocvtosis or to inhibit it only at concentrations far beyond those required to interfere with microtubule.37 The very tentatixe conclusions drawn from this incomplete survey are that in the random locomotion of neutrophils, microfilaments are required. but microtubules are not; in fact, they may actually inhibit. In contrast, directed locomotion (chemotaxis), seems to require both microfilaments and microtubules. Lysosomal enzyme secretion does not require microfilaments; on the contrary, their presence may be somew-hat inhibitorv. There is suggestive but conflicting evidence that enzyme secretion depends upon microtubules, but more wvork is required before this conclusion is acceptable. Phagocytosis requires microfilament activity, but probably does not require microtubules, although the latter possibly may aid the process. References Becker EL. Henson PM. Shovell HJ. Hsu LS: The ability of chemotactic factors to induce l-sosomal enzy-me release. I. The characteristics of the release. the importance of surfaces and the relation of the enzyme release to chemotactic responsiveness. J Immunol 112:2047-20554 1974 2. Goldstein MNI. Brai \M. Osler AG. Weissmann G: Lysosomal enzyme release from human leukocvtes: Mediation by the alternate pathwvay of complement activation. J 1.
Immunol 109::3:3-37. 197:3 :3. Musson R. Becker EL: The inhibitory effect of chemotactic factors on erythrophagocvtosis by human neutrophils. J Immunol 117:43:3-439, 197-6 4. Schiffmann E. Corcoran BA. WNahl SM: N-Formvlmethionine peptides as chemoattractants for leucocytes. Proc Natl Acad Sci USA 72:1059-1062. 197-5
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6. 7.
8. 9.
10. 11. 12. 13. 14. 15.
16. 17. 18. 19. 20. 21.
22. 23. 24.
25.
26.
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Showell HJ. Freer RJl Zigmond SH. Schiffmann S. Aswankumar S, Corcoran B. Becker EL: The structure activity relations of synthetic peptides as chemotactic factors and inducers of lvsosomal enzyme secretion for neutrophils. J Exp NMed 143:1154-1169, 19 7-6 Zigmond SH, Hirsch HG: Leukocyte locomotion and chemotaxis: A new method for evaluation and demonstration of cell derived chemotactic factor. J Exp Med 137 :387-410, 1973 Henson PM: Mechanisms of activation and secretion by platelets and neutrophils. Progress in Immunology, V'ol. 2 Edited by L Brent. J Holborow. Amsterdam-Oxford, North-Holland Press, 1974, pp 95-105 Wilkinson PC: Chemotaxis and Inflammation. Edinburgh & London. Churchill and Livingstone 1974 Hook WNA, Schiffmann E, Aswankumar S, Siraganian RP: Histamine release from basophils by formyl-methionine peptides. Fed Proc 35:864, 1976 Becker EL, Henson PM: In vitro studies of immunologically induced secretion of mediators from cells and related phenomena. Adv Immunol 17:93-193, 1973 Stossel TP: Phagocytosis: Recognition and ingestion. Semin Hematol 12:83-116, 1975 Pollard TD, Weihing RA: Actin and myosin and cell movement. CRC Crit Rev Biochem 2:1-65, 1974 Boxer L-, Hedle%--Whv%hte ET, Stossel TP: Neutrophil action dysfunction and abnormal neutrophil behavior. N Engl J Med 291:1093-1099, 1974 Oliver JM. Zurier RB, Berlin RD: Concanavalin A cap formation on polvmorphonuclear leucocytes of normal and beige (Chediak-Higashi) mice. Nature 253:471-473, 1975 Clark RA. Kimball HR: Defective granulocyte chemotaxis in the Chediak-Higashi syndrome. J Clin Invest 50:2645-2652, 197-1 Stossel TP, Root RK, Vaughn MI: Phagocytosis in chronic granulomatous disease and the Chediak-Higashi syndrome. N Engl J Med 286:120-123, 1972 Becker EL Davis AT, Estenson RD, Quie PG: Cytochalasin B. IV'. Inhibition and stimulation of chemotaxis of rabbit and human polymorphonuclear leukocytes. J Immunol 108:396-402, 1972 Borel JF, Stahlein H: Effects of cytochalasin B on chemotaxis and immune reactions. Experentia 28:745, 1972 Zigmond SH, Hirsch JG: Effects of cvtochalasin B on pol-morphonuclear leucocyte locomotion, phagocytosis and glycolysis. Exp Cell Res 73:383-393. 1972 Canarozzi NA. Malawista SE: Phagocvtosis of human blood leukocytes measured by the uptake of31 I-labeled human serum albumin: Inhibitory and stimulatorv effects of cytochalasin B. Yale J Biol Med 46:117-189. 1973 Zurier RB, Hoffstein S, Weissmann G: Cvtochalasin B: Effect on lysosomal enz-me release from human leukocvtes: Proc Nati Acad Sci USA 70:844-848, 1973 (Abstr) Becker EL Showell HJ: The ability of chemotactic factors to induce lysosomal enzyme release. II. The mechanism of the release. J Immunol 112:2055-2062. 1974 Goldstein I, Hoffstein S. Gallin J. Weissmann G: Mechanisms of lysosomal enzyme release from human leukocv-tes: Microtubule assembly and membrane fusion induced by a component of complement. Proc Natl Acad Sci USA 70:2916-2920. 1973 Gallin JIA Rosenthal AS: The regulator- role of divalent cations in human granuloc-te chemotaxis: Evidence for an association between calcium exchanges and microtubule assembly. J Cell Biol 62:594-60, 1974 Bhisey AN, Freed JN: Altered movement of endosomes in colchicine-treated cultured macrophages. Exp Cell Res 64:430-438. 1971 Allison AC, David P, dePetris S: Role of contractile microfilaments in macrophage movement and endocytosis. Nature [New Biol. ] 2.32:153-155, 197 1
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:33. 34. ,3..
:36. 37.
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Bandmann U. Rydgren L, Norbert B: The difference betu-een random moveement and chemotaxis. Exp Cell Res 88:6,3-73, 1974 Ramsey- WS. Harris A: Leukocyte locomotion and its inhibition by antimitotic drugs. Exp Cell Res 82:262-270, 1973 Caner JE Colchicine inhibition of chemotaxis. Arthritis Rheum 8:757-764, 1965 Phelps P, McCarty DJ Jr: Crystal induced arthritis. Postgrad Med 45:87-93. 1969 WN'ard PA: Leukotactic factors in health and disease. Am J Pathol 64:521-530, 1971 Wreissmann G, Goldstein I, Hoffstein S, Tsung PK: Reciprocal effects of AMP and GMP on microtubule-dependent release of l-sosomal enzymes. Ann NY Acad Sci 253:750-762. 1975 Henson PM: Pathologic mechanisms in neutrophil-mediated injury. Am J Pathol 68:593-612. 1972 Haw-kins D: Inhibition of neutrophil lysosomal release. Fed Proc 31:748, 1972 Hoffstein S: Microtubule assembly and secretion in human polymorphonuclear leukoc-tes. Fed Proc 34:868, 1975 Stossel TP, Mason RJ, Hartwig J, V'aughn N: Quantitative studies of phagocvtosis by polymorphonuclear leukocv-tes: Use of emulsions to measure the initial rate of phagoc'vtosis. J Clin Ivest 51:615-625, 1972 Malawista SE: Action of colchicine in acute gouty arthritis. Arthritis Rheum 18:8.35-846, 1975 Lehrer RI: Effects of colchicine and chloramphenicol on the oxidative metabolism and phagocvtic activity of human neutrophils. J Infect Dis 127:40-48, 1973
Sy nthetic oligopeptides of appropriate structure stimulate neutrophil random locomotion, chemotaxis, lysosomal enzyme secretion, and phagocytosis. The structure-activity relationships found for enhanced migration and ly-sosomal enzyme secretion strongly suggest that the peptides bind to a structurally specific receptor on the neutrophil surface. It is further suggested that the binding of a peptide to the same receptor initiates all of these neutrophil functions. It is postulated that this is accomplished by the receptor-peptide combination initiating a series of parallel but coordinated and interdependent biochemical sequences leading to either microfilament or microtubule activation in addition to other processes. The various functions of the neutrophil differentially utilize the microfilament and microtubule systems. (Am J Pathol
85:385-394, 1976)
C HE\IOTAXIS.
PH XGOCY TOSIS. AN D LYSOSONIAL ENZY\M E degranula-
tion or secretion are all involved in the important roles the neutrophil play s in inflammation. Thus it is not amiss that wve consider the neutrophil functions in this Symposium. The bases of the w-ork that I shall discuss are sev-eral. We and others have show-n that a variety of chemotactic factors can induce l-sosomal enz-me secretion.'2 There is evidence that under the proper circumstances chemotactic factors can inhibit phagocytosis.3 Schiffmann and his co-wvorkers have demonstrated that simple formylmethiony l peptides stimulated neutrophil movement and mvere presumablv chemotactic.4 Based on this latter finding. %ve have synthesized some 24 peptides. most of them formyl-methionyl peptides and mostly di- and tripeptides, with a few- tetrapeptides. W7e are using these peptides to study their activity as l-sosomal enzyme releasers and enhancers of neutrophil movement 5 and their effect on phagocytosis. In w-hat follows, I shall first described the results of our systematic study of the relation of the structure of these peptides to their ability to enhance both chemotactic and random mo-ement of neutrophils and to induce secretion of their lvsosomal enzymes.5 I shall also showv that w-hen in solution these peptides From the Department of Patholog%-. Universitv of Connecticut Health Center. Farmington. Connecticut Presented at the Sixtieth Annual MIeeting of the Federation of American Societies for Experimental Bioloyv. Xnaheim. Calif. Xpril 11. 1976 Supported bx- Grants AI-06945 and Contract NIDR-N-01-DE-52477 from the US Public Health Serx ice. Xddress reprint requests to Dr Elmer Becker. Department of Pathology. Unixersity of ConnecticuJt Health Center. Farmington CT 06032 385
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inhibit phagocvtosis, and when placed on latex particles they enhance phagocvtosis. I shall then suggest some inferences about the primary interaction of these peptides with the neutrophil surface in relation to the initiation of these various functions. Finally, I shall speculate on the nature of the interrelations of the final pathways in these neutrophil functions. In the work itself, rabbit peritoneal neutrophils were employed throughout and the Boyden chamber technique was utilized to study the ability of the peptides to stimulate their movement. In this technique, neutrophils are placed above a Millipore filter and peptide placed below; the stimulated movement is measured by counting the number of neutrophils induced to penetrate into the pores of the filter after a suitable incubation time. Various substances increase penetration of cells into the filter either by enhancing the cells' random locomotion or their directedthat is, chemotactic-motion or by both means. Zigmond and Hirsch have described a technique for determining if the movement of a cell into the filter under the influence of different gradients is greater than can be expected on the basis of increased random locomotion and to that extent is due to a true chemotactic response.6 We used their technique to test two representative peptides, F-Met-Met-Phe and F-Met-Met-Met. Both peptides were found to stimulate random movement and also to induce a directed, that is, a chemotactic response.5 Thus, the initial conclusion is that these peptides stimulate both random and chemotactic movement of neutrophils. The migration-enhancing activity of the various peptides is assayed by determining dose-response curves for each peptide. For this purpose, the measured stimulated activity is plotted against the logarithm of the concentration. Within a given experiment (and for most experiments), the dose-response curves are sigmoid, reasonably parallel in their linear portions, and have the same maxima. We, therefore, describe the activities of the different peptides in terms of their EDs, i.e., the concentration of peptide giving 50% of the maximum activity.5 The peptides in the presence of 5 Mg/ml of cytochalasin B induce the release of the lysosomal enzvmes, W-glucuronidase and lysozyme, but cause no release of the cytoplasmic marker enzyme, lactic dehydrogenase (LDH). Parallel, sigmoid log dose-response curves are obtained for the release of both enzvmes, allowing the enzyme-secreting activity of the peptides also to be expressed as an ED. for each enzyme. The peptides are highly specific in their activity, small changes in structure being capable of leading to large changes in activity, whether the activity is measured as migration enhancement or the induction of
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Table 1-Effect of Formyl (F) Group on the Activity of Formyl-Methionyl Peptides in Stimulating Migration and Releasing Lysosomal Enzymes*
F-Met-Leu-Phe Met-Leu-Phe F-Met-Phe-Leu Met-Phe-Leu F-Met-Met-Met Met-Met-Met *
EDO
±
Migration-stimulating activity
Lysozyme
3-Glucuronidase
7.0 ± 1.7 x 10 " 6.7 1.9 x 10-7 5.4 1.9 x 108 2.4 ± 1 x 10' 5.1 e0.62 x 10 9 1.0 ± 0.39 x 10'
2.4 ± 0.31 x 10 10 8.9 ± 1.4 x 10-7 3.3 0.7 x 10-7 >1 X 103 4.3 1.4 x 10 3 8.8 4.4 x 10'4
2.6 ± 0.32 x 10 10 9.7 ± 0.12 x 10 7 3.5 1.6 x 10 >1 X 103 3.1 ±0.63 x 10 9 7.2 ± 1.3 x 10 4
SE, molar concentration.
Iysosomal enzyme secretion. This illustrated in Table 1 -here wve see, in confirmation of the findings of Schiffmann et al.,j that the presence of a formyl group on the methionine leads to a 3,000 to 30,000 increase in activity. The approximately 500-fold greater activity of the F-Met-LeuPhe compared to the F-Met-Phe-Leu, -here the position of the last t wo amino acids is simply inverted, is one illustration of the specificity of the peptides. Also apparent in Table 1 is the high activity of some of the peptides: the ED,, for migration enhancement of F-Met-Leu-Phe is 7 X 10-" NI. This peptide is easily detected at 1 x 10- ' NM and wvith sufficiently active cells at 10-12 NI. Yet, we believe that this peptide is not the most active obtainable. The specificity depends not only on the nature of the amino acid but also on the position of the amino acid in the peptide chain. This is illustrated by the structural requirements for increased activity of amino acids in the second position from the NH2-terminal end compared to those in the third position. Table 2 shows that in the second position from the NH2-terminal end, leucine, methionine, and phenylalanine are essentially equivalent; methionine possibly contributing slightly less to the activity than the other tw-o.5 Other work has showvn that F-Met-His, with its positively charged histidine, and F-Met-Glu, wvith its negatively charged glutamic acid, are distinctly less active than the dipeptides in Table 2. Table 2-Structural Requirements for Migration-Stimulating and Enzyme-Releasing Activity of Amino Acids in the Second Position in N-Formyl-Methionyl Peptides*
Migration-stimulating F-Met F-Met-Leu F-Met-Phe F-Met-Met 0
2.1 4.0 4.1 8.8
activity 0.6 x ± 0.45 x ± 0.95 X ± 2.3 x
ED50 ± SE, molar concentration.
10' 10' 10-7 10-7
Enzyme-releasing activity 3-Gkucuronidase Lysozyme >1 X 102 >1.0 X 102 1.7 = 0.17 x 10' 1.9 t 0.2 x 10' 1.5 ± 0.39 x 10 ' 2.0 ± 0.4 x 10'6 2.9 ± 0.47 x 10 ' 7.5 ± 3.5 x 10'6
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Table 3-Structural Requirements for Migration-Stimulating and Enzyme-Releasing Activity of Amino Acids in the Third Position in N-Formyl-Methionyl Peptides*
Migration-stimulating activity F-Met-Leu F-Met-Leu-Phe F-Met-Leu-Leu F-Met-Leu-Glu F-Met-Leu-Arg F-Met-Phe-Leu F-Met-Met F-Met-Met-Ala F-Met-Met-Met F-Met-Met-Phe F-Met-Phe-Met *
4.0 ± 0.45 x 10'7 7.0 ± 0.17 x 10 4.8 0.13 x 106 1.3 0.38 x 10' 3.6 1.0 x 107 5.4 ± 1.9 x 106 8.8 ± 2.3 x 10' 5.4 ± 1.8 x 10 7 5.1 0.62 x 109 2.1 0.4 x 1010 1.5 + 0.33 x 109
Enzyme-releasing activity Lysozyme 1.7 0.17 2.4 ± 0.31 2.8 0.41 5.0 ± 1.3 2.2 0.45 3.3 0.7 2.9 ± 0.47 1.5 ±0.5 4.3 + 1.4 1.9 0.4 2.6 ±0.5
x 10-6 x 10 1"
x 10' x 10' x 10'
x 107 x 10-6 x 10'
x 108 x 109 x 109
3-Glucuronidase 1.9 ± 0.21 x 106 2.6 0.32 x 10"'° 4.5 2.1 x 10' 7.2 ± 0.29 x 10 6 2.6 0.7 x 10' 3.5 ± 1.6 X 107 7.5 ± 3.5 x 10 ' 1.5 ± 0.45 x 10' 3.1 0.63 x 106 1.8 0.24 x 109 3.5 + 0.6 x 106
ED,O ± SE, molar concentration.
This suggests that in the second position, any neutral, sufficiently hydrophobic amino acid is sufficient for maximal or nearly maximal activity. Table 3 show-s that this is not true for amino acids in the third position of the peptide. F-Met-Leu-Phe is approximately 300 times more active than F-Met-Leu-Leu (Table 3). In other words, adding phenvlalanine to F-Met-Leu increased activity much more than adding leucine. This is in contrast to the dipeptides where the contribution of the two amino acids to activitv was the same. Similarly, F-Met-Met-Phe is approximately 25 times more active than F-Met-Met-Met: that is, adding phenylalanine to F-MIet-Mlet increases activity much more than the addition of methionine, -whereas. the addition of phenvlalanine or methionine to F-Met leads to compounds -ith essentially the same activity. Thus, when phenylalanine is in the third position of a tripeptide it contributes much more to the activity than when it is in the second position of either a dipeptide or tripeptide. Turning to consider the ly-sosomal enzyme-releasing activity of the peptides. -ve see from Tables 1-3 that the ability of the various peptides to release either ly sozyme or 0-glucuronidase correlates very well with their ability to stimulate neutrophil movement. In fact, -vhen the logarithm of the ED50 for migration stimulation -vas plotted against the logarithm of the EDsos for either lysozvme or ,B-glucuronidase release, an essentially perfect correlation was found.5 The correlation coefficient for both enzvrmes was 0.98. In this test all 19 peptides which possessed sufficient activitv to act as enzyme releasers as well as stimulators of movement were employed. As is also evident from the data in Tables 1-3, a higher concentration of peptide is required to induce lysosomal enzyme release than the corresponding degree of stimulation of movement. On the aver-
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age, the ED50 for lysozyme release is 7.5 times and that for 0-glucuronidase release is nine times more than the ED50 for movement. In addition to their stimulation of random locomotion, chemotaxis, and ly-sosomal enzyme secretion, these peptides also affect phagocvtosis in two w-ays. Mr. Robert Musson in my laboratory has shown that various chemotactic factors added to a suspension of human neutrophils and complement containing sheep erythrocytes (EAC1423) inhibit the initial rate of erythrophagocytosis but not the final extent of ingestion.3 There is some evidence compatible with the inhibition being competitive, but not enough work has been done to be sure of the mechanism. Table 4, made from data furnished by M r. M usson, showvs that the chemotactically active F-M.et-Mlet and F-Met-Leu inhibit the initial rate of erythrophagocytosis to essentially the same extent, but the chemotacticallv much less active, unformvlated Met-Met has no effect on erythrophagocytosis at a 100-fold higher concentration. More directly, in very preliminary wvork we have demonstrated that the synthetic peptide F-NMet-Leu-Phe, w-hen adsorbed to latex particles, greatly enhances their phagocytosis. The latex spherules were placed in 1 x 106 NI F-Met-Leu-Phe for 1 hour at 37 C, washed thoroughly, and resuspended in buffer. The presence of F-Met-Leu-Phe on the latex spherule increased the initial rate of ingestion approximately 5.5-fold and the final extent of ingestion approximately 3.5 times. These results are similar to the unpublished results of Henson (referred to in Henson 7) that C5a adsorbed to latex beads enhances their ability to be phagocytosed. Summary of Experimental Results 1. Synthetic peptides of appropriate structure can stimulate random locomotion, chemotaxis, I-sosomal enzyme secretion, and phagocvtosis. 2. There is an exact correlation between the ability of the peptides to enhance neutrophil moveement and their ability to induce lvsosomal secretion; how-ever, greater amounts are required for the latter. Table 4-Effect of Synthetic Peptides on the Initial Rate of Phagocytosis of EAC1423 by Human Neutrophils
Rate of ingestion* z SE 1.9 ± 0.1 1.4 ± 0.04t 1.3 ± 0.03t 1.9 ± 0.1 1.9 + 0.1 Rate of ingestion (10k EAC1423/10' PMN/min). t Significantly different from control (P < 0.05). Peptide
Percent inhibition
None 10 5M F-Met-Leu 10-5M F-Met-Met 105M Met-Met 10-3M Met-Met
27 31 0 0
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3. The relation of structure to these activities is extremely specific; verv small changes in structure can make large changes in activity. Moreover, this specificity exhibits a definite regularity and pattern; the activity of a given peptide depends not only on the constituent amino acids but also on their precise position in the peptide chain. Some Interpetive H eses
An attempt at an explanation of these experimental findings will be done in terms of the following series of hypotheses. 1. The primary interaction of neutrophil and synthetic chemotactic peptide is a binding of the peptide with a structurally specific receptor on the neutrophil surface. This is the simplest hypothesis explaining the nature of the relationships between structure and activity just described, both the great specificity of the structural requirements and the manner in which the activity depends upon the position of the amino acid in the peptide chain. The further work necessary to establish this hypothesis, the binding studies, the isolation of the putative receptor, etc., has yet to be done. In fact, the incentive to do such work is one of the virtues of this hypothesis. A wide variety of substances have been reported to be chemotactic.5 Whether some or all of them act through different receptors or, as suggested, bind through nonstructurally specific receptors can not be answered at this time. 2. All the neutrophil functions just described-random locomotion, chemotaxis, lysosomal enzyme secretion and phagocytosis, and probably others-can arise through stimulation of the same receptor. In this view, interaction at a single type of receptor is sufficient to trigger the various neutrophil functions, the conditions under which the triggering occurs, determining the nature of the reaction which ensues. Chemotactic factors stimulate random locomotion at the same concentrations they induce chemotaxis in the few instances where this has been looked at specifically. '6 This provides at least some evidence that random and directed locomotion may be stimulated by the same peptide-receptor interaction. The essentially exact correlation between migration-enhancing and exzyme-secreting activities5 makes plausible the suggestion that enzvme secretion from both primary and specific granules can be triggered bv the same receptor supposedlv responsible for chemotaxis and random locomotion. The demonstration that a chemotactic peptide can enhance phagocytosis is compatible with the single receptor hypothesis. We are in the process of extending this work to see if the structure-activity relations for phagocytosis enhancement are the same as for the other functions of the neutrophil we have studied.
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The fact that the chemotactically inactive Met-Met did not inhibit ervthrophagocytosis, whereas the chemotactically active F-Met-Met did, suggests that in phagocytosis the same structure-activity relationships might hold; here also, however, much more evidence is required. The relationship of the findings cited here to the recent report of Hook et al. that certain of the F-methionyl peptides induce noncytotoxic histamine release from human basophils, another type of granulocyte is unknown.9 3. The various neutrophil responses to the chemotactic peptides result from the triggering of parallel biochemical sequences. The various neutrophil functions can be disassociated from each other in a number of different ways (reviewed in Becker and Henson 1). This suggests that the interaction of the peptide with the putative neutrophil receptor initiates a series of parallel, interdependent, and coordinated biochemical sequences which lead to one or another neutrophil response. 4. The various neutrophil responses are the result of microtubule and /or microfilament involvement. All of the neutrophil functions are forms of cell movement.' Microfilaments and microtubules are generally considered to be involved in cell movement.1'11"2 There are greater or lesser accumulations of evidence suggesting that these two systems are differentially involved in the various neutrophil functions. The evidence is all indirect consisting of either observations of the behavior of neutrophils considered to have a defect of microfilament or microtubule activity or the effects on the various cell functions of inhibitors of one or the other system. Boxer et al. found that the neutrophils from a patient subject to recurrent infections were deficient in their random locomotion and chemotactic and phagocvtic responses but did show enhanced degranulation."3 The number of microfilaments in these neutrophils were fewer and the actin isolated from these neutrophils polymerized to a lesser extent than actin isolated from normal neutrophils. As Dr. Oliver will describe later in this Symposium, a defect in microtubule assembly is thought to be the basic defect in the neutrophils of animals or humans with the Chediak-Higashi svndrome.'4 The cells are defective in their chemotactic responsiveness and ability to degranulate although their random locomotion and phagocytosis is reportedly unaffected or even increased. Cvtochalasin B, a drug reported to inhibit microfilament action as well as other cell responses, inhibits chemotaxis and random locomotion 17-20 and phagocytosis,19'20 but enhances Iysosomal enzyme secretion.21'2 C5a, a chemotactic factor, induces assembly of neutrophil microtubules. "'2' Based on the use of colchicine and other antimicrotubule agents, a number of workers have suggested that microtubules are re-
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quired in directional movement (chemotaxis) but not in random locomotion 25,27
Although there is general agreement that random locomotion is unaffected and even enhanced by colchicine and similar agents, 2.28 wide variations have been reported in the concentrations required to inhibit chemotaxis.27.9-31 Colchicine is reported to inhibit lsosomal enzyme secretion, but frequently, concentrations wvere employed much above those reported to inhibit microtubules.32 In some instances, high concentrations of colchicine Xvere found to have no effect on lvsosomal enzyme release.33 ' A major defect in all of these and similar investigations is that no concomitant ultrastructural studies were done to correlate the inhibitory or lack of inhibitory effect of colchicine on the microtubule system with the effect of the drug on lysosomal enzyme secretion. How-ever, Hoffstein et al.. although not using colchicine, related microtubule assembly to l-sosomal enzyme release under a variety of conditions and concluded that microtubule assembly w as a necessarv but not sufficient condition for secretion.35 Except for the report of Stossel et al., colchicine has been found either not to affect phagocvtosis or to inhibit it only at concentrations far beyond those required to interfere with microtubule.37 The very tentatixe conclusions drawn from this incomplete survey are that in the random locomotion of neutrophils, microfilaments are required. but microtubules are not; in fact, they may actually inhibit. In contrast, directed locomotion (chemotaxis), seems to require both microfilaments and microtubules. Lysosomal enzyme secretion does not require microfilaments; on the contrary, their presence may be somew-hat inhibitorv. There is suggestive but conflicting evidence that enzyme secretion depends upon microtubules, but more wvork is required before this conclusion is acceptable. Phagocytosis requires microfilament activity, but probably does not require microtubules, although the latter possibly may aid the process. References Becker EL. Henson PM. Shovell HJ. Hsu LS: The ability of chemotactic factors to induce l-sosomal enzy-me release. I. The characteristics of the release. the importance of surfaces and the relation of the enzyme release to chemotactic responsiveness. J Immunol 112:2047-20554 1974 2. Goldstein MNI. Brai \M. Osler AG. Weissmann G: Lysosomal enzyme release from human leukocvtes: Mediation by the alternate pathwvay of complement activation. J 1.
Immunol 109::3:3-37. 197:3 :3. Musson R. Becker EL: The inhibitory effect of chemotactic factors on erythrophagocvtosis by human neutrophils. J Immunol 117:43:3-439, 197-6 4. Schiffmann E. Corcoran BA. WNahl SM: N-Formvlmethionine peptides as chemoattractants for leucocytes. Proc Natl Acad Sci USA 72:1059-1062. 197-5
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6. 7.
8. 9.
10. 11. 12. 13. 14. 15.
16. 17. 18. 19. 20. 21.
22. 23. 24.
25.
26.
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Showell HJ. Freer RJl Zigmond SH. Schiffmann S. Aswankumar S, Corcoran B. Becker EL: The structure activity relations of synthetic peptides as chemotactic factors and inducers of lvsosomal enzyme secretion for neutrophils. J Exp NMed 143:1154-1169, 19 7-6 Zigmond SH, Hirsch HG: Leukocyte locomotion and chemotaxis: A new method for evaluation and demonstration of cell derived chemotactic factor. J Exp Med 137 :387-410, 1973 Henson PM: Mechanisms of activation and secretion by platelets and neutrophils. Progress in Immunology, V'ol. 2 Edited by L Brent. J Holborow. Amsterdam-Oxford, North-Holland Press, 1974, pp 95-105 Wilkinson PC: Chemotaxis and Inflammation. Edinburgh & London. Churchill and Livingstone 1974 Hook WNA, Schiffmann E, Aswankumar S, Siraganian RP: Histamine release from basophils by formyl-methionine peptides. Fed Proc 35:864, 1976 Becker EL, Henson PM: In vitro studies of immunologically induced secretion of mediators from cells and related phenomena. Adv Immunol 17:93-193, 1973 Stossel TP: Phagocytosis: Recognition and ingestion. Semin Hematol 12:83-116, 1975 Pollard TD, Weihing RA: Actin and myosin and cell movement. CRC Crit Rev Biochem 2:1-65, 1974 Boxer L-, Hedle%--Whv%hte ET, Stossel TP: Neutrophil action dysfunction and abnormal neutrophil behavior. N Engl J Med 291:1093-1099, 1974 Oliver JM. Zurier RB, Berlin RD: Concanavalin A cap formation on polvmorphonuclear leucocytes of normal and beige (Chediak-Higashi) mice. Nature 253:471-473, 1975 Clark RA. Kimball HR: Defective granulocyte chemotaxis in the Chediak-Higashi syndrome. J Clin Invest 50:2645-2652, 197-1 Stossel TP, Root RK, Vaughn MI: Phagocytosis in chronic granulomatous disease and the Chediak-Higashi syndrome. N Engl J Med 286:120-123, 1972 Becker EL Davis AT, Estenson RD, Quie PG: Cytochalasin B. IV'. Inhibition and stimulation of chemotaxis of rabbit and human polymorphonuclear leukocytes. J Immunol 108:396-402, 1972 Borel JF, Stahlein H: Effects of cytochalasin B on chemotaxis and immune reactions. Experentia 28:745, 1972 Zigmond SH, Hirsch JG: Effects of cvtochalasin B on pol-morphonuclear leucocyte locomotion, phagocytosis and glycolysis. Exp Cell Res 73:383-393. 1972 Canarozzi NA. Malawista SE: Phagocvtosis of human blood leukocytes measured by the uptake of31 I-labeled human serum albumin: Inhibitory and stimulatorv effects of cytochalasin B. Yale J Biol Med 46:117-189. 1973 Zurier RB, Hoffstein S, Weissmann G: Cvtochalasin B: Effect on lysosomal enz-me release from human leukocvtes: Proc Nati Acad Sci USA 70:844-848, 1973 (Abstr) Becker EL Showell HJ: The ability of chemotactic factors to induce lysosomal enzyme release. II. The mechanism of the release. J Immunol 112:2055-2062. 1974 Goldstein I, Hoffstein S. Gallin J. Weissmann G: Mechanisms of lysosomal enzyme release from human leukocv-tes: Microtubule assembly and membrane fusion induced by a component of complement. Proc Natl Acad Sci USA 70:2916-2920. 1973 Gallin JIA Rosenthal AS: The regulator- role of divalent cations in human granuloc-te chemotaxis: Evidence for an association between calcium exchanges and microtubule assembly. J Cell Biol 62:594-60, 1974 Bhisey AN, Freed JN: Altered movement of endosomes in colchicine-treated cultured macrophages. Exp Cell Res 64:430-438. 1971 Allison AC, David P, dePetris S: Role of contractile microfilaments in macrophage movement and endocytosis. Nature [New Biol. ] 2.32:153-155, 197 1
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28. 29.
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:36. 37.
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Bandmann U. Rydgren L, Norbert B: The difference betu-een random moveement and chemotaxis. Exp Cell Res 88:6,3-73, 1974 Ramsey- WS. Harris A: Leukocyte locomotion and its inhibition by antimitotic drugs. Exp Cell Res 82:262-270, 1973 Caner JE Colchicine inhibition of chemotaxis. Arthritis Rheum 8:757-764, 1965 Phelps P, McCarty DJ Jr: Crystal induced arthritis. Postgrad Med 45:87-93. 1969 WN'ard PA: Leukotactic factors in health and disease. Am J Pathol 64:521-530, 1971 Wreissmann G, Goldstein I, Hoffstein S, Tsung PK: Reciprocal effects of AMP and GMP on microtubule-dependent release of l-sosomal enzymes. Ann NY Acad Sci 253:750-762. 1975 Henson PM: Pathologic mechanisms in neutrophil-mediated injury. Am J Pathol 68:593-612. 1972 Haw-kins D: Inhibition of neutrophil lysosomal release. Fed Proc 31:748, 1972 Hoffstein S: Microtubule assembly and secretion in human polymorphonuclear leukoc-tes. Fed Proc 34:868, 1975 Stossel TP, Mason RJ, Hartwig J, V'aughn N: Quantitative studies of phagocvtosis by polymorphonuclear leukocv-tes: Use of emulsions to measure the initial rate of phagoc'vtosis. J Clin Ivest 51:615-625, 1972 Malawista SE: Action of colchicine in acute gouty arthritis. Arthritis Rheum 18:8.35-846, 1975 Lehrer RI: Effects of colchicine and chloramphenicol on the oxidative metabolism and phagocvtic activity of human neutrophils. J Infect Dis 127:40-48, 1973
