The American Journal of PATHOLOGY SEPTEMBER 1976 * VOLUME 84, NUMBER 3
Immune Complex Receptors on Cell Surfaces II. Cytochemical Evaluation of Their Abundance on Different Immune Cells: Distribution, Uptake, and Regeneration Paul E. McKeever, MD, PhD, A. Julian Garvin, MD, PhD, Deborah H. Hardin, and Samuel S. Spicer, MD
A recently developed method for ultrastructural demonstration of cell surface receptors for immune complexes is applied to evaluation of these receptors on various cell tvpes. The method entailing incubation with a complex of horseradish peroxidase (HRP) and antibody to HRP (anti-HRP) disclosed dense foci indicative of immune complex receptors distributed at 30- to 120-mu intervals over macrophage surfaces. Invaginations, loop-like evaginations, and pinocytotic vesicles stained prominently. The number of stained immune complex receptors averaged 200,000 per oil-induced macrophage and 120,000 per noninduced macrophage, as determined from counts of focal deposits in electron micrographs. Receptor periodicity on giant cells present in oil-induced exudates resembled that on macrophages, but the larger giant cells contained an estimated 1.5 million sites. Although receptor periodicity on eosinophils and neutrophils equaled that on macrophages, the staining was lighter and was interrupted bv intervals of unstained membrane. Neutrophils averaged 28,000 and eosinophils 35,000 receptors per cell, whereas those lymphocytes with receptors averaged 3,500 per cell. Viable cells incubated with anti-HRP and HRP sequentially exhibited about half as many reactive sites as did cells incubated with immune complex. When warmed to 37 C, viable macrophages and eosinophils pinocytosed soluble immune complexes almost completely within 30 minutes and phagocytosed insoluble complexes more slowly. The endocytosed soluble immune complexes were sequestered within tubulovesicular structures in addition to the expected phagocytic vacuoles. Receptors appeared fully active on macrophages that were restained with soluble, cold immune complex after they had endocytosed immune complex in the course of a 30-minute warming interval. (Am J Pathol 84:437-456, 1976)
From the Department of
Carolina
Pathology. Mledical Universit%
of South Carolina. Charleston. South
Supported by a Research Project Grant from the Southern \Medical Association. Grants AM\-10956 and A\1-1 1028 from the National Institutes of Health, and Training Grant TR-168a from the \Veterans Administration Accepted for publication May 4. 1976. Address reprint requests to Dr S S Spicer. Department of Pathology. Medical Unisersity of South Carolina. 80 Barre Street. Charleston. SC 29401 437
438
MCKEEVER ETAL
American Journal of Pathology
MACROPHAGES. EOSINOPHILS. AND NEUTROPHILS are recognized participants in the endocvtic phase of the host immune response and are capable of interacting with and sequestering antigens, immune complexes, microorganisms; and diverse particulates. The primary interaction in this process entails adherence of such components to the cell surface. Phagocytes have an affinity for immune complexes, ordinarily mediated by a receptor for the crystallizable (Fc) portion of immunoglobulin." NMorphologic demonstration of this receptor depended initially on the binding of immunoglobulin-coated erythrocvtes to the cell surface.2'3 Precise definition of the prevalence and distribution of the immune complex receptor was not possible wvith this method because of the large size of the marker and its failure to identify individual binding sites on the cell surface. A recently developed method replacing sensitized erythrocytes with a soluble complex composed of horseradish peroxidase (HRP) and antibody to HRP (anti-HRP) has allow-ed precise localization of immune complex receptors at the ultrastructural level.7'8 Optimal demonstration of receptors was accomplished by staining unfixed cells at 4 C with soluble immune complex. The studies described here apply this method to appraising the relative abundance and distribution of these receptors on different normal or altered cell types. The binding of immune complex to macrophages is compared with that of immune globulin alone. The microendocvtic uptake of bound complexes and subsequent regeneration of surface receptors are also investigated.
Materials and Methods Peritoneal Cels Peritoneal cells were obtained bx- lavage from 2 to 4 kg New- Zealand albino rabbits 8 to 12 davs after induction of a peritoneal exudate by injection of 5 ml kg of mineral oil (Nujol. Plaugh. Inc.. New York). Cells were also harvested by lavaging the peritoneal cavitv without prior induction with mineral oil. Cells were harvested by aspiration of 250 ml of lavage fluid consisting of phosphate-buffered saline (PBS).9 The cells were rinsed twice by suspension in PBS and sedimentation at 5OOg for 13 minutes and were then resuspended in 10 ml of PBS and counted in a standard hemocytometer. Antiserum to Horseradish Peroxidase The antiserum to HRP was prepared in rabbits, monitored for specificity. and assessed for potency as previously described.7 Soluble immune complex was prepared according to
the method detailed previouslx 7 generally employing a dilution of one volume of antiserum w-ith 100 parts of a 1 mg 100 ml solution of HRP in PBS.
Staining Metiod The staining method referred to formerl 7 and again in this report as the standard procedure or standard incubation entailed suspending 1 to 2 million unfixed pelletized
Vol. 84, No. 3 September 1976
IMMUNE COMPLEX RECEPTORS
439
cells in 1.0 ml of freshly prepared HRP-anti-HRP soluble complex for 30 minutes at 4 C. The cells were then rinsed, incubated for 15 minutes in 3,3'-diaminobenzidine + H202 (DAB) substrate," rinsed, fixed 60 minutes at 4 C in 2% buffered osmium tetroxide, rinsed, dehydrated, and embedded in Epon 812. Heterologous binding of immune complex was evaluated by staining human buffy coat leukocytes with the standard soluble immune complex procedure described above employing rabbit antibody. As a control against nonspecific protein adsorption, red blood cells from the donor rabbit were stained by the standard soluble immune complex procedure. Controls for immunostaining included incubating the cells in the first step with HRP alone or with HRP plus serum from the donor of the peritoneal wash cells or another rabbit. The above standard incubation with soluble complex was varied to assess the biologic characteristics of the immune complex receptor. The receptor affinity for antibody alone was tested by a sequential incubation with anti-HRP followed by HRP at 4 C in place of immune complex as the first step of the procedure. For this purpose, cells were incubated with 1 part anti-HRP to 100 parts PBS, rinsed in PBS, incubated with 1 mg/100 ml HRP, rinsed, incubated in DAB substrate medium, rinsed, and postosmicated. After a final rinse with distilled water, the cells were dehydrated and embedded. In addition, the requirement for complement was evaluated by standard incubation with soluble immune complex prepared from antiserum which either had been supplemented with guinea pig complement or had been decomplemented by heating at 56 C for 30 minutes.
Fn-oytoSis O Immu Comles NW Peitmocytic Receptors Endocytosis of immune complexes and postendocytic receptors was investigated by warming the cells to 37 C either during the initial 30 minutes incubation with immune complex or for 10 or 30 minutes or 8 hours after the usual triple rinse which followed the initial incubation with immune complex at 4 C and preceded exposure to DAB. These temperature increases were carried out before, rather than after, incubation with DAB to assess the binding and uptake of immune complex as separate from the predictable phagocytic response to clumps of DAB reaction product. To test for receptor activity following endocytosis of immune complexes, cells were warmed for 30 minutes after the triple rinse which followed the initial exposure to immune complexes in the cold. A 30-minute warming interval was selected to allow nearly complete internalization of the initial complex. The cells were then reincubated with fresh soluble immune complex at 4 C for 30 minutes, triple rinsed, exposed to DAB, fixed with osmium tetroxide, and processed for electron microscopy. Cells incubated as described except for deletion of specific antiserum from the 30-minute reincubation served as controls.
_UDrasbuctw For ultrastructural examination, thin sections from blocks having satisfactorv morphologv in toluidine blue-stained thick sections were viewed with or without prior uranvl acetate and lead citrate staining in an Hitachi 12-A with a goniometer stage and Hitaci HS-8 electron microscope. To assess the binding of immune complexes compared with immunoglobulin alone and the effects of elevated temperature on bound complexes, randomlv selected cells from triplicate runs of these various incubations were photographed at low magnification. Ratios of stained to total plasma membrane for 25 cells per group were tallied blindly. Means of these ratios were tabulated and compared by unpaired method of proportions, and probabilities of significant differences expressed as P.'s Means were compared with the standard incubation (Tables 1 and 2).
440
MCKEEVER ET AL
American Joumal of Pathology
Table 1-Macrophage Binding of Immune Complex Compared With Immunoglobulin Alone and With Decomplted Immune Complex P eof m surface stained Probability of difference Method (mean ± SD) from standard incubation Standard procedure with cold soluble immune complex* 87.6 ± 13.5 Sequential incubation of cells with anti-HRP followed by HRP 45.0 ± 9.1 P < 0.05 Decompkemented soluble complex 86.5 ± 13.7 No significant difference * See Materials and Methods section.
Qaitation of Immue Comple Receptors Quantitation of immune complex receptors on the macrophage surface was undertaken
bv computing the ratio of the whole cell's plasma membrane surface area to its surface area within Epon thin section. As derived and more fully described in the Discussion, this ratio is 2 r/h, where r is the radius of the cell and h is the heighth of the cylindrical portion of cell in thin section (i.e., the 40-mu thickness of the section). The reported macrophage radius of 7.5 was used in these calculations and confirmed by light microscopy of whole peritoneal macrophages.ll.U2 The number of stained immune complexes on the thin section profile of the cell surface for 15 randomly selected (except for exclusion of obviously tangential sections) macrophage profiles from four or more standard incubations was counted. The total number of receptors on the whole cell's surface was then calculated for each cellular profile by multiplying the number of receptors in the thin section by 2 r/h. Receptor numbers for these whole cells were then tabulated, and the mean ± standard error was calculated. The number of immune complex receptors on the other peritoneal exudate cell tvpes was estimated with the same procedure, assuming that the eosinophil, neutrophil, and lymphocvte approximate spheres of 6.5, 5.5, and 4 gu radii, respectively.U2 Tabulated numbers of stained receptors on different cell types were compared by t test, and P < 0.05 was considered to represent significant difference."3 Table 2-Internalization of Bound Immune Complexes at Elevated Temperature Percenta of macrophag surface stained Probability of difference Method (mean ± SD) from standard incubation Standard procedure with cold soluble 87.6 ± 13.5 immune complex Modification of standard procedure Elevafion of temperature to 37 C during incubation with immune 12.5 ± 6.6 P < 0.001 complex Elevation of ternperature to 37 C for 34.8 ± 7.5 P < 0.05 10 minutes before DAB Elevation of temperature to 37 C for 1.5 ± 8 P < 0.001 30 minutes before DAB Elevation of temperature to 37 C for 30 minutes,recooling and reincubation with cold soluble immune 83.9 ± 14.9 No significant difference complex before DAB * As described in Materials and Methods.
Vol. 84, No. 3 September 1976
IMMUNE COMPLEX RECEPTORS
441
Results Staining of Immune Complex Receptors and Immunoglobulin Receptors on Macrophages
Macrophages stained prior to fixation with soluble immune complex at 4 C. according to the standard procedure, disclosed reaction product indicative of immune complex receptors in periodic foci over the entire cell surface (Figure 1). These foci wvere separated by 30- to 120-mg intervals and covered about 85%c of the cell surface (Table 1). Controls incubated with HRP alone or a mixture of HRP and normal rabbit serum, including that from an exudate donor, disclosed no surface staining on leukocytes. A conspicuous feature of the macrophage surface consisted of round cytoplasmic exvaginations \vith a diameter of 0.2 to 1.5 aand a uniformlytranslucent center. The evaginations protruded from both control and stained macrophages processed at 4 C (Figures 2 and 3) and occurred less frequently in those processed at 37 C. These generally circular profiles were composed of tw o membranes -hich were 20 to 35 mp apart and connected by periodic thin bridges. Continuity between the intermembranous space and the macrophage cytoplasm Xwas evident in appropriately sectioned evaginations (Figure 3). Both internal and external membranes stained for soluble immune complex, indicating that both sides of the evaginations communicated with or had recently communicated with the extracellular space. Attempts to isolate these structures by multiple velocity centrifugations of supernatant over the range of 1.000 to 25.OOOg proved unsuccessful. Similar evaginations were not present on the few eosinophils. neutrophils. or lymphocytes encountered. Varying the procedure for localizing immune complex by first incubating cells with uncomplexed antiserum to HRP, then rinsing and exposing to HRP, produced less surface staining than did exposure to soluble immune complex and similar rinsing (Table 1). Decomplementing the anti-HRP serum, or adding guinea pig complement to it. did not alter staining of macrophages (Table 1). Quantitation of Receptors on Various Cell Types
The number of immune complex receptor sites on the cell surface was estimated by counting the focal precipitates on the cell profile and extrapolating mathematically to the surface area of the \vhole cell. Fifteen oilinduced macrophages randomly selected from those incubated with soluble immune complexes at 4 C averaged 200,000 ± 30,000 (standard error) stained sites indicative of the immune complex receptor per cell. Small stained structures, possibly caveolae or tubular invaginations of the surface or pinocvtic vesicles, lay near the plasma membrane (Figures
442
MCKEEVER ET AL
American Journal of Pathology
4-6). Although these structures were present when all immunostaining steps had been performed at 4 C, they were too small and infrequent to alter significantly the staining periodicity. A comparable sample of macrophages obtained without induction of an exudate by mineral oil injection disclosed somewhat fewer receptors. Thus, the noninduced macrophages averaged 120,000 ± 10,000 receptor sites. The noninduced macrophages also contained fewer lysosomes and were slightly smaller than the induced cells. Multinucleate giant cells with diameters in the range of 18 to 50 y occurred in the oil-induced peritoneal exudate, but were not encountered among the noninduced cells. The giant cells consistentlv stained like macrophages, showing similar periodicity and receptors covering cvtoplasmic invaginations and evaginations as well as intervening plasmalemma (Figure 7). The round cytoplasmic evaginations with translucent centers (Figures 2 and 3) were less common on giant cells than macrophages. The number of receptor sites on the surface of the larger giant cells was estimated mathematically at 1.5 ± 0.6 million per cell. Eosinophil and neutrophil immune complex receptors stained with periodicity as did those of macrophages; however, unstained regions of membrane were more frequent, and the receptors were more weaklv stained compared with those of macrophages (Figures 8 and 9). The average number of receptor sites per cell was 28,000 ± 4,000 on neutrophils and 35,000 ± 6,000 on eosinophils. Receptors were very sparsely distributed on the majority of the lvmphocytes (Figure 10) and essentially absent from some lymphocyte profiles (Figure 11). Stained sites on lymphocytes having receptors numbered 3,500 ± 450 per cell and varied little according to the presence or absence of complement. When the numbers of stained receptors on all of the cell types previously described were compared by the unpaired t test, the differences in means were significant at P < 0.05 in all comparisons except the neutrophils and eosinophils. Light microscopic measurement of whole cells in suspension revealed radii of mean dimensions similar to those previously reported.l'"l The largest range in cellular radius was from 5 to 15 Ai in oil-induced macrophages. Electron micrographs of obviously tangential sections were excluded from the group counted. The mean radius from electron micrographs was an expected 10% lower than values obtained from measuring whole cells. Heterologous staining of human monocytes, granulocytes, and lymphocytes resembled the staining of the comparable rabbit cell lines in both morphology and relative intensity. Homologous (Figure 12) and
Vol. 84, No.3 September 1976
IMMUNE COMPLEX RECEPTORS
443
heterologous erythrocytes lacked surface staining after processing with soluble immune complex by the standard procedure. Endocytosis of hmnme Compees and Posteotic Receptors
The endocytic uptake of immune complex bound to the extemal plasmalemma was assessed by warming the cells to 37 C during incubation with immune complex or between incubation with immune complex and with DAB (Table 2). Incubation with immune complex at the elevated temperature largely prevented staining of receptors. Pinocytosis removed much to nearly all of the bound soluble immune complexes from the macrophages warmed 10 to 30 minutes prior to exposure to DAB (Figures 14 and 15). After either 37 C incubation with soluble immune complex or warming 10 minutes before the DAB step, stretches of stained surface alternated with 0.1- to 20-M lengths of unstained membrane, apparently as a result of active pinocytosis. The endocytosed soluble immune complex was sequestered in moderately large cytoplasmic dense bodies of obvious heterophagic nature. Notably, the soluble complex was also sequestered in tubulovesicular structures. Some macrophages incorporated immune complex mainly into the dense bodies (Figure 14) and others mainly into tubulovesicular structures (Figure 15). Peroxidase activity gradually disappeared within heterophagic dense bodies, but a small proportion remained within tubulovesicular structures after warming for 8 hours. Eosinophils and lymphocytes internalized surface-bound, soluble immune complex by pinocytosis as did macrophages. After 30 minutes, the majority of the complex in eosinophils was enclosed in large vacuoles presumed to be secondary lysosomes (Figure 13). These vacuoles were distinctly separate from the crystalloid granules which appeared not to contain endocytosed immune complex. Macrophages internalized more complex than eosinophils; lymphocytes endocytosed much less. Neutrophil morphology rapidly deteriorated when these unfixed cells were exposed to soluble complex at physiologic temperature. Macrophages exposed to immune complex, warmed for 30 minutes to remove complex from the surface by endocytosis, and immediately reincubated with cold, soluble immune complex disclosed surface staining similar to cells processed by the standard procedure (Figure 16 and Table 2). Control cells with specific antiserum deleted during reincubation disclosed almost no surface staining. With the method employed here, the major portion of the immune complex receptors on immune cells is visualized and quantified, and the internalization of immune complex bound to receptors is traced in vitro.
444
MCKEEVER ET AL
American Journal of Pathology
Determining the true periodicity of macrophage immune complex receptor sites is complicated by the endocytosis of the labeling moiety. Initial experiments 7 showed that decreasing the incubation temperature markedly increased the amount and uniformity of surface staining, presumably because of the corresponding decrease in pinocytic activity. As shown here, low temperature incubation did not entirely eliminate pinocytic internalization of immune complex, since stained profiles suggestive of pinocytic vesicles occasionally lay close beneath the plasmalemma. Nevertheless, the periodic stained foci spaced 30 my apart over macrophage surfaces are considered to reflect the actual distribution of immune complex receptors. The following observations support this interpretation. Those membrane sites least susceptible to pinocytosis, i.e., the thin cytoplasmic evaginations stained consistently in this way. Mechanically disrupted membranes of viable cells, presumably devoid of endocytic capacity, disclosed this finely periodic staining, as did occasional areas of cells fixed before incubation. The more patchy distribution of stained sites on cells warmed during or after incubation with immune complex observed in the present and other 6investigations probably resulted from endocytic uptake of bound complexes. This surface staining, which was encountered on all of the cell types after incubation at elevated temperature, suggests that complexes are internalized in patches rather than by a capping process. The observations on immune complex internalization during staining afforded no indication of migration of the receptors to a polar cap, as occurs prior to endocytosis of surface immunoglobulin and its specific antiimmunoglobulin on lymphocytes.14 The capacity of a soluble immune complex, presumably containing a single immunoglobulin molecule, to bind only monovalently to a single Fc receptor possibly explains the occurrence of patching rather than capping in this system. The cytoplasmic evaginations observed on macrophages resembled similar cytoplasmic evaginations termed loops on guinea pig exudate peritoneal macrophages.15 Membrane-to-membrane cross bridges reported to stabilize similar structures in other cell types 16 were evident also in cells examined here. The occasionally observed separation of these immune complex-rich structures from cells suggests that they may detach from macrophages and that these cells in migrating could leave behind a trail of immune complexes. The sequestering of immune complex on the inner surface of these structures might temporarily protect them from degradation. We have observed the same evaginations on macrophages at sites of injured human tissue. In the present study, the number of evaginations appeared increased by cold incubation, and they might result
Vol. 84, No. 3 September 1976
IMMUNE COMPLEX RECEPTORS
445
from an imbalance between accretion and depletion of plasmalemma in the cold. Recent studies have demonstrated the ability of soluble immune complexes to inhibit macrophage migration."7 The complexes thus restrain macrophages at an inflammatory site, acting like the migration inhibitorv factor of T lymphocytes. The extensive surface staining and pinocytotic activity observed in the present study provide a plausible explanation of this phenomenon. A surface coat of immune complex, or the undirectional pinocytosis of this complex, may directly impede cellular motility. The method of quantitating immune complex receptors is not standard and requires further explanation. A thin section of a cell contains a discrete portion of the whole cell's plasma membrane surface area within it. The whole cell's surface area is a specific multiple of the surface area within the thin section. If the cell is a sphere with radius r, its surface area within thin section is 2irrh, where h is the heighth of the nearlv cylindrical portion of cell in section. The whole surface area of this same cell is 47rr2. Thus, the multiple relating the whole cell's surface area to its surface area within thin section is 4xrr2/2wrh or, more simply, 2r/h. If the number of stained receptors counted on the cell's surface within thin section is multiplied by 2r/h, the number of stained receptors on the whole cell's surface can be calculated mathematically. The same analytic scheme may be applied to cells containing ruffled and invaginated membranes, providing they are spherical and their surface irregularities are uniformly distributed. Uniform distribution insures that receptors counted in thin sections will include the count variations produced by surface irregularities so that these variations will automatically be incorporated into the calculation of receptor numbers on the whole cell. Scanning electron microscopy (SEM) of macrophages stained with our standard procedure and similarly fixed as a pellet 16 and SEM of macrophages specifically stained with crystalline reaction product localizing immune complex receptors 19 have shown spherical cells with a uniform distribution of surface irregularities (pseudopods and veils), providing spreading on coverslips is avoided. Our experience has been similar with neutrophils and lymphocytes, except for the minor interference of uropods in the latter. The number of receptors on rabbit alveolar macrophages has been measured with radiolabeled soluble immune complexes.20 The number estimated by this method exceeds by fivefold that obtained in the present study by counting individual precipitates of reaction product in thin sections and extrapolating to the entire surface area. Like the peritoneal macrophage, the rabbit alveolar macrophage pinocytoses all of its soluble
446
MCKEEVER ET AL
American Jourma of Pathology
immune complex within 30 minutes at 37 C.8 Since the radiolabeled immune complex binding study was performed at 37 C for 30 minutes, pinocytosed and surface-bound radiolabeled immune complexes were probably counted and the number of receptors may have been overestimated. On the other hand, the number of receptors may have been underestimated in the present study either because of failure of some receptors to stain-for example, because of competitive binding with immunoglobulins other than anti-HRP in the antiserum-or because stained foci observed here represent more than one receptor. The regular periodicity of these foci at 30-ma intervals in many areas argues against the latter consideration. The counting procedure introduced in this study is susceptible to a 5 to 20% underestimation of surface counts from thin sections which missed the equatorial plane of the cells, although we avoided counting obviously tangential sections and, thereby, minimally altered the distribution of cell sizes counted. The several leukocyte lines varied widely in prevalence of immune complex receptors, decreasing in order from macrophage to granulocyte to lymphocyte. This order of receptor abundance correlated with the relative capacity of these cells to endocytose immune complexes. The receptor content also corresponds with the capacity of the different cell types to incorporate small particles such as colloidal gold spherules by microendocytosis.21'* Apparently, the latter ability to internalize such inert particles concerns a property or component of the cell membrane correlated in amount with the immune complex receptor. Fc receptors have been demonstrated on B lymphocytes by light and electron microscopic immunostaining and autoradiography.2>28 Receptors for aggregated immunoglobulin have been demonstrated on both B and T cells.25 26 In the present study, the majority of lymphocytes washed from the peritoneal cavity bound immune complex, but the cells varied in abundance of receptors possibly in relation to their identity as B or T lymphocytes. The prevalence of immunoglobulins visualized by immunostaining , on the lymphocyte surface apparently exceeds that of immune complex receptors demonstrated here. The abundance of immunostained histocompatability antigens on lymphocytes 1 also greatly exceeds that of immune complex receptors. The immune complex receptor thus appears to comprise a different component from either the bound immunoglobulin or the histocompatibility antigens on the lymphocyte surface. That the relative sparsity of foci on lymphocytes reactive with HRP-anti-HRP complex reflects incomplete staining of these receptors seems unlikely, since the more numerous receptors of the macrophage were clearly visualized over its entire surface.
Vol. 84, No. 3 September 1976
IMMUNE COMPLEX RECEPTORS
447
The existence on macrophages of separate receptors for immune complex and for immunoglobulin is suggested by the greater abundance on these cells of sites stained with immune complex than of sites stained with the sequence of anti-HRP and then HRP. Further experiments with double labeling techniques perhaps would clarify whether this recognized 2 difference can be attributed to weaker binding of immunoglobulin than of immune complex or to the existence of different receptors for immunoglobulin alone and for immune complex on the macrophage surface. The observed differences conceivably relate to the known variable binding affinities of leukocytes for subelasses of immunoglobulin n and to a possible preponderance of high affinity immunoglobulin types in the immune complex employed. The ratio of immune complex binding sites to bound immunoglobulin appears to be reversed for macrophages compared with lymphocytes, in that macrophages show abundant immune complex receptors with the procedure described here, and lymphocytes show abundant membranebound immunoglobulins when stained with labeled antibody to immunoglobulin. A question remains whether the latter lymphocyte surface immunoglobulin, which presumably originates endogenously within the lymphocyte, is bound by the same mechanism as exogenous immunoglobulin demonstrated ultrastructurally with radiolabeled 2'*- or ferritinlabeled immunoglobulin, and whether the term Fc receptor applies to the binding site on lymphocytes for endogenous or exogenous immunoglobulin or both. Beyond this question, the marked difference between binding immunoglobulin compared with that of immune complex on both macrophages and lymphocytes would favor designating the immune complex and the immunoglobulin binding sites as different entities. Notably, immune complex receptors were not demonstrable on cell types other than immune cells and specifically were absent from ervthrocvtes.
Macrophages were found to pinocytose almost completely a surface complement of immune complex within 30 minutes at 37 C. Some pinocytosed soluble immune complexes were sequestered within tubulovesicular structures which appeared morphologically distinct from the large phagocytic vacuoles that sequester and digest insoluble complex 6 and other substances. The tubulovesicular structures possibly comprise a separate endocytic system and could perhaps function in antigen processing to enhance the immune response. Some macrophages contained tubulovesicular structures predominantly and might differ from those with only large heterophagic vacuoles. The present findings also indicate full receptor capacity 30 minutes following apparent internalization of 90 to 99% of immune complex
448
MCKEEVER ET AL
American Journal of Pathology
bound to the majority of the macrophage plasma membrane. Other studies which perturbed immunoglobulin receptors have demonstrated receptor regeneration slo'wer than the phenomenon described here. For example, receptor activity recovers slowly over a 2-day culture interval after removal of an antimembrane antibody wvhich inhibits macrophage receptor activity." Phagocytosis of large particles uncoated wvith antibody inhibits by 50% the subsequent attachment or phagocytosis of antibodycoated ervthrocytes over a 2-hour interval." The present system emphasizes the capacity of the immune complex receptor to relatively rapidly accommodate more immune complex when perturbed specifically and solely by a thin surface-coat of immune complex. It, therefore, differs from the aforementioned systems which demonstrated the more prolonged effects of antimembrane antibodies and a phagocvtic load. Possible explanations for this surprisingly rapid accommodation of a second surface-coat of complex include pinocytosis of complex alone, leaving receptors behind in the plasma membrane; initial staining or internalization of only a small portion of receptors; and rapid regeneration of pinocvtosed receptors. These possibilities may be susceptible to further study with techniques of tracing external plasma membrane components during endocvtosis. Rabbit giant cells possessed more receptor sites for homologous immune complex than any other cell tested. Their staining differs remarkably from murine giant cells studied at 37 C which demonstrated few receptor sites for heterologous ferritin-labeled IgG.5 The different binding to giant cells of antigen-antibody complexes compared with ferritin-labeled antibody may contribute to this wide diversity. Different pinocytotic rates between 4 C and 37 C could have also contributed. References 1. Arend \\NP. Niannik N1 In vitro adherence of soluble immune complexes to macrophages. J Exp Med 136:514-531. 1972 2. Berken A. Benacerraf B: Properties of antibodies cytophilic for macrophages. J Exp Med 12:3:119-144. 1966 :3. Box-den SV: Cytophilic antibody in guinea pigs w%ith delayed-type hypersensitivity. Immunology 7:474-483. 1964 4. Lies FYN The binding of the F, fragment of guinea pig cytophilic antibody to peritoneal macrophages. Immunology 20:S17-829. 1971 5. Papadimitriou J\: Detection of macrophage receptors for heterologous IgG by scanning and transmission electron microscopy- J Pathol 110:213-220. 197:3 6. Steinman RNM. Cohn ZA: The interaction of particulate horseradish peroxidase (HRP)-anti-HRP immune complexes wvith mouse peritoneal macrophages in vitro. J Cell Biol .55:616 -&34. 1972
Vol. 84, No. 3 September 1976
IMMUNE COMPLEX RECEPTORS
449
7. McKeever PE, Garvin AJ, Spicer SS: Immune complex receptors on cell surfaces. I. Ultrastructural demonstration. J Histochem Cvtochem 24 (In press) 8. McKeever PE, Garvin AJ: Ultrastructural localization of immune complexes binding to living and fixed macrophages. Am J Pathol 78:55a, 1975 (Abstr) 9. Mason TE, Phifer RF, Spicer SS, Swallow RA, Dreskin RB: An immunoglobulinenzvme bridge method for localizing tissue antigens. J Histochem Cytochem 17:563-4569, 1969 10. Graham RC Jr, Karnovsky MJ: The early stages of absorption of injected horseradish peroxidase in the proximal tubules of mouse kidnev: Ultrastructural cvtochemistry by a new technique. J Histochem Cytochem 14:291-302, 1966 11. Carr I: The Macrophage: A Review of Ultrastructure and Function. New York, Academic Press, Inc., 1973, pp 5-7 12. Rhodin JAG: Histology: A Text and Atlas. New York, Oxford University Press, 1974, pp 98, 100, 106, 156, 388 13. Annitage P: Statistical NMethods in NMedical Research. New York, John Wiley and Sons, 1973, pp 104-114 14. de Petris S, Raff MC: Normal distribution, patching and capping of lymphocyte surface immunoglobulin studied by electron microscopy. Nature [New Biol] 241:257-259, 1973
15. Daems WT, Brederoo P: Electron microscopical studies on the structure, phagocytic properties and peroxidatic activity of resident and exudate peritoneal macrophages in the guinea pig. Z Zellforsh Mikrosk Anat 144:247-297, 1973 16. Franke WW, Kartenbeck J, Zentgraf H, Scheer U, Falk H: Membrane-to-membrane cross-bridges: A means to orientation and interaction of membrane faces. J Cell Biol 51:881-888, 1971 17. Kotkes P, Pick E: Studies on the inhibition of macrophage migration induced by soluble antigen-antibody complexes. Clin Exp Immunol 19:105-120, 1975 18. McKeever PE, Garvin AJ, Spicer SS: Unpublished observations 19. McKeever PE, Garvin AJ, Brissie NT, Spicer SS: Crystalline markers for scanning electron microscopy: Visualization of immune complex receptors on macrophage surfaces. J Reticuloendothel Soc 18:30b, 1975 (Abstr) 20. Arend WP, Mannik M: The macrophage receptor for IgG: Number and affinity of binding sites. J Immunol 110:1455-1463, 1973 21. Komivama A, Spicer SS, Bank H, Farrington J: Induction of autophagic vacuoles in peritoneal cells. J Reticuloendothel Soc 17:146-161, 1975 22. Komiyama A, Spicer SS: Microendocytosis in eosinophil leukocvtes. J Cell Biol 64:622-635, 1975 23. Basten A, Miller JFAP, Sprent J, Pye J: A receptor for antibody on B lymphocytes. I. Method of detection and functional significance. J Exp Med 135:610-626, 1972 24. Basten A, Warner NL, Mandel T: A receptor for antibody on B lymphocytes. II. Immunochemical and electron microscopy characteristics. J Exp Med 135:627-642, 1972 25. Van Boxel JA, Rosenstreich DL: Binding of aggregated Y -globulin to activated T lymphocytes in the guinea pig. J Exp Med 139:1002-1012, 1974 26. Anderson CL, Grey HM: Receptors for aggregated IgG on mouse lymphocytes: Their presence on thymocvtes, thvmus-derived, and bone marrow-derived lymphocytes. J Exp Med 139:1175-1188, 1974 27. Dickler HB: Studies of the human lymphocyte receptor for heat-aggregated or antigen-complexed immunoglobulin. J Exp Med 140:508-522, 1974 28. Theofilopoulos AN, Wilson CB, Bokisch VA, Dixon FJ: Binding of soluble immune complexes to human lymphoblastoid cells. J Exp Med 140:1230-1244, 1974 29. Bretton R, Ternvnck T, Avrameas S: Comparison of peroxidase and ferritin labelling of cell surface antigens. Exp Cell Res 71:145-155, 1972
450
MCKEEVER ET AL
American Joumal of Pathology
30. Reves F, Lejonc JL, Gourdin MF, Mannoni P, Drevfus B: The surface morpholog'y of human B lymphocytes as revealed by immunoelectron microscopy. j Exp Med 141:392-410, 1975
31. Willingham MC, Spicer SS, Graber CD: Immunocytologic labeling of calf and human lymphocyte surface antigens. Lab Invest 25:211-219, 1971 32. Lawrence DA, Weigle WO, Spiegelberg HL: Immunoglobulins cytophilic for human lymhpocytes, monocytes and neutrophils. J Clin Invest 55:368-376, 1975 33. Holland P, Holland NH, Cohn ZA: The selective inhibition of macrophage phagocytic receptors by anti-membrane antibodies. J Exp Med 135:458-475, 1972 34. Schmidt ME, Douglas SD: Disappearance and recovery of human monocyte IgG receptor activity after phagocvtosis. J Immunol 109:914-917, 1972 35. De Pierre J, Kamovskv ML: Ectoenzvmes, sialic acid, and the internalization of cell membrane during phagocytosis. Inflammation: Mechanisms and Control. Edited bv IH Lepow, PA Ward. New York, Academic Press, Inc., 1972, pp 55-70 36. Hubbard AL, Cohn ZA: Externally disposed plasma membrane proteins. II. Metabolic fate of iodinated polypeptides of mouse L cells. J Cell Biol 64:461-479, 1975 The authors would like to express their appreciation for the generous advice and support of Drs. G. R. Hennigar, J. D. Balentine, and J. M. Powers; the skilled technical assistance of Nancy Brissie and Betty Hall; the dependable photographic assistance of Jim Nicholson and his staff; and the efficient secretarial assistance of Karen Beaufort.
IPRJM06,17. t
F.
e 'S
de
r.
{
_#_,, s
! :.
u., *
; r
db
v@s=. .%
t 4,
+
.:
:
Fgu 1-Macrophages from peritoneal exudate exhibit deposits of reaction product indicative of immune complex receptors in periodic foci over the surface. Stained with soluble immune complex in cold by the standard procedure. (x 9000)
Figues 2 and 3-Like Figure 1 except thin sections were stained with uranyl acetate and lead citrate. Internal and external membrane surfaces of macrophage evaginations are separated by a constant 30-mM distance and are both heavily stained for immune complex receptors. Continuity with macrophage cytoplasm (arrows) and cross bridges between membranes are evident. (x 45,000)
Figures 4-6-Surface area in cells treated like that in Figure 1. Stained profiles suggestive of pinocytic vesicles (short arrows) lie near the plasmalemma often dosely associated with a surface invagination (long arrow). Unstained vesicles are in the proximity (arrowhead). (4 md 5, x 32,000; 6, x 18,200)
Figure 7-Giant cell treated like cell in Figure 1. Its entire surface stains uniformly for immune complex. (x 5250)
3
2
4
5
6
7
f
'r
.
...4..40411
-11
-
--A lml..
.
-.-
41,
".
--4 .,. --&
a
.
1
%Ait
.a
I
n,
140 -4
-,
.:.
t;
'. 11
8
I
I7
:% ,l
a
N,_:-Iw- .
!-d.W
J
10
.4-
JO
40 :6.
". .k
Is 4tL
-
Feum 8-11-Staining for immune complex receptor on other cell types occasionally encountered in the rabbit peritoneal exudate. In the eosinophil (E) (8 and i), the plasmalemma contains relatively sparse foci of reaction product indicative of receptor staining widely distributed along its surface. Intrinsic peroxidase activity accounts for the density of the eosinophil crystalloid granules. The neutrophil (N) (9 and itset) exhibits light similar surface staining and faint staining indicative of intrinsic peroxidase in cytoplasmic primary granules. The lymphocyte (L) (10) contains very sparse reaction product in an uneven distribution along its surface, but the lymphocyte (L) (11) almost totally lacks surface staining. The characteristically abundant reactive foci on neighboring macrophages contrast with the sparsity of such staining on the other leukocytes. Soluble immune complex standard procedure. (Unstained thin sections, 8, x 7600; d%, x 17,400; 9, x 9800; kiset, x 14,000; 10, x 8800; 11, X 9800)
9
; 12
13 13
12
s-
14
t 'A
Figure 12-The red blood cell (R) lacks surface reaction product in contrast to the stained neighboring macrophages. Soluble immune complex standard procedure applied to a mixture of Figure 13-This eosinophil was peritoneal wash cells and homologous erythrocytes. (x 7200) processed as in the standard procedure except for warming to 37 C for 30 minutes between exposure to immune complex and incubation with DAB. Surface staining has been eliminated by pinocytosis. Most of the internalized immune complex is sequestered in secondary lysosomes (arrow), but a trace remains in pinocytotic vesicles. Crystalloid granules appearing uniformly dense from content of intrinsic peroxidase, afford no evidence of alteration through having incorporated immune complex and appear not to participate in microendocytosis. (x Figures 14 and 15-Cells treated like that in Figure 13. Surface staining has been 11,800) eliminated by pinocytosis except at the tips of occasional macrophage processes (arrowheads). The cells display two sites of antigen sequestration: one in relatively large dense bodies (arrows, 14), and the other in small tubulovesicular structures (arrows, 15). These macrophages differ in containing mainly one or the other type of endosome. (Unstained thin sections, x 6700)
15
i, . i!i ,=F
m\t4!
M,. .
.I.
Figure 16-These macrophages were processed as in the standard procedure except for warming to 37 C for 30 minutes and then reincubating with cold soluble immune complex before exposure to DAB. Endocytosed antigen (arrowheads) is sequestered as in Figures 14 and 15. The surface staining (arrow) comparable to that in Figure 1 indicates the macrophages have regained the receptor capability, following endocytosis of the initially applied immune complex. (Unstained thin section; x 10.500)
Immune Complex Receptors on Cell Surfaces II. Cytochemical Evaluation of Their Abundance on Different Immune Cells: Distribution, Uptake, and Regeneration Paul E. McKeever, MD, PhD, A. Julian Garvin, MD, PhD, Deborah H. Hardin, and Samuel S. Spicer, MD
A recently developed method for ultrastructural demonstration of cell surface receptors for immune complexes is applied to evaluation of these receptors on various cell tvpes. The method entailing incubation with a complex of horseradish peroxidase (HRP) and antibody to HRP (anti-HRP) disclosed dense foci indicative of immune complex receptors distributed at 30- to 120-mu intervals over macrophage surfaces. Invaginations, loop-like evaginations, and pinocytotic vesicles stained prominently. The number of stained immune complex receptors averaged 200,000 per oil-induced macrophage and 120,000 per noninduced macrophage, as determined from counts of focal deposits in electron micrographs. Receptor periodicity on giant cells present in oil-induced exudates resembled that on macrophages, but the larger giant cells contained an estimated 1.5 million sites. Although receptor periodicity on eosinophils and neutrophils equaled that on macrophages, the staining was lighter and was interrupted bv intervals of unstained membrane. Neutrophils averaged 28,000 and eosinophils 35,000 receptors per cell, whereas those lymphocytes with receptors averaged 3,500 per cell. Viable cells incubated with anti-HRP and HRP sequentially exhibited about half as many reactive sites as did cells incubated with immune complex. When warmed to 37 C, viable macrophages and eosinophils pinocytosed soluble immune complexes almost completely within 30 minutes and phagocytosed insoluble complexes more slowly. The endocytosed soluble immune complexes were sequestered within tubulovesicular structures in addition to the expected phagocytic vacuoles. Receptors appeared fully active on macrophages that were restained with soluble, cold immune complex after they had endocytosed immune complex in the course of a 30-minute warming interval. (Am J Pathol 84:437-456, 1976)
From the Department of
Carolina
Pathology. Mledical Universit%
of South Carolina. Charleston. South
Supported by a Research Project Grant from the Southern \Medical Association. Grants AM\-10956 and A\1-1 1028 from the National Institutes of Health, and Training Grant TR-168a from the \Veterans Administration Accepted for publication May 4. 1976. Address reprint requests to Dr S S Spicer. Department of Pathology. Medical Unisersity of South Carolina. 80 Barre Street. Charleston. SC 29401 437
438
MCKEEVER ETAL
American Journal of Pathology
MACROPHAGES. EOSINOPHILS. AND NEUTROPHILS are recognized participants in the endocvtic phase of the host immune response and are capable of interacting with and sequestering antigens, immune complexes, microorganisms; and diverse particulates. The primary interaction in this process entails adherence of such components to the cell surface. Phagocytes have an affinity for immune complexes, ordinarily mediated by a receptor for the crystallizable (Fc) portion of immunoglobulin." NMorphologic demonstration of this receptor depended initially on the binding of immunoglobulin-coated erythrocvtes to the cell surface.2'3 Precise definition of the prevalence and distribution of the immune complex receptor was not possible wvith this method because of the large size of the marker and its failure to identify individual binding sites on the cell surface. A recently developed method replacing sensitized erythrocytes with a soluble complex composed of horseradish peroxidase (HRP) and antibody to HRP (anti-HRP) has allow-ed precise localization of immune complex receptors at the ultrastructural level.7'8 Optimal demonstration of receptors was accomplished by staining unfixed cells at 4 C with soluble immune complex. The studies described here apply this method to appraising the relative abundance and distribution of these receptors on different normal or altered cell types. The binding of immune complex to macrophages is compared with that of immune globulin alone. The microendocvtic uptake of bound complexes and subsequent regeneration of surface receptors are also investigated.
Materials and Methods Peritoneal Cels Peritoneal cells were obtained bx- lavage from 2 to 4 kg New- Zealand albino rabbits 8 to 12 davs after induction of a peritoneal exudate by injection of 5 ml kg of mineral oil (Nujol. Plaugh. Inc.. New York). Cells were also harvested by lavaging the peritoneal cavitv without prior induction with mineral oil. Cells were harvested by aspiration of 250 ml of lavage fluid consisting of phosphate-buffered saline (PBS).9 The cells were rinsed twice by suspension in PBS and sedimentation at 5OOg for 13 minutes and were then resuspended in 10 ml of PBS and counted in a standard hemocytometer. Antiserum to Horseradish Peroxidase The antiserum to HRP was prepared in rabbits, monitored for specificity. and assessed for potency as previously described.7 Soluble immune complex was prepared according to
the method detailed previouslx 7 generally employing a dilution of one volume of antiserum w-ith 100 parts of a 1 mg 100 ml solution of HRP in PBS.
Staining Metiod The staining method referred to formerl 7 and again in this report as the standard procedure or standard incubation entailed suspending 1 to 2 million unfixed pelletized
Vol. 84, No. 3 September 1976
IMMUNE COMPLEX RECEPTORS
439
cells in 1.0 ml of freshly prepared HRP-anti-HRP soluble complex for 30 minutes at 4 C. The cells were then rinsed, incubated for 15 minutes in 3,3'-diaminobenzidine + H202 (DAB) substrate," rinsed, fixed 60 minutes at 4 C in 2% buffered osmium tetroxide, rinsed, dehydrated, and embedded in Epon 812. Heterologous binding of immune complex was evaluated by staining human buffy coat leukocytes with the standard soluble immune complex procedure described above employing rabbit antibody. As a control against nonspecific protein adsorption, red blood cells from the donor rabbit were stained by the standard soluble immune complex procedure. Controls for immunostaining included incubating the cells in the first step with HRP alone or with HRP plus serum from the donor of the peritoneal wash cells or another rabbit. The above standard incubation with soluble complex was varied to assess the biologic characteristics of the immune complex receptor. The receptor affinity for antibody alone was tested by a sequential incubation with anti-HRP followed by HRP at 4 C in place of immune complex as the first step of the procedure. For this purpose, cells were incubated with 1 part anti-HRP to 100 parts PBS, rinsed in PBS, incubated with 1 mg/100 ml HRP, rinsed, incubated in DAB substrate medium, rinsed, and postosmicated. After a final rinse with distilled water, the cells were dehydrated and embedded. In addition, the requirement for complement was evaluated by standard incubation with soluble immune complex prepared from antiserum which either had been supplemented with guinea pig complement or had been decomplemented by heating at 56 C for 30 minutes.
Fn-oytoSis O Immu Comles NW Peitmocytic Receptors Endocytosis of immune complexes and postendocytic receptors was investigated by warming the cells to 37 C either during the initial 30 minutes incubation with immune complex or for 10 or 30 minutes or 8 hours after the usual triple rinse which followed the initial incubation with immune complex at 4 C and preceded exposure to DAB. These temperature increases were carried out before, rather than after, incubation with DAB to assess the binding and uptake of immune complex as separate from the predictable phagocytic response to clumps of DAB reaction product. To test for receptor activity following endocytosis of immune complexes, cells were warmed for 30 minutes after the triple rinse which followed the initial exposure to immune complexes in the cold. A 30-minute warming interval was selected to allow nearly complete internalization of the initial complex. The cells were then reincubated with fresh soluble immune complex at 4 C for 30 minutes, triple rinsed, exposed to DAB, fixed with osmium tetroxide, and processed for electron microscopy. Cells incubated as described except for deletion of specific antiserum from the 30-minute reincubation served as controls.
_UDrasbuctw For ultrastructural examination, thin sections from blocks having satisfactorv morphologv in toluidine blue-stained thick sections were viewed with or without prior uranvl acetate and lead citrate staining in an Hitachi 12-A with a goniometer stage and Hitaci HS-8 electron microscope. To assess the binding of immune complexes compared with immunoglobulin alone and the effects of elevated temperature on bound complexes, randomlv selected cells from triplicate runs of these various incubations were photographed at low magnification. Ratios of stained to total plasma membrane for 25 cells per group were tallied blindly. Means of these ratios were tabulated and compared by unpaired method of proportions, and probabilities of significant differences expressed as P.'s Means were compared with the standard incubation (Tables 1 and 2).
440
MCKEEVER ET AL
American Joumal of Pathology
Table 1-Macrophage Binding of Immune Complex Compared With Immunoglobulin Alone and With Decomplted Immune Complex P eof m surface stained Probability of difference Method (mean ± SD) from standard incubation Standard procedure with cold soluble immune complex* 87.6 ± 13.5 Sequential incubation of cells with anti-HRP followed by HRP 45.0 ± 9.1 P < 0.05 Decompkemented soluble complex 86.5 ± 13.7 No significant difference * See Materials and Methods section.
Qaitation of Immue Comple Receptors Quantitation of immune complex receptors on the macrophage surface was undertaken
bv computing the ratio of the whole cell's plasma membrane surface area to its surface area within Epon thin section. As derived and more fully described in the Discussion, this ratio is 2 r/h, where r is the radius of the cell and h is the heighth of the cylindrical portion of cell in thin section (i.e., the 40-mu thickness of the section). The reported macrophage radius of 7.5 was used in these calculations and confirmed by light microscopy of whole peritoneal macrophages.ll.U2 The number of stained immune complexes on the thin section profile of the cell surface for 15 randomly selected (except for exclusion of obviously tangential sections) macrophage profiles from four or more standard incubations was counted. The total number of receptors on the whole cell's surface was then calculated for each cellular profile by multiplying the number of receptors in the thin section by 2 r/h. Receptor numbers for these whole cells were then tabulated, and the mean ± standard error was calculated. The number of immune complex receptors on the other peritoneal exudate cell tvpes was estimated with the same procedure, assuming that the eosinophil, neutrophil, and lymphocvte approximate spheres of 6.5, 5.5, and 4 gu radii, respectively.U2 Tabulated numbers of stained receptors on different cell types were compared by t test, and P < 0.05 was considered to represent significant difference."3 Table 2-Internalization of Bound Immune Complexes at Elevated Temperature Percenta of macrophag surface stained Probability of difference Method (mean ± SD) from standard incubation Standard procedure with cold soluble 87.6 ± 13.5 immune complex Modification of standard procedure Elevafion of temperature to 37 C during incubation with immune 12.5 ± 6.6 P < 0.001 complex Elevation of ternperature to 37 C for 34.8 ± 7.5 P < 0.05 10 minutes before DAB Elevation of temperature to 37 C for 1.5 ± 8 P < 0.001 30 minutes before DAB Elevation of temperature to 37 C for 30 minutes,recooling and reincubation with cold soluble immune 83.9 ± 14.9 No significant difference complex before DAB * As described in Materials and Methods.
Vol. 84, No. 3 September 1976
IMMUNE COMPLEX RECEPTORS
441
Results Staining of Immune Complex Receptors and Immunoglobulin Receptors on Macrophages
Macrophages stained prior to fixation with soluble immune complex at 4 C. according to the standard procedure, disclosed reaction product indicative of immune complex receptors in periodic foci over the entire cell surface (Figure 1). These foci wvere separated by 30- to 120-mg intervals and covered about 85%c of the cell surface (Table 1). Controls incubated with HRP alone or a mixture of HRP and normal rabbit serum, including that from an exudate donor, disclosed no surface staining on leukocytes. A conspicuous feature of the macrophage surface consisted of round cytoplasmic exvaginations \vith a diameter of 0.2 to 1.5 aand a uniformlytranslucent center. The evaginations protruded from both control and stained macrophages processed at 4 C (Figures 2 and 3) and occurred less frequently in those processed at 37 C. These generally circular profiles were composed of tw o membranes -hich were 20 to 35 mp apart and connected by periodic thin bridges. Continuity between the intermembranous space and the macrophage cytoplasm Xwas evident in appropriately sectioned evaginations (Figure 3). Both internal and external membranes stained for soluble immune complex, indicating that both sides of the evaginations communicated with or had recently communicated with the extracellular space. Attempts to isolate these structures by multiple velocity centrifugations of supernatant over the range of 1.000 to 25.OOOg proved unsuccessful. Similar evaginations were not present on the few eosinophils. neutrophils. or lymphocytes encountered. Varying the procedure for localizing immune complex by first incubating cells with uncomplexed antiserum to HRP, then rinsing and exposing to HRP, produced less surface staining than did exposure to soluble immune complex and similar rinsing (Table 1). Decomplementing the anti-HRP serum, or adding guinea pig complement to it. did not alter staining of macrophages (Table 1). Quantitation of Receptors on Various Cell Types
The number of immune complex receptor sites on the cell surface was estimated by counting the focal precipitates on the cell profile and extrapolating mathematically to the surface area of the \vhole cell. Fifteen oilinduced macrophages randomly selected from those incubated with soluble immune complexes at 4 C averaged 200,000 ± 30,000 (standard error) stained sites indicative of the immune complex receptor per cell. Small stained structures, possibly caveolae or tubular invaginations of the surface or pinocvtic vesicles, lay near the plasma membrane (Figures
442
MCKEEVER ET AL
American Journal of Pathology
4-6). Although these structures were present when all immunostaining steps had been performed at 4 C, they were too small and infrequent to alter significantly the staining periodicity. A comparable sample of macrophages obtained without induction of an exudate by mineral oil injection disclosed somewhat fewer receptors. Thus, the noninduced macrophages averaged 120,000 ± 10,000 receptor sites. The noninduced macrophages also contained fewer lysosomes and were slightly smaller than the induced cells. Multinucleate giant cells with diameters in the range of 18 to 50 y occurred in the oil-induced peritoneal exudate, but were not encountered among the noninduced cells. The giant cells consistentlv stained like macrophages, showing similar periodicity and receptors covering cvtoplasmic invaginations and evaginations as well as intervening plasmalemma (Figure 7). The round cytoplasmic evaginations with translucent centers (Figures 2 and 3) were less common on giant cells than macrophages. The number of receptor sites on the surface of the larger giant cells was estimated mathematically at 1.5 ± 0.6 million per cell. Eosinophil and neutrophil immune complex receptors stained with periodicity as did those of macrophages; however, unstained regions of membrane were more frequent, and the receptors were more weaklv stained compared with those of macrophages (Figures 8 and 9). The average number of receptor sites per cell was 28,000 ± 4,000 on neutrophils and 35,000 ± 6,000 on eosinophils. Receptors were very sparsely distributed on the majority of the lvmphocytes (Figure 10) and essentially absent from some lymphocyte profiles (Figure 11). Stained sites on lymphocytes having receptors numbered 3,500 ± 450 per cell and varied little according to the presence or absence of complement. When the numbers of stained receptors on all of the cell types previously described were compared by the unpaired t test, the differences in means were significant at P < 0.05 in all comparisons except the neutrophils and eosinophils. Light microscopic measurement of whole cells in suspension revealed radii of mean dimensions similar to those previously reported.l'"l The largest range in cellular radius was from 5 to 15 Ai in oil-induced macrophages. Electron micrographs of obviously tangential sections were excluded from the group counted. The mean radius from electron micrographs was an expected 10% lower than values obtained from measuring whole cells. Heterologous staining of human monocytes, granulocytes, and lymphocytes resembled the staining of the comparable rabbit cell lines in both morphology and relative intensity. Homologous (Figure 12) and
Vol. 84, No.3 September 1976
IMMUNE COMPLEX RECEPTORS
443
heterologous erythrocytes lacked surface staining after processing with soluble immune complex by the standard procedure. Endocytosis of hmnme Compees and Posteotic Receptors
The endocytic uptake of immune complex bound to the extemal plasmalemma was assessed by warming the cells to 37 C during incubation with immune complex or between incubation with immune complex and with DAB (Table 2). Incubation with immune complex at the elevated temperature largely prevented staining of receptors. Pinocytosis removed much to nearly all of the bound soluble immune complexes from the macrophages warmed 10 to 30 minutes prior to exposure to DAB (Figures 14 and 15). After either 37 C incubation with soluble immune complex or warming 10 minutes before the DAB step, stretches of stained surface alternated with 0.1- to 20-M lengths of unstained membrane, apparently as a result of active pinocytosis. The endocytosed soluble immune complex was sequestered in moderately large cytoplasmic dense bodies of obvious heterophagic nature. Notably, the soluble complex was also sequestered in tubulovesicular structures. Some macrophages incorporated immune complex mainly into the dense bodies (Figure 14) and others mainly into tubulovesicular structures (Figure 15). Peroxidase activity gradually disappeared within heterophagic dense bodies, but a small proportion remained within tubulovesicular structures after warming for 8 hours. Eosinophils and lymphocytes internalized surface-bound, soluble immune complex by pinocytosis as did macrophages. After 30 minutes, the majority of the complex in eosinophils was enclosed in large vacuoles presumed to be secondary lysosomes (Figure 13). These vacuoles were distinctly separate from the crystalloid granules which appeared not to contain endocytosed immune complex. Macrophages internalized more complex than eosinophils; lymphocytes endocytosed much less. Neutrophil morphology rapidly deteriorated when these unfixed cells were exposed to soluble complex at physiologic temperature. Macrophages exposed to immune complex, warmed for 30 minutes to remove complex from the surface by endocytosis, and immediately reincubated with cold, soluble immune complex disclosed surface staining similar to cells processed by the standard procedure (Figure 16 and Table 2). Control cells with specific antiserum deleted during reincubation disclosed almost no surface staining. With the method employed here, the major portion of the immune complex receptors on immune cells is visualized and quantified, and the internalization of immune complex bound to receptors is traced in vitro.
444
MCKEEVER ET AL
American Journal of Pathology
Determining the true periodicity of macrophage immune complex receptor sites is complicated by the endocytosis of the labeling moiety. Initial experiments 7 showed that decreasing the incubation temperature markedly increased the amount and uniformity of surface staining, presumably because of the corresponding decrease in pinocytic activity. As shown here, low temperature incubation did not entirely eliminate pinocytic internalization of immune complex, since stained profiles suggestive of pinocytic vesicles occasionally lay close beneath the plasmalemma. Nevertheless, the periodic stained foci spaced 30 my apart over macrophage surfaces are considered to reflect the actual distribution of immune complex receptors. The following observations support this interpretation. Those membrane sites least susceptible to pinocytosis, i.e., the thin cytoplasmic evaginations stained consistently in this way. Mechanically disrupted membranes of viable cells, presumably devoid of endocytic capacity, disclosed this finely periodic staining, as did occasional areas of cells fixed before incubation. The more patchy distribution of stained sites on cells warmed during or after incubation with immune complex observed in the present and other 6investigations probably resulted from endocytic uptake of bound complexes. This surface staining, which was encountered on all of the cell types after incubation at elevated temperature, suggests that complexes are internalized in patches rather than by a capping process. The observations on immune complex internalization during staining afforded no indication of migration of the receptors to a polar cap, as occurs prior to endocytosis of surface immunoglobulin and its specific antiimmunoglobulin on lymphocytes.14 The capacity of a soluble immune complex, presumably containing a single immunoglobulin molecule, to bind only monovalently to a single Fc receptor possibly explains the occurrence of patching rather than capping in this system. The cytoplasmic evaginations observed on macrophages resembled similar cytoplasmic evaginations termed loops on guinea pig exudate peritoneal macrophages.15 Membrane-to-membrane cross bridges reported to stabilize similar structures in other cell types 16 were evident also in cells examined here. The occasionally observed separation of these immune complex-rich structures from cells suggests that they may detach from macrophages and that these cells in migrating could leave behind a trail of immune complexes. The sequestering of immune complex on the inner surface of these structures might temporarily protect them from degradation. We have observed the same evaginations on macrophages at sites of injured human tissue. In the present study, the number of evaginations appeared increased by cold incubation, and they might result
Vol. 84, No. 3 September 1976
IMMUNE COMPLEX RECEPTORS
445
from an imbalance between accretion and depletion of plasmalemma in the cold. Recent studies have demonstrated the ability of soluble immune complexes to inhibit macrophage migration."7 The complexes thus restrain macrophages at an inflammatory site, acting like the migration inhibitorv factor of T lymphocytes. The extensive surface staining and pinocytotic activity observed in the present study provide a plausible explanation of this phenomenon. A surface coat of immune complex, or the undirectional pinocytosis of this complex, may directly impede cellular motility. The method of quantitating immune complex receptors is not standard and requires further explanation. A thin section of a cell contains a discrete portion of the whole cell's plasma membrane surface area within it. The whole cell's surface area is a specific multiple of the surface area within the thin section. If the cell is a sphere with radius r, its surface area within thin section is 2irrh, where h is the heighth of the nearlv cylindrical portion of cell in section. The whole surface area of this same cell is 47rr2. Thus, the multiple relating the whole cell's surface area to its surface area within thin section is 4xrr2/2wrh or, more simply, 2r/h. If the number of stained receptors counted on the cell's surface within thin section is multiplied by 2r/h, the number of stained receptors on the whole cell's surface can be calculated mathematically. The same analytic scheme may be applied to cells containing ruffled and invaginated membranes, providing they are spherical and their surface irregularities are uniformly distributed. Uniform distribution insures that receptors counted in thin sections will include the count variations produced by surface irregularities so that these variations will automatically be incorporated into the calculation of receptor numbers on the whole cell. Scanning electron microscopy (SEM) of macrophages stained with our standard procedure and similarly fixed as a pellet 16 and SEM of macrophages specifically stained with crystalline reaction product localizing immune complex receptors 19 have shown spherical cells with a uniform distribution of surface irregularities (pseudopods and veils), providing spreading on coverslips is avoided. Our experience has been similar with neutrophils and lymphocytes, except for the minor interference of uropods in the latter. The number of receptors on rabbit alveolar macrophages has been measured with radiolabeled soluble immune complexes.20 The number estimated by this method exceeds by fivefold that obtained in the present study by counting individual precipitates of reaction product in thin sections and extrapolating to the entire surface area. Like the peritoneal macrophage, the rabbit alveolar macrophage pinocytoses all of its soluble
446
MCKEEVER ET AL
American Jourma of Pathology
immune complex within 30 minutes at 37 C.8 Since the radiolabeled immune complex binding study was performed at 37 C for 30 minutes, pinocytosed and surface-bound radiolabeled immune complexes were probably counted and the number of receptors may have been overestimated. On the other hand, the number of receptors may have been underestimated in the present study either because of failure of some receptors to stain-for example, because of competitive binding with immunoglobulins other than anti-HRP in the antiserum-or because stained foci observed here represent more than one receptor. The regular periodicity of these foci at 30-ma intervals in many areas argues against the latter consideration. The counting procedure introduced in this study is susceptible to a 5 to 20% underestimation of surface counts from thin sections which missed the equatorial plane of the cells, although we avoided counting obviously tangential sections and, thereby, minimally altered the distribution of cell sizes counted. The several leukocyte lines varied widely in prevalence of immune complex receptors, decreasing in order from macrophage to granulocyte to lymphocyte. This order of receptor abundance correlated with the relative capacity of these cells to endocytose immune complexes. The receptor content also corresponds with the capacity of the different cell types to incorporate small particles such as colloidal gold spherules by microendocytosis.21'* Apparently, the latter ability to internalize such inert particles concerns a property or component of the cell membrane correlated in amount with the immune complex receptor. Fc receptors have been demonstrated on B lymphocytes by light and electron microscopic immunostaining and autoradiography.2>28 Receptors for aggregated immunoglobulin have been demonstrated on both B and T cells.25 26 In the present study, the majority of lymphocytes washed from the peritoneal cavity bound immune complex, but the cells varied in abundance of receptors possibly in relation to their identity as B or T lymphocytes. The prevalence of immunoglobulins visualized by immunostaining , on the lymphocyte surface apparently exceeds that of immune complex receptors demonstrated here. The abundance of immunostained histocompatability antigens on lymphocytes 1 also greatly exceeds that of immune complex receptors. The immune complex receptor thus appears to comprise a different component from either the bound immunoglobulin or the histocompatibility antigens on the lymphocyte surface. That the relative sparsity of foci on lymphocytes reactive with HRP-anti-HRP complex reflects incomplete staining of these receptors seems unlikely, since the more numerous receptors of the macrophage were clearly visualized over its entire surface.
Vol. 84, No. 3 September 1976
IMMUNE COMPLEX RECEPTORS
447
The existence on macrophages of separate receptors for immune complex and for immunoglobulin is suggested by the greater abundance on these cells of sites stained with immune complex than of sites stained with the sequence of anti-HRP and then HRP. Further experiments with double labeling techniques perhaps would clarify whether this recognized 2 difference can be attributed to weaker binding of immunoglobulin than of immune complex or to the existence of different receptors for immunoglobulin alone and for immune complex on the macrophage surface. The observed differences conceivably relate to the known variable binding affinities of leukocytes for subelasses of immunoglobulin n and to a possible preponderance of high affinity immunoglobulin types in the immune complex employed. The ratio of immune complex binding sites to bound immunoglobulin appears to be reversed for macrophages compared with lymphocytes, in that macrophages show abundant immune complex receptors with the procedure described here, and lymphocytes show abundant membranebound immunoglobulins when stained with labeled antibody to immunoglobulin. A question remains whether the latter lymphocyte surface immunoglobulin, which presumably originates endogenously within the lymphocyte, is bound by the same mechanism as exogenous immunoglobulin demonstrated ultrastructurally with radiolabeled 2'*- or ferritinlabeled immunoglobulin, and whether the term Fc receptor applies to the binding site on lymphocytes for endogenous or exogenous immunoglobulin or both. Beyond this question, the marked difference between binding immunoglobulin compared with that of immune complex on both macrophages and lymphocytes would favor designating the immune complex and the immunoglobulin binding sites as different entities. Notably, immune complex receptors were not demonstrable on cell types other than immune cells and specifically were absent from ervthrocvtes.
Macrophages were found to pinocytose almost completely a surface complement of immune complex within 30 minutes at 37 C. Some pinocytosed soluble immune complexes were sequestered within tubulovesicular structures which appeared morphologically distinct from the large phagocytic vacuoles that sequester and digest insoluble complex 6 and other substances. The tubulovesicular structures possibly comprise a separate endocytic system and could perhaps function in antigen processing to enhance the immune response. Some macrophages contained tubulovesicular structures predominantly and might differ from those with only large heterophagic vacuoles. The present findings also indicate full receptor capacity 30 minutes following apparent internalization of 90 to 99% of immune complex
448
MCKEEVER ET AL
American Journal of Pathology
bound to the majority of the macrophage plasma membrane. Other studies which perturbed immunoglobulin receptors have demonstrated receptor regeneration slo'wer than the phenomenon described here. For example, receptor activity recovers slowly over a 2-day culture interval after removal of an antimembrane antibody wvhich inhibits macrophage receptor activity." Phagocytosis of large particles uncoated wvith antibody inhibits by 50% the subsequent attachment or phagocytosis of antibodycoated ervthrocytes over a 2-hour interval." The present system emphasizes the capacity of the immune complex receptor to relatively rapidly accommodate more immune complex when perturbed specifically and solely by a thin surface-coat of immune complex. It, therefore, differs from the aforementioned systems which demonstrated the more prolonged effects of antimembrane antibodies and a phagocvtic load. Possible explanations for this surprisingly rapid accommodation of a second surface-coat of complex include pinocytosis of complex alone, leaving receptors behind in the plasma membrane; initial staining or internalization of only a small portion of receptors; and rapid regeneration of pinocvtosed receptors. These possibilities may be susceptible to further study with techniques of tracing external plasma membrane components during endocvtosis. Rabbit giant cells possessed more receptor sites for homologous immune complex than any other cell tested. Their staining differs remarkably from murine giant cells studied at 37 C which demonstrated few receptor sites for heterologous ferritin-labeled IgG.5 The different binding to giant cells of antigen-antibody complexes compared with ferritin-labeled antibody may contribute to this wide diversity. Different pinocytotic rates between 4 C and 37 C could have also contributed. References 1. Arend \\NP. Niannik N1 In vitro adherence of soluble immune complexes to macrophages. J Exp Med 136:514-531. 1972 2. Berken A. Benacerraf B: Properties of antibodies cytophilic for macrophages. J Exp Med 12:3:119-144. 1966 :3. Box-den SV: Cytophilic antibody in guinea pigs w%ith delayed-type hypersensitivity. Immunology 7:474-483. 1964 4. Lies FYN The binding of the F, fragment of guinea pig cytophilic antibody to peritoneal macrophages. Immunology 20:S17-829. 1971 5. Papadimitriou J\: Detection of macrophage receptors for heterologous IgG by scanning and transmission electron microscopy- J Pathol 110:213-220. 197:3 6. Steinman RNM. Cohn ZA: The interaction of particulate horseradish peroxidase (HRP)-anti-HRP immune complexes wvith mouse peritoneal macrophages in vitro. J Cell Biol .55:616 -&34. 1972
Vol. 84, No. 3 September 1976
IMMUNE COMPLEX RECEPTORS
449
7. McKeever PE, Garvin AJ, Spicer SS: Immune complex receptors on cell surfaces. I. Ultrastructural demonstration. J Histochem Cvtochem 24 (In press) 8. McKeever PE, Garvin AJ: Ultrastructural localization of immune complexes binding to living and fixed macrophages. Am J Pathol 78:55a, 1975 (Abstr) 9. Mason TE, Phifer RF, Spicer SS, Swallow RA, Dreskin RB: An immunoglobulinenzvme bridge method for localizing tissue antigens. J Histochem Cytochem 17:563-4569, 1969 10. Graham RC Jr, Karnovsky MJ: The early stages of absorption of injected horseradish peroxidase in the proximal tubules of mouse kidnev: Ultrastructural cvtochemistry by a new technique. J Histochem Cytochem 14:291-302, 1966 11. Carr I: The Macrophage: A Review of Ultrastructure and Function. New York, Academic Press, Inc., 1973, pp 5-7 12. Rhodin JAG: Histology: A Text and Atlas. New York, Oxford University Press, 1974, pp 98, 100, 106, 156, 388 13. Annitage P: Statistical NMethods in NMedical Research. New York, John Wiley and Sons, 1973, pp 104-114 14. de Petris S, Raff MC: Normal distribution, patching and capping of lymphocyte surface immunoglobulin studied by electron microscopy. Nature [New Biol] 241:257-259, 1973
15. Daems WT, Brederoo P: Electron microscopical studies on the structure, phagocytic properties and peroxidatic activity of resident and exudate peritoneal macrophages in the guinea pig. Z Zellforsh Mikrosk Anat 144:247-297, 1973 16. Franke WW, Kartenbeck J, Zentgraf H, Scheer U, Falk H: Membrane-to-membrane cross-bridges: A means to orientation and interaction of membrane faces. J Cell Biol 51:881-888, 1971 17. Kotkes P, Pick E: Studies on the inhibition of macrophage migration induced by soluble antigen-antibody complexes. Clin Exp Immunol 19:105-120, 1975 18. McKeever PE, Garvin AJ, Spicer SS: Unpublished observations 19. McKeever PE, Garvin AJ, Brissie NT, Spicer SS: Crystalline markers for scanning electron microscopy: Visualization of immune complex receptors on macrophage surfaces. J Reticuloendothel Soc 18:30b, 1975 (Abstr) 20. Arend WP, Mannik M: The macrophage receptor for IgG: Number and affinity of binding sites. J Immunol 110:1455-1463, 1973 21. Komivama A, Spicer SS, Bank H, Farrington J: Induction of autophagic vacuoles in peritoneal cells. J Reticuloendothel Soc 17:146-161, 1975 22. Komiyama A, Spicer SS: Microendocytosis in eosinophil leukocvtes. J Cell Biol 64:622-635, 1975 23. Basten A, Miller JFAP, Sprent J, Pye J: A receptor for antibody on B lymphocytes. I. Method of detection and functional significance. J Exp Med 135:610-626, 1972 24. Basten A, Warner NL, Mandel T: A receptor for antibody on B lymphocytes. II. Immunochemical and electron microscopy characteristics. J Exp Med 135:627-642, 1972 25. Van Boxel JA, Rosenstreich DL: Binding of aggregated Y -globulin to activated T lymphocytes in the guinea pig. J Exp Med 139:1002-1012, 1974 26. Anderson CL, Grey HM: Receptors for aggregated IgG on mouse lymphocytes: Their presence on thymocvtes, thvmus-derived, and bone marrow-derived lymphocytes. J Exp Med 139:1175-1188, 1974 27. Dickler HB: Studies of the human lymphocyte receptor for heat-aggregated or antigen-complexed immunoglobulin. J Exp Med 140:508-522, 1974 28. Theofilopoulos AN, Wilson CB, Bokisch VA, Dixon FJ: Binding of soluble immune complexes to human lymphoblastoid cells. J Exp Med 140:1230-1244, 1974 29. Bretton R, Ternvnck T, Avrameas S: Comparison of peroxidase and ferritin labelling of cell surface antigens. Exp Cell Res 71:145-155, 1972
450
MCKEEVER ET AL
American Joumal of Pathology
30. Reves F, Lejonc JL, Gourdin MF, Mannoni P, Drevfus B: The surface morpholog'y of human B lymphocytes as revealed by immunoelectron microscopy. j Exp Med 141:392-410, 1975
31. Willingham MC, Spicer SS, Graber CD: Immunocytologic labeling of calf and human lymphocyte surface antigens. Lab Invest 25:211-219, 1971 32. Lawrence DA, Weigle WO, Spiegelberg HL: Immunoglobulins cytophilic for human lymhpocytes, monocytes and neutrophils. J Clin Invest 55:368-376, 1975 33. Holland P, Holland NH, Cohn ZA: The selective inhibition of macrophage phagocytic receptors by anti-membrane antibodies. J Exp Med 135:458-475, 1972 34. Schmidt ME, Douglas SD: Disappearance and recovery of human monocyte IgG receptor activity after phagocvtosis. J Immunol 109:914-917, 1972 35. De Pierre J, Kamovskv ML: Ectoenzvmes, sialic acid, and the internalization of cell membrane during phagocytosis. Inflammation: Mechanisms and Control. Edited bv IH Lepow, PA Ward. New York, Academic Press, Inc., 1972, pp 55-70 36. Hubbard AL, Cohn ZA: Externally disposed plasma membrane proteins. II. Metabolic fate of iodinated polypeptides of mouse L cells. J Cell Biol 64:461-479, 1975 The authors would like to express their appreciation for the generous advice and support of Drs. G. R. Hennigar, J. D. Balentine, and J. M. Powers; the skilled technical assistance of Nancy Brissie and Betty Hall; the dependable photographic assistance of Jim Nicholson and his staff; and the efficient secretarial assistance of Karen Beaufort.
IPRJM06,17. t
F.
e 'S
de
r.
{
_#_,, s
! :.
u., *
; r
db
v@s=. .%
t 4,
+
.:
:
Fgu 1-Macrophages from peritoneal exudate exhibit deposits of reaction product indicative of immune complex receptors in periodic foci over the surface. Stained with soluble immune complex in cold by the standard procedure. (x 9000)
Figues 2 and 3-Like Figure 1 except thin sections were stained with uranyl acetate and lead citrate. Internal and external membrane surfaces of macrophage evaginations are separated by a constant 30-mM distance and are both heavily stained for immune complex receptors. Continuity with macrophage cytoplasm (arrows) and cross bridges between membranes are evident. (x 45,000)
Figures 4-6-Surface area in cells treated like that in Figure 1. Stained profiles suggestive of pinocytic vesicles (short arrows) lie near the plasmalemma often dosely associated with a surface invagination (long arrow). Unstained vesicles are in the proximity (arrowhead). (4 md 5, x 32,000; 6, x 18,200)
Figure 7-Giant cell treated like cell in Figure 1. Its entire surface stains uniformly for immune complex. (x 5250)
3
2
4
5
6
7
f
'r
.
...4..40411
-11
-
--A lml..
.
-.-
41,
".
--4 .,. --&
a
.
1
%Ait
.a
I
n,
140 -4
-,
.:.
t;
'. 11
8
I
I7
:% ,l
a
N,_:-Iw- .
!-d.W
J
10
.4-
JO
40 :6.
". .k
Is 4tL
-
Feum 8-11-Staining for immune complex receptor on other cell types occasionally encountered in the rabbit peritoneal exudate. In the eosinophil (E) (8 and i), the plasmalemma contains relatively sparse foci of reaction product indicative of receptor staining widely distributed along its surface. Intrinsic peroxidase activity accounts for the density of the eosinophil crystalloid granules. The neutrophil (N) (9 and itset) exhibits light similar surface staining and faint staining indicative of intrinsic peroxidase in cytoplasmic primary granules. The lymphocyte (L) (10) contains very sparse reaction product in an uneven distribution along its surface, but the lymphocyte (L) (11) almost totally lacks surface staining. The characteristically abundant reactive foci on neighboring macrophages contrast with the sparsity of such staining on the other leukocytes. Soluble immune complex standard procedure. (Unstained thin sections, 8, x 7600; d%, x 17,400; 9, x 9800; kiset, x 14,000; 10, x 8800; 11, X 9800)
9
; 12
13 13
12
s-
14
t 'A
Figure 12-The red blood cell (R) lacks surface reaction product in contrast to the stained neighboring macrophages. Soluble immune complex standard procedure applied to a mixture of Figure 13-This eosinophil was peritoneal wash cells and homologous erythrocytes. (x 7200) processed as in the standard procedure except for warming to 37 C for 30 minutes between exposure to immune complex and incubation with DAB. Surface staining has been eliminated by pinocytosis. Most of the internalized immune complex is sequestered in secondary lysosomes (arrow), but a trace remains in pinocytotic vesicles. Crystalloid granules appearing uniformly dense from content of intrinsic peroxidase, afford no evidence of alteration through having incorporated immune complex and appear not to participate in microendocytosis. (x Figures 14 and 15-Cells treated like that in Figure 13. Surface staining has been 11,800) eliminated by pinocytosis except at the tips of occasional macrophage processes (arrowheads). The cells display two sites of antigen sequestration: one in relatively large dense bodies (arrows, 14), and the other in small tubulovesicular structures (arrows, 15). These macrophages differ in containing mainly one or the other type of endosome. (Unstained thin sections, x 6700)
15
i, . i!i ,=F
m\t4!
M,. .
.I.
Figure 16-These macrophages were processed as in the standard procedure except for warming to 37 C for 30 minutes and then reincubating with cold soluble immune complex before exposure to DAB. Endocytosed antigen (arrowheads) is sequestered as in Figures 14 and 15. The surface staining (arrow) comparable to that in Figure 1 indicates the macrophages have regained the receptor capability, following endocytosis of the initially applied immune complex. (Unstained thin section; x 10.500)
