EXPERIMENTAL
AND
hIOLECULAR
26, 85-91 (1977)
PATHOLOGY
lmmunocytochemical
Evidence for Antibody Cell Granules
Mast
J, A. V. SIMSON, D. S. HINIZ,
A. M. MUNSTER,
Binding
AND
S.
S.
to
SPICER
Departments of Anatomy, Pathology and Surgeq, Medical University of South Carolina, Colleges of Medicine and Dentistry, Charleston, South Carolina 29401, and Veterans Administration Hospital, Charleston, South Carolina 29401 Received
June 3, 1976,
and in revised
form
August
30, 1976
Immunocytochemical studies of lymphoid tissues of the rat indicate that immunoglobulins possess strong affinity for some component of mast cell granules. In appropriately fixed tissue, binding of antibody to mast cell granules was stronger and more consistent than binding to any other tissue component examined. Both heterologous and homologous antibody bound to mast cell granules. Several crude antiserum preparations as well as purified IgG fraction exhibited binding. Antibody was capable of binding to mast cell granules in thin sections of Epon-embedded material.
INTRODUCTION Mast cells are known to play a role in immunological responsiveness as effector cells mediating an inflammatory reaction via the release of vasoactive amines ( Mongar and Schild, 1962; Mota, 1963). Immunoglobulin E ( IgE ), the classical reagin, binds to receptors at the cell surface (Ishizaka et nl., 1966; Ishizaka and Ishizaka, 1971), and, when challenged with appropriate antigen, triggers the release of several mast cell granule components mediating immediate-type hypersensitivity or local anaphylaxis ( Mongar and Schild, 1962; Orr, 1973). There is evidence that immunoglobulins other than IgE also bind to mast cells, since immunoglobulin G ( IgG) fractions have been shown to elicit passive cutaneous anaphylaxis in mice, rats and humans (Vas, 1971; Bloch and Ohman, 1971; Parish, 1970; Stanworth and Smith, 1972). In this laboratory, we have been investigating cellular production and localization of immunoglobulins in human and rat tissues (Garvin et al., 1974) employing an immunoglobulin-enzyme bridge technique with horseradish peroxidase (HRP) as the enzyme marker (Mason et al., 1969). During these studies, staining was observed which indicated binding of immunoglobulins to mast cell granules (Simson and Hintz, 1975). We have investigated this immunoglobulin binding to mast cells in the present study with the aim of obtaining information about the site of binding, its lability to fixation, and the nature of the bound antibody. MATERIALS Tissue and cell processing. 200 and 250 gm were utilixrcl
Copyright All rights
0 1977 by Academic Press, of reproduction in any form
AND
Female
Inc. reserved.
for
these
METHODS
Sprague-Dawley experiments.
rats weighing between Lymphoid tissue excised
ISSN
0014-4800
86
SIMSON ET AL.
from pentobarbital-anesthetized animals was immediately placed in one of the following fixatives: (a) calcium-acetate buffered formalin (CAF); (b) cacodylate buffered 3% glutaraldehyde; (c) Carnoy’s alcohol-acetic acid, or (d) CAF with 0.5% glutaraldehyde. Only the latter fixative without postosmication was utilized for electron microscopy. Free, unstimulated cells from the rat peritoneal cavity were harvested by a method described previously (Simson et al., 1973) and fixed with the same fixatives. After selected fixation times, tissues or cells were processed either for light or electron microscopy by routine methods. Light microscopy studies were performed on tissues fixed for times ranging from 30 min to 24 hr; fixation time for electron microscopy was 4 hr. Tissue or cells were embedded in paraffin for light microscopy and in Epon 812 for electron microscopy. Sections for light microscopy were cut at about 3 ,J and mounted on glass slides. Thin sections for electron microscopy were between 500 A and 800 A (based upon interference colors), and were picked up on stainless steel grids. lmmunoglobulin bridge technique. Sections were reacted with the immunoglobulin-enzyme bridge sequence. This technique, originally described by Mason et al., (1969) consisted of the following reaction sequence: (a) rabbit antiserum to tissue antigen, (b) goat antiserum to rabbit immunoglobulin (G anti-R Ig), (c) rabbit antiserum to horseradish peroxidase (R anti-HRP), (d) horseradish peroxidase (HRP). The latter enzyme, bound to the tissue by the immunoglobulin bridge, was demonstrated utilizing diaminobenzidine (DAB) and hydrogen peroxide ( HaOz) (Graham and Karnovsky, (1966). The oxidized, precipitated DAB was further “blackened” by treatment with osmium tetroxide, exposing the sections 5 set for light microscopy, or 10 min for electron microscopy. Thorough rinsing with phosphate-buffered saline between each step was essential. Controls consisted of normal rabbit serum (NRS) substituted for specific antiserum in the bridge (control for “nonspecific” binding) or DAB/H202 alone (control for endogenous peroxidase). In addition to the complete bridge, described above, several modifications and abbreviations of the bridge have been used in the present study as indicated in the text and table. After initial evaluation of mast cell binding of antiserum with modifications of the bridge sequence, a “truncated” bridge, consisting of [anti-HRP]-[HRP]-[DAB/H202] was used for most of the remaining studies. Sections were viewed without counterstaining. Antisera. The antisera to HRP were made in this laboratory by giving an initial antigen dose, mixed with complete Freund’s adjuvant, subcutaneously, followed in 1 month and again 2 weeks later by boosters in incomplete Freund’s adjuvant. Other injection routes were also used in the rat, including intramuscular and intraperitoneal injections. Animals were bled at several intervals after the final injection, and sera were separated following room temperature coagulation. Most antisera were not further purified. Antisera raised in rabbits, rats, guinea pigs, and goats were tested, although the majority of the studies utilized rabbit antiHRP. A purified IgG fraction of anti-HRP antiserum was obtained from Cappel Laboratories ( Downingtown, Pennsylvania). RESULTS AND DISCUSSION The binding of horseradish peroxidase to mast cell granules was observed when serum from normal or immunized rabbits was applied to the tissue as the first step of the complete immunoglobulin-peroxidase bridge. Sequential elimination
MAST
CELL
GRANULE
BINDING
OF ANTIBODY
87
of various steps of the bridge (Table I) disclosed that mast cells were stained by the sequence: [anti-HRP]-[HRP]-[DAB/HZOZ] (Fig. 1); but not by [HRP][DAB/H,O,] or [DAB/H,O,] a1one (Fig. 2). These results demonstrated that antibody was necessary for staining, and excluded the possibility that either cndogenous peroxidase or nonspecific adsorption of HRP were responsible for the reaction product. If NRS was interposed between [anti-HRP] and [HRP], the final peroxidase activity was reduced considerably, suggesting displacement of anti-HRP by immunoglobulins with other specificities. The localization of reactivity to mast cells was confirmed by staining adjacent 3 p serial sections, one with the [anti-HRP]-[HRP]-[DAB/HZOZ] sequence and the next with a reference toluidine blue stain for mast cell metachromasia (Figs. 3 and 4). The observable binding was restricted to mast cells. The staining filled the cytoplasm and generally appeared to be localized to mast cell granules rather than the cell periphery. No other cells stained in the lymphoid tissue, peritoneal fluid, and numerous other tissues examined except those cells which exhibited endogenous peroxidase in control sections as well. Conditions under which anti-HRP was capable of binding to rat mast cells were investigated by either varying tissue fixations or heating or diluting the antiserum (Table I). A short fixation time with aldehydes (2 hr or less) was essential for preservation of mast cell affinity for immunoglobulin, whereas binding was observed after 24 hr fixation with Carnoy’s fixative. Heating the antiserum to 60” for 30 min or 4 hr did not appreciably reduce binding affinity. This suggested that the binding was more likely attributable to IgG than to IgE, inasmuch as the latter is heat labile. Dilution of the anti-HRP serum with NRS resulted in rapid diminution of specific binding. A 1:25 dilution of our highest titre anti-HRP antiserum with NRS resulted in complete loss of demonstrable binding. On the other hand, dilution of the same anti-HRP serum with phosphate-buffered saline (PBS) resulted in diminished binding only at a 1:lOO dilution and loss of binding at a 1:400 dilution. This suggested competition for binding sites by immunoglobulins not reactive for HRP which were present in NRS but not in PBS. A purified IgG fraction of anti-HRP bomid as well as did the unpurified antisera prepared in this laboratory, suggesting that the immunoglobulin which binds to the granules may be an IgG subfraction rather than IgE. Although the results described apply for the most part to antisera produced in rabbits, anti-HRP raised in rats with intraperitoneal immunization also yielded good binding, indicating that this cytochemically demonstrable binding of immunoglobulin to mast cell granules was not simply a heterocytotropic phenomenon. To determine whether immunoglobulin was bound to the granule membrane or to the granule matrix, thin epoxy sections of tissue which had been fixed with the formaldehyde-glutaraldehyde mixture without postosmication, were reacted seq uence for ultrastructural examinawith the [anti-HRP]-[HRP]-[DAB/H30L’] tion. This procedure imparted electron density selectively to the mast cell granule matrix whether the antiserum utilized was purified IgG anti-HRP (Fig. 5) or the routine anti-HRP antiserum. The density in the small condensed granules exceeded that in the larger cytoplasmic granules in which reaction product was distributed in a coiled thread pattern. All of the controls utilized at the light
SIMSON
88
TABLE Mast Cell Binding
Methodological
ET AL. I
of Anti-HRP: the Effect of Immunocytochemical Tissue Fixation, Heating, or Antiserum Dilutiona
variant
Bridge Sequence,
Immunoglobulin binding (reaction product)b
Bridge Sequence” [NItSI-[G
anti-It Ig]-[R [G anti-R Ig>[R
anti-HRP]-[HRP+[DAB/H&l anti-HRP]-[HRP]-[DAB/H2021 [anti-IIRP>[HRP]-[DAB/H&21 [NItSI-[HRP~[DAB/HzOz] [HRP-J[DAB/HzOJ [DAB/HzOzl [anti-HRP]-[NRS)-[HRP]-[DAB/HzOJ [anti-HRP]-[HRP>[NRS]-[DAB/HzO?] [NRS]-[anti-HRP]-[HRP]-[DAB/HzOJ
2 4+ 2 4+ 2 4+ 0 0 0 0 -1+ 1 -3+ 2 -4+
Fixation Calcium-acetate buffered formalin (CAF) 30 min, 1 hr, 2 hr 24 hr 3’s Glutaraldehyde (cacodylate buffered) 1 hr 2 hr 24 hr CSF + 0.5y0 glutaraldehyde 1 hr Carnoy’s alrohol-acetic acid 1 hr 24 hr Antiserum
2 4+ 0 -2fd 1 -3f 0 -1+ 0 2 -4+ 3 4+ 3 4t
heating
60” for 30 min or 4 hr Antiserum
dilutions
With With With With With With
NRS-1:s NRS-1: PBS-1 PBS--l PBS”-1 PBSe-1
25 :5 : 25 :200 :400
2 -3f
1 -a+ 0 3 -4+ 2 -3+ 0 -2+ 0
a The tissue utilized in all of these studies was rat lymph node. The data represent light microscopic observations. b The intensity of the final DAB reaction product was graded on a lf to 4+ basis and assessed by the color developed as follows: straw to tan = l+; tan to light brown = 2f; dark brown = 3+; black = 4f. In all procedures except those in which the bridge sequence was varied, figures represent the intensity of reaction product with the [anti-HRP>[HRP]-[DAB/HzOz] sequence blackened in 2y0 0~0, for 5 sec. In all procedures, appropriate controls were performed. c All of these variants were performed on &sue fixed with 49;1 formaldehyde, 0.5% glutaraldehyde in calcium-acetate buffer, pH 7.0 for 1 hr. Certain of the variants were also performed on tissue fixed with the above fixative (CAF) without glutaraldehyde, with identical results. d The mast, cells in t.he siromn snrrolmdillg I he lymph node rr~t:~irlocl some bindinK cnpacaity, lmt t.hose in the body of the nc~le did not, e Phosphate buffered saline,
MAST
CELL
GRANULE
BINDING
OF ANTIBODY
FIG. 1. Light micrograph of rat peritoneal cells to which the sequence [anti-HRP]-[HRP][DAB/Hz02] was applied. The two large cells (XI) are mast cells showing reactivity; the small cells with reaction product are monocytes or eosinophils which possess endogenous peroxidase (see Fig. 2). X450. FIG. 2. Light micrograph of same preparation as Fig. 1, reacted with DAB/H202 alone, to demonstrate endogenous peroxidase. Some small cells (monocytes and eosinophils) are reactive; however, mast cells show no reactivity. The clear “holes” (X) probably represent unstained mast cells. Faintly refractile granules were seen microscopically in the lower clear area, but they are not clearly visible in the photograph. x450. FIG. 3. Light micrograph of the capsule of a lymph node. The section was reacted with [anti-HRP]-[HRP]-[DAB/H?OJ. Erythrocytes in the blood vessel (BV) show some endogenous peroxidatic activity. The two mast cells possessing reaction product (arrows) correspond to two Toluidine-blue metachromatic cells in Fig. 4. X450. FIG. 4. Light micrograph of a section serial to that in Fig. 3 stained with Toluidine blue. A green filter was used to enhance the density of the highly metachromatic mast cells (arrows). Erythrocytes in the blood vessel (BV) are unstained but refractile. X450. FIG. 5. An electron micrograph of mast cell granules illustrating reaction product over the granule matrix. This section was treated with a purified IgC fraction of anti-HRP followed by [HRPI-[DAB/ROJ X28,000. FIG. 6. An electron micrograph illustrating mast cell granules following a control procedure (DAB/H,O?). x24,000.
level were performed on thin sections (Fig. 6); none gave evidence either of binding of HRP alone to granules or of intrinsic perosidase activity. The significance of immunoglobulin binding by mast cell granules remains to 1~ elucidated. Such binding could partly account for the several hour delay that is required between administration of antibody and the capacity of an animal
90
SIMSON
ET
AL.
to manifest passive cutaneous anaphylaxis. The uptake of exogenously administered material into mast cell granules has been amply demonstrated by Padawer ( 1968, 1971), who h as suggested that mast cell granules may concentrate antibodies in viva (1974). Granule-bound antibody could react with specific antigen either before or after granule discharge from mast cells, contributing to the release of inflammatory mediators, such as histamine, from the granules. Antigen (ferritin) binding to discharged mast cell granules has been demonstrated ( Movat et al., 1966). Heparin, a normal mast cell granule constituent, stimulates endocytosis (Cohn and Parks, 1967), and appears important for the activity of some opsonic proteins (Allen et al., 1973). Discharged mast cell granules are avidly taken up by nearby macrophages (Riley, 1963; Padawer, 1970). In addition, heparin has an inhibitory effect on some hydrolytic enzymes (Brittinger et al., 1968; de Chatelet et al., 1972) which might be expected to degrade phagocytosed antigen. Thus, mast cell granule-bound antigen-antibody complexes could be ideal immunogens. ACKNOWLEDGMENTS We appreciate the helpful discussions and wish to thank Dr. H. H. Fudenberg This work was supported by NIH grants appropriation 22700-A401, ‘73-‘74.
of Dr. A. J. Garvin during for reviewing the manuscript AM-10956 and AM-11028
the course of this study, prior to its submission. and South Carolina State
REFERENCES ALLEN, C., SABA, T. M., and MOLNAR, J. (1973). Isolation, purification, and characterization of opsonic protein. J. Reticuloendothel. Sot. 13, 410-423. BLOCH, K. J., and OHMAN, J. L. ( 1971). The stable homocytotropic antibodies of guinea pig, mouse and rat, and some indirect evidence for the in vioo interaction of homocytotropic antibodies of two different rat immunoglobulin classes at a common receptor on target cells. In “Biochemistry of the Acute Allergic Reactions,” K. F. Austen and E. L. Becker, eds. p. 45-64, Blackwell, Oxford. BRITTINGER, G., HIRSCHHORN, R., DOUGLAS, S. D., and WEISSMANN, G. (1968). Studies on lysosomes. XI. Characterization of a hydrolase-rich fraction from human lymphocytes. j. Cell Biol. 37, 394411. COHN, Z. A., and PARKS, E. ( 1967). The regulation of pinocytosis in mouse macrophages. II. Factors inducing vesicle formation. .I. Exp. Med. 125, 213-230. DECHATELET, L. R., MCCALL, C. E., COOPER, M. R., and SHIRLEY, P. (1972). Inhibition of leukocyte acid phosphatase by heparin. Clin. Chem. 18, 1532-1534. GARVIN, A. J., SPICER, S. S., PAR~ILEY, R. T., and MUNSTER, A. M. (1974). Immunohistochemical demonstration of IgG in Reed-Stemberg and other cells in Hodgkin’s disease. j. Exp. Med. 139, 1077-1083. GRAHAX, R. C., and KARNOVSKY, M. J. ( 1966). The early stages of absorption of injected horseradish peroxidase in the proximal tubules of mouse kidney. Ultrastructural cytochemistry by a new technique. J. Histochem. Cytochem. 14, 291-302. ISHIZAKA, K., and ISHIZAICA, T. (1971). IgE and reaginic hypersensitivity. Ann, N.Y. Acad. Sci. 190, 443-456. ISHIZAKA, K., ISHIZAIU, T., and HORNBROOK, M. M. ( 1966). Physicochemical properties of human reaginic antibody. IV. Presence of a unique immunoglobulin as a carrier of reaginic activity. J. Immunol. 97, 75-85. MASON, T. E., PHIFER, R. F., SPICER, S. S., SWALLOW, R. A., and DHESKIN, R. B. (1969). An immunoglobulin-enzyme bridge method for localizing tissue antigens, j. Histochem. Cytochem. 17, 563-569. hIoxGAH, J. R., and SCIIILD, H. 0. ( 1962). Cellular mechanisms in anaphylaxis. Physiol. RN. 42, 226-270.
MAST
CELL
GRANULE
BINDING
OF
ANTIBODY
01
hlw~, I. (1963). Mast cells and anaphylaxis. Ann. N.Y. Acad. Sk. 103, 264-277. Movar, H. Z., LOVETT, C. A., and TAICHMAN, N. S. (1966). Demonstration of antigrn ou the surface of sensitized rat mast cells. Nature 212, 851-853. Onn, T. S. C. ( 1973). Mast cells and allergic asthma. Brit. J. Uis. Chest 67, 87-IOG. PADAWER, J. ( 1968). Ingestion of colloidal gold by mast cells. l’roc. Sot. L'x~J. Bid. I\fcd. 129, 905-907. PADAWER, J. ( 1970). The reaction of rat mast cells to polylysine. J. Cell Biol. 47, 352-372. PADAWR, J. ( 1971). Phagocytosis of particulate substances by mast cells. Lab. Inwst. 25, 320-330. PA~AWER, J. (1974). The ins and outs of mast cell function. Am. J. Anat. 141, 299-302. PARISH, W. E. (1970). Short-term anaphylactic IgG antibodies in human sera. Lancct 2, 591-592. RILEY, J. F. ( 1963). Functional significance of histamine and heparin in tissue mast cells. Ann. N.Y. Acad. Sci. 103, 151-161. SIHSO~T, J. A. V., and HINTZ, D. S. (1975). Evidence for immunoglobulin binding to rat mast cell granules. Anat. Rec. 181, 480 (abstract). SIMSOX, J. A. V., SPICER, S. S., and WRIGHT, N. J. (1973). Unusual cellular aggregates from rat peritoneal cavity. J. Reticdoendothel. Sot. 14, 361-370. STANWOHTH, D. R., and SMITH, A. K. ( 1972). Human IgG subclass with primate skinbinding activity. Lancet 2, 491. VAZ, N. M., ( 1971). Anaphylactic sensitization of mouse tissues with IgGL and reaginic antibodies. In “Biochemistry of the Acute Allergic Reactions,” K. F. ,4usten and E. L. Becker, eds. p. 91-109, Blackwell, Oxford.
AND
hIOLECULAR
26, 85-91 (1977)
PATHOLOGY
lmmunocytochemical
Evidence for Antibody Cell Granules
Mast
J, A. V. SIMSON, D. S. HINIZ,
A. M. MUNSTER,
Binding
AND
S.
S.
to
SPICER
Departments of Anatomy, Pathology and Surgeq, Medical University of South Carolina, Colleges of Medicine and Dentistry, Charleston, South Carolina 29401, and Veterans Administration Hospital, Charleston, South Carolina 29401 Received
June 3, 1976,
and in revised
form
August
30, 1976
Immunocytochemical studies of lymphoid tissues of the rat indicate that immunoglobulins possess strong affinity for some component of mast cell granules. In appropriately fixed tissue, binding of antibody to mast cell granules was stronger and more consistent than binding to any other tissue component examined. Both heterologous and homologous antibody bound to mast cell granules. Several crude antiserum preparations as well as purified IgG fraction exhibited binding. Antibody was capable of binding to mast cell granules in thin sections of Epon-embedded material.
INTRODUCTION Mast cells are known to play a role in immunological responsiveness as effector cells mediating an inflammatory reaction via the release of vasoactive amines ( Mongar and Schild, 1962; Mota, 1963). Immunoglobulin E ( IgE ), the classical reagin, binds to receptors at the cell surface (Ishizaka et nl., 1966; Ishizaka and Ishizaka, 1971), and, when challenged with appropriate antigen, triggers the release of several mast cell granule components mediating immediate-type hypersensitivity or local anaphylaxis ( Mongar and Schild, 1962; Orr, 1973). There is evidence that immunoglobulins other than IgE also bind to mast cells, since immunoglobulin G ( IgG) fractions have been shown to elicit passive cutaneous anaphylaxis in mice, rats and humans (Vas, 1971; Bloch and Ohman, 1971; Parish, 1970; Stanworth and Smith, 1972). In this laboratory, we have been investigating cellular production and localization of immunoglobulins in human and rat tissues (Garvin et al., 1974) employing an immunoglobulin-enzyme bridge technique with horseradish peroxidase (HRP) as the enzyme marker (Mason et al., 1969). During these studies, staining was observed which indicated binding of immunoglobulins to mast cell granules (Simson and Hintz, 1975). We have investigated this immunoglobulin binding to mast cells in the present study with the aim of obtaining information about the site of binding, its lability to fixation, and the nature of the bound antibody. MATERIALS Tissue and cell processing. 200 and 250 gm were utilixrcl
Copyright All rights
0 1977 by Academic Press, of reproduction in any form
AND
Female
Inc. reserved.
for
these
METHODS
Sprague-Dawley experiments.
rats weighing between Lymphoid tissue excised
ISSN
0014-4800
86
SIMSON ET AL.
from pentobarbital-anesthetized animals was immediately placed in one of the following fixatives: (a) calcium-acetate buffered formalin (CAF); (b) cacodylate buffered 3% glutaraldehyde; (c) Carnoy’s alcohol-acetic acid, or (d) CAF with 0.5% glutaraldehyde. Only the latter fixative without postosmication was utilized for electron microscopy. Free, unstimulated cells from the rat peritoneal cavity were harvested by a method described previously (Simson et al., 1973) and fixed with the same fixatives. After selected fixation times, tissues or cells were processed either for light or electron microscopy by routine methods. Light microscopy studies were performed on tissues fixed for times ranging from 30 min to 24 hr; fixation time for electron microscopy was 4 hr. Tissue or cells were embedded in paraffin for light microscopy and in Epon 812 for electron microscopy. Sections for light microscopy were cut at about 3 ,J and mounted on glass slides. Thin sections for electron microscopy were between 500 A and 800 A (based upon interference colors), and were picked up on stainless steel grids. lmmunoglobulin bridge technique. Sections were reacted with the immunoglobulin-enzyme bridge sequence. This technique, originally described by Mason et al., (1969) consisted of the following reaction sequence: (a) rabbit antiserum to tissue antigen, (b) goat antiserum to rabbit immunoglobulin (G anti-R Ig), (c) rabbit antiserum to horseradish peroxidase (R anti-HRP), (d) horseradish peroxidase (HRP). The latter enzyme, bound to the tissue by the immunoglobulin bridge, was demonstrated utilizing diaminobenzidine (DAB) and hydrogen peroxide ( HaOz) (Graham and Karnovsky, (1966). The oxidized, precipitated DAB was further “blackened” by treatment with osmium tetroxide, exposing the sections 5 set for light microscopy, or 10 min for electron microscopy. Thorough rinsing with phosphate-buffered saline between each step was essential. Controls consisted of normal rabbit serum (NRS) substituted for specific antiserum in the bridge (control for “nonspecific” binding) or DAB/H202 alone (control for endogenous peroxidase). In addition to the complete bridge, described above, several modifications and abbreviations of the bridge have been used in the present study as indicated in the text and table. After initial evaluation of mast cell binding of antiserum with modifications of the bridge sequence, a “truncated” bridge, consisting of [anti-HRP]-[HRP]-[DAB/H202] was used for most of the remaining studies. Sections were viewed without counterstaining. Antisera. The antisera to HRP were made in this laboratory by giving an initial antigen dose, mixed with complete Freund’s adjuvant, subcutaneously, followed in 1 month and again 2 weeks later by boosters in incomplete Freund’s adjuvant. Other injection routes were also used in the rat, including intramuscular and intraperitoneal injections. Animals were bled at several intervals after the final injection, and sera were separated following room temperature coagulation. Most antisera were not further purified. Antisera raised in rabbits, rats, guinea pigs, and goats were tested, although the majority of the studies utilized rabbit antiHRP. A purified IgG fraction of anti-HRP antiserum was obtained from Cappel Laboratories ( Downingtown, Pennsylvania). RESULTS AND DISCUSSION The binding of horseradish peroxidase to mast cell granules was observed when serum from normal or immunized rabbits was applied to the tissue as the first step of the complete immunoglobulin-peroxidase bridge. Sequential elimination
MAST
CELL
GRANULE
BINDING
OF ANTIBODY
87
of various steps of the bridge (Table I) disclosed that mast cells were stained by the sequence: [anti-HRP]-[HRP]-[DAB/HZOZ] (Fig. 1); but not by [HRP][DAB/H,O,] or [DAB/H,O,] a1one (Fig. 2). These results demonstrated that antibody was necessary for staining, and excluded the possibility that either cndogenous peroxidase or nonspecific adsorption of HRP were responsible for the reaction product. If NRS was interposed between [anti-HRP] and [HRP], the final peroxidase activity was reduced considerably, suggesting displacement of anti-HRP by immunoglobulins with other specificities. The localization of reactivity to mast cells was confirmed by staining adjacent 3 p serial sections, one with the [anti-HRP]-[HRP]-[DAB/HZOZ] sequence and the next with a reference toluidine blue stain for mast cell metachromasia (Figs. 3 and 4). The observable binding was restricted to mast cells. The staining filled the cytoplasm and generally appeared to be localized to mast cell granules rather than the cell periphery. No other cells stained in the lymphoid tissue, peritoneal fluid, and numerous other tissues examined except those cells which exhibited endogenous peroxidase in control sections as well. Conditions under which anti-HRP was capable of binding to rat mast cells were investigated by either varying tissue fixations or heating or diluting the antiserum (Table I). A short fixation time with aldehydes (2 hr or less) was essential for preservation of mast cell affinity for immunoglobulin, whereas binding was observed after 24 hr fixation with Carnoy’s fixative. Heating the antiserum to 60” for 30 min or 4 hr did not appreciably reduce binding affinity. This suggested that the binding was more likely attributable to IgG than to IgE, inasmuch as the latter is heat labile. Dilution of the anti-HRP serum with NRS resulted in rapid diminution of specific binding. A 1:25 dilution of our highest titre anti-HRP antiserum with NRS resulted in complete loss of demonstrable binding. On the other hand, dilution of the same anti-HRP serum with phosphate-buffered saline (PBS) resulted in diminished binding only at a 1:lOO dilution and loss of binding at a 1:400 dilution. This suggested competition for binding sites by immunoglobulins not reactive for HRP which were present in NRS but not in PBS. A purified IgG fraction of anti-HRP bomid as well as did the unpurified antisera prepared in this laboratory, suggesting that the immunoglobulin which binds to the granules may be an IgG subfraction rather than IgE. Although the results described apply for the most part to antisera produced in rabbits, anti-HRP raised in rats with intraperitoneal immunization also yielded good binding, indicating that this cytochemically demonstrable binding of immunoglobulin to mast cell granules was not simply a heterocytotropic phenomenon. To determine whether immunoglobulin was bound to the granule membrane or to the granule matrix, thin epoxy sections of tissue which had been fixed with the formaldehyde-glutaraldehyde mixture without postosmication, were reacted seq uence for ultrastructural examinawith the [anti-HRP]-[HRP]-[DAB/H30L’] tion. This procedure imparted electron density selectively to the mast cell granule matrix whether the antiserum utilized was purified IgG anti-HRP (Fig. 5) or the routine anti-HRP antiserum. The density in the small condensed granules exceeded that in the larger cytoplasmic granules in which reaction product was distributed in a coiled thread pattern. All of the controls utilized at the light
SIMSON
88
TABLE Mast Cell Binding
Methodological
ET AL. I
of Anti-HRP: the Effect of Immunocytochemical Tissue Fixation, Heating, or Antiserum Dilutiona
variant
Bridge Sequence,
Immunoglobulin binding (reaction product)b
Bridge Sequence” [NItSI-[G
anti-It Ig]-[R [G anti-R Ig>[R
anti-HRP]-[HRP+[DAB/H&l anti-HRP]-[HRP]-[DAB/H2021 [anti-IIRP>[HRP]-[DAB/H&21 [NItSI-[HRP~[DAB/HzOz] [HRP-J[DAB/HzOJ [DAB/HzOzl [anti-HRP]-[NRS)-[HRP]-[DAB/HzOJ [anti-HRP]-[HRP>[NRS]-[DAB/HzO?] [NRS]-[anti-HRP]-[HRP]-[DAB/HzOJ
2 4+ 2 4+ 2 4+ 0 0 0 0 -1+ 1 -3+ 2 -4+
Fixation Calcium-acetate buffered formalin (CAF) 30 min, 1 hr, 2 hr 24 hr 3’s Glutaraldehyde (cacodylate buffered) 1 hr 2 hr 24 hr CSF + 0.5y0 glutaraldehyde 1 hr Carnoy’s alrohol-acetic acid 1 hr 24 hr Antiserum
2 4+ 0 -2fd 1 -3f 0 -1+ 0 2 -4+ 3 4+ 3 4t
heating
60” for 30 min or 4 hr Antiserum
dilutions
With With With With With With
NRS-1:s NRS-1: PBS-1 PBS--l PBS”-1 PBSe-1
25 :5 : 25 :200 :400
2 -3f
1 -a+ 0 3 -4+ 2 -3+ 0 -2+ 0
a The tissue utilized in all of these studies was rat lymph node. The data represent light microscopic observations. b The intensity of the final DAB reaction product was graded on a lf to 4+ basis and assessed by the color developed as follows: straw to tan = l+; tan to light brown = 2f; dark brown = 3+; black = 4f. In all procedures except those in which the bridge sequence was varied, figures represent the intensity of reaction product with the [anti-HRP>[HRP]-[DAB/HzOz] sequence blackened in 2y0 0~0, for 5 sec. In all procedures, appropriate controls were performed. c All of these variants were performed on &sue fixed with 49;1 formaldehyde, 0.5% glutaraldehyde in calcium-acetate buffer, pH 7.0 for 1 hr. Certain of the variants were also performed on tissue fixed with the above fixative (CAF) without glutaraldehyde, with identical results. d The mast, cells in t.he siromn snrrolmdillg I he lymph node rr~t:~irlocl some bindinK cnpacaity, lmt t.hose in the body of the nc~le did not, e Phosphate buffered saline,
MAST
CELL
GRANULE
BINDING
OF ANTIBODY
FIG. 1. Light micrograph of rat peritoneal cells to which the sequence [anti-HRP]-[HRP][DAB/Hz02] was applied. The two large cells (XI) are mast cells showing reactivity; the small cells with reaction product are monocytes or eosinophils which possess endogenous peroxidase (see Fig. 2). X450. FIG. 2. Light micrograph of same preparation as Fig. 1, reacted with DAB/H202 alone, to demonstrate endogenous peroxidase. Some small cells (monocytes and eosinophils) are reactive; however, mast cells show no reactivity. The clear “holes” (X) probably represent unstained mast cells. Faintly refractile granules were seen microscopically in the lower clear area, but they are not clearly visible in the photograph. x450. FIG. 3. Light micrograph of the capsule of a lymph node. The section was reacted with [anti-HRP]-[HRP]-[DAB/H?OJ. Erythrocytes in the blood vessel (BV) show some endogenous peroxidatic activity. The two mast cells possessing reaction product (arrows) correspond to two Toluidine-blue metachromatic cells in Fig. 4. X450. FIG. 4. Light micrograph of a section serial to that in Fig. 3 stained with Toluidine blue. A green filter was used to enhance the density of the highly metachromatic mast cells (arrows). Erythrocytes in the blood vessel (BV) are unstained but refractile. X450. FIG. 5. An electron micrograph of mast cell granules illustrating reaction product over the granule matrix. This section was treated with a purified IgC fraction of anti-HRP followed by [HRPI-[DAB/ROJ X28,000. FIG. 6. An electron micrograph illustrating mast cell granules following a control procedure (DAB/H,O?). x24,000.
level were performed on thin sections (Fig. 6); none gave evidence either of binding of HRP alone to granules or of intrinsic perosidase activity. The significance of immunoglobulin binding by mast cell granules remains to 1~ elucidated. Such binding could partly account for the several hour delay that is required between administration of antibody and the capacity of an animal
90
SIMSON
ET
AL.
to manifest passive cutaneous anaphylaxis. The uptake of exogenously administered material into mast cell granules has been amply demonstrated by Padawer ( 1968, 1971), who h as suggested that mast cell granules may concentrate antibodies in viva (1974). Granule-bound antibody could react with specific antigen either before or after granule discharge from mast cells, contributing to the release of inflammatory mediators, such as histamine, from the granules. Antigen (ferritin) binding to discharged mast cell granules has been demonstrated ( Movat et al., 1966). Heparin, a normal mast cell granule constituent, stimulates endocytosis (Cohn and Parks, 1967), and appears important for the activity of some opsonic proteins (Allen et al., 1973). Discharged mast cell granules are avidly taken up by nearby macrophages (Riley, 1963; Padawer, 1970). In addition, heparin has an inhibitory effect on some hydrolytic enzymes (Brittinger et al., 1968; de Chatelet et al., 1972) which might be expected to degrade phagocytosed antigen. Thus, mast cell granule-bound antigen-antibody complexes could be ideal immunogens. ACKNOWLEDGMENTS We appreciate the helpful discussions and wish to thank Dr. H. H. Fudenberg This work was supported by NIH grants appropriation 22700-A401, ‘73-‘74.
of Dr. A. J. Garvin during for reviewing the manuscript AM-10956 and AM-11028
the course of this study, prior to its submission. and South Carolina State
REFERENCES ALLEN, C., SABA, T. M., and MOLNAR, J. (1973). Isolation, purification, and characterization of opsonic protein. J. Reticuloendothel. Sot. 13, 410-423. BLOCH, K. J., and OHMAN, J. L. ( 1971). The stable homocytotropic antibodies of guinea pig, mouse and rat, and some indirect evidence for the in vioo interaction of homocytotropic antibodies of two different rat immunoglobulin classes at a common receptor on target cells. In “Biochemistry of the Acute Allergic Reactions,” K. F. Austen and E. L. Becker, eds. p. 45-64, Blackwell, Oxford. BRITTINGER, G., HIRSCHHORN, R., DOUGLAS, S. D., and WEISSMANN, G. (1968). Studies on lysosomes. XI. Characterization of a hydrolase-rich fraction from human lymphocytes. j. Cell Biol. 37, 394411. COHN, Z. A., and PARKS, E. ( 1967). The regulation of pinocytosis in mouse macrophages. II. Factors inducing vesicle formation. .I. Exp. Med. 125, 213-230. DECHATELET, L. R., MCCALL, C. E., COOPER, M. R., and SHIRLEY, P. (1972). Inhibition of leukocyte acid phosphatase by heparin. Clin. Chem. 18, 1532-1534. GARVIN, A. J., SPICER, S. S., PAR~ILEY, R. T., and MUNSTER, A. M. (1974). Immunohistochemical demonstration of IgG in Reed-Stemberg and other cells in Hodgkin’s disease. j. Exp. Med. 139, 1077-1083. GRAHAX, R. C., and KARNOVSKY, M. J. ( 1966). The early stages of absorption of injected horseradish peroxidase in the proximal tubules of mouse kidney. Ultrastructural cytochemistry by a new technique. J. Histochem. Cytochem. 14, 291-302. ISHIZAKA, K., and ISHIZAICA, T. (1971). IgE and reaginic hypersensitivity. Ann, N.Y. Acad. Sci. 190, 443-456. ISHIZAKA, K., ISHIZAIU, T., and HORNBROOK, M. M. ( 1966). Physicochemical properties of human reaginic antibody. IV. Presence of a unique immunoglobulin as a carrier of reaginic activity. J. Immunol. 97, 75-85. MASON, T. E., PHIFER, R. F., SPICER, S. S., SWALLOW, R. A., and DHESKIN, R. B. (1969). An immunoglobulin-enzyme bridge method for localizing tissue antigens, j. Histochem. Cytochem. 17, 563-569. hIoxGAH, J. R., and SCIIILD, H. 0. ( 1962). Cellular mechanisms in anaphylaxis. Physiol. RN. 42, 226-270.
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hlw~, I. (1963). Mast cells and anaphylaxis. Ann. N.Y. Acad. Sk. 103, 264-277. Movar, H. Z., LOVETT, C. A., and TAICHMAN, N. S. (1966). Demonstration of antigrn ou the surface of sensitized rat mast cells. Nature 212, 851-853. Onn, T. S. C. ( 1973). Mast cells and allergic asthma. Brit. J. Uis. Chest 67, 87-IOG. PADAWER, J. ( 1968). Ingestion of colloidal gold by mast cells. l’roc. Sot. L'x~J. Bid. I\fcd. 129, 905-907. PADAWER, J. ( 1970). The reaction of rat mast cells to polylysine. J. Cell Biol. 47, 352-372. PADAWR, J. ( 1971). Phagocytosis of particulate substances by mast cells. Lab. Inwst. 25, 320-330. PA~AWER, J. (1974). The ins and outs of mast cell function. Am. J. Anat. 141, 299-302. PARISH, W. E. (1970). Short-term anaphylactic IgG antibodies in human sera. Lancct 2, 591-592. RILEY, J. F. ( 1963). Functional significance of histamine and heparin in tissue mast cells. Ann. N.Y. Acad. Sci. 103, 151-161. SIHSO~T, J. A. V., and HINTZ, D. S. (1975). Evidence for immunoglobulin binding to rat mast cell granules. Anat. Rec. 181, 480 (abstract). SIMSOX, J. A. V., SPICER, S. S., and WRIGHT, N. J. (1973). Unusual cellular aggregates from rat peritoneal cavity. J. Reticdoendothel. Sot. 14, 361-370. STANWOHTH, D. R., and SMITH, A. K. ( 1972). Human IgG subclass with primate skinbinding activity. Lancet 2, 491. VAZ, N. M., ( 1971). Anaphylactic sensitization of mouse tissues with IgGL and reaginic antibodies. In “Biochemistry of the Acute Allergic Reactions,” K. F. ,4usten and E. L. Becker, eds. p. 91-109, Blackwell, Oxford.
