J.COMP.PATH.~~~~.VOL.~~.
DISTRIBUTION TISSUES
611
BY
OF IMMUNOGLOBULINS IN INDIRECT IMMUNOFLUORESCENCE
EQUINE
BY
S. A. KHALEEL University
and R. M.
of Pennsylvania
School of Veterinary Medicine, Kennett Square, Pa. 19348, U.S.A.
P. z.
KENNEY ,~eul Bolton Center,
ALLER'
Department of Microbiology, Division of Immunolopl. University of Rochester School of Medicine and Dentistry, Rochester, New York 14642, U.S.A.
INTRODUCTIOX Heterogeneity in the molecular forms and properties of equine immunoglobulins has been revealed by immunochemical studies on equine antibodies (Rockey, Klinman and Karush, 1964; Klinman, Rockey and Karush, 1965 ; Klinman, Rockey, Frauenberger and Karush, 1966; Klinman and Karush, 1967; Rockey, 1967 ; Raynaud and Iscaki, 1970; Rockey, Montgomery and Dorrington, 1970; Helms and Allen, 1970, 197 1; Allen and Johnson, 1972; Audibert and Sandor, 1972; Allen and Dalton, 1975). Although at least 10 antigenically distinct equine immunoglobulins have been recognized, only 7 equine immunoglobulins designated IgGa, IgGt,, IgG,, and IgM have been isolated and studied. I@, k-4 WV’) IgM appears to predominate among serum antibodies made early in the immune response of the horse against carbohydrate components of bacterial vaccines (Hill and Cebra, 1965; Kim and Karush, 1973). Prolonged or continued immunization with bacterial vaccines, however, leads to the production of relatively large amounts of IgB anti-carbohydrate (Zolla and Goodman, 1968; Sandor and Audibert, 1970; Lavergne and Raynaud, 1973). In contrast to this IgB response, hyperimmunization of horses with protein antigens usually leads to the prevalence of IgG(T) antibodies (Raynaud and Iscaki, 1970; Audibert and Sandor, 1972). and IgM in equine secretions was demonstrated The occurrence of IgG, IgG(T) by Genco, Yecies and Karush (1969), who described their isolation from equine parotid fluid and colostrum. Although evidence for a secretory IgA system was not obtained by these workers, the occurrence of IgA and secretory component (SC) in equine secretions has been established by several studies indicating the existence of an IgA secretory system in the horse analogous to that described for other species. Audibert and Sandor (1968) isolated an antigenically distinct immunoglobulin from equine colostrum and milk characterized as IgA on the basis of its electrophoretic mobility, behaviour with zinc ions, carbohydrate content and its selective Heremans and VanKerkhoven (1969), secretion by exocrine glands. Vaerman, Orlans and Feinstein (1971) and McGuire and Crawford (1972) identified horse serum IgA by demonstrating its immunological cross reaction with specific antihuman alpha chain reagents. Employing monospecific anti-horse IgA reagent, quantitative studies have shown IgA to be the major immunoglobulin present in equine milk, tears, saliva, nasal secretions and spermatic fluid, but a minor component of colostrum (Vaerman, Querinjean and Heremans, 197 1; McGuire and Crawford, 1972; Pahud and Mach, 1972). Comparable to findings in other species, the intestinal mucosa of the horse has also
612
s. A.
KHALEEL
et
al.
been identified as a site of local IgA synthesis (Vaerman, 1973). By means of immunofluorescent staining, IgA-containing plasma cells were identified in horse duodenum and proximal jejunum where they were found by Vaerman et al. (1971) to be more abundant than IgM-containing cells. Studies on the in vitro synthesis of immunoglobulins by salivary gland have identified the submandibular gland of the horse as another site of local IgA synthesis. Fluids from tissue cultures of the horse submandibular gland examined by radioimmunoelectrophoresis were found by Hurlim.ann and Darling (1971) to show the selective synthesis of a secretory IgA-like globulin when compared to lymph node cultures.
Immunofluorescent studies on the distribution of specific classes and subclassesof equine immunoglobulin in horse salivary glands and lymph nodes have not been previously reported. In the present investigation, monospecific rabbit antisera prepared against the heavy chains of equine IgG,, IgG,, IgG,, and IgM were used as reagents for the indirect immunok-4 IgB, W(T) fluorescent staining of tissue immunoglobulins. The identification of cells containing specific immunoglobulin and their distribution in horse parotid and submaxillary salivary glands was compared with that obtained for regional parotid and mesenteric lymph nodes as well as duodenum. In addition, tissue extracts prepared from horse salivary glands and lymph nodes were assayed by immunodiffusion to estimate the relative amount of the various immunoglobulins present. MATERIALS
AND
METHODS
Tissues. Parotid and submaxillary salivary glands; submandibular lymph nodes; mesenteric lymph nodes and duodenum were obtained from 7 horses within 5 min. after death. Slices 4 mm. thick were immediately quenched in isopentane chilled with liquid nitrogen or placed into one of several fixatives. Tissue extracts. Soluble tissue extracts were made from frozen tissues stored at -20 “C. Each gram of tissue was homogenized at 4 “C in a Servall tissue homogenizer, with 1 ml. of 0.1 M phosphate buffer pH 7.4, containing 0.9 per cent. sodium chloride. Homogenates were centrifuged at 10 000 g for 2 h. The clear supernatant fluid provided soluble tissue extract for immunodiffusion analysis. Protein concentration of clear extracts was determined by micro-Kjeldahl nitrogen analysis, assuming a nitrogen content of 16 per cent. for proteins. Antisera. The preparation and characterization of monospecific rabbit antisera against horse IgGa, IgG, IgGc, IgB, IgA, IgG(T), IgM and a polyvalent rabbit antiserum against horse (IgG, + Ig& + IgG,) h ave been previously described (Allen and Dalton, 1975 ; Allen and Johnson, 1972; Martineau and Allen, 1973). All antisera employed in immunofluorescent localization were absorbed with horse tissue powders of fascia lata, liver and tendon to remove nonspecific binding components. Tissue powders were prepared by the method of Mellors (1959). Goat antirabbit IgG globulin was obtained from a commercial source (Cappel Laboratories, Downingtown, Penna., U.S.A.). Fl uorescein conjugated goat anti-rabbit 7S-globulin was also purchased from a commercial source (Hyland Division, Travenol Laboratories, Inc., Costa Mesa, California, U.S.A.). Immunodi$usion analysis. Double diffusion in plates was carried out as previously described (Johnston and Allen, 1968). Double diffusion in microtubes by the method of Preer as described by Finger ( 197 I), was used to obtain a relative estimate of the amount of immunoglobulin present in tissue extracts. A single, commercial, pooled lot of normal horse serum (Hyland Laboratories, Los Angeles, California, U.S.A.) was used as a primary reference standard for all immunoglobulins. Serial, two-fold dilutions of reference serum standard or tissue extract were diffused through a 5 mm.
DISTRIBUTION
OF IMMUNOGLOBULINS
IN EQUINE
TISSUES
613
column of agar against 0.02 ml. of each monospecific anti-horse immunoglobulin reagent. The position of each precipitin band relative to the agar column height was measured daily for 5 days. The ratio of the dilutions of extract and serum standard producing a band at comparable positions was used to provide an estimate of the amount of immunoglobulin present in extracts relative to that of reference serum standard. IgM did not penetrate the intermediate agar column beyond 20 per cent. Difficulty was encountered in efforts to assay IgM by this method since the IgM band position did not show significant displacement on doubling dilution and thus quantitation of IgM was not carried out. Tissue extracts were qualitatively tested for the presence of IgM by both plate and Preer methods. Fixation. Post fixation: frozen material was sectioned (3 to 5 pm.) in a cryostat at - 15 “C. and each section was postfixed for 10 min. in one of the following solutions: acetone; 70 per cent. ethanol; 95 per cent. ethanol; 95 per cent. methanol; 95 per cent. methanol and 5 per cent. acetic acid. In addition, one or two sections from each tissue were stained with HE. Liquid fixation: tissues were embedded in paraffin and sectioned at 4 pm. after fixing the sections overnight at 4 “C. in one of the following fixatives: cold acetone; cold 70 per cent. ethanol; cold 95 per cent. ethanol; cold 10 per cent. neutral buffered saline; cold methanol; cold 95 per cent. methanol and 5 per cent. acetic acid and Bouin’s solution. Immunojkorescent staining procedure. Cryostat and paraffin embedded sections were incubated with unconjugated monospecific rabbit anti-equine immunoglobulin reagents in a moist chamber at 37 “C. for 40 min. They were individually rinsed in 0.01 M-phosphate buffered saline (PBS) pH 7.2 3 times (3 min. each wash) with continuous, gentle, agitation. Sections were air dried, incubated with fluorescein isothiocyanate (FITC) conjugated goat anti-rabbit serum in a moist chamber at 37 “C. for 40 min., then washed, air dried as described above and mounted in 90 per cent. glycerine in PBS. Controls. The specificity of staining reactions was controlled by: (1) the determination of the degree of blocking of the reaction with unconjugated goat anti-rabbit serum prior to incubation with conjugated goat anti-rabbit serum; (2) treating sections with normal rabbit serum before incubating with FITC-conjugated reagents; (3) treating sections with a heterologous unrelated antibody (guinea pig anti-hamster immunoglobulin obtained from Cappel Laboratories, Downingtown, Penna., U.S.A.) before staining with FITC-conjugates; (4) treating sections with trypsin or colsections were treated with a lagenase before staining. For collagenase treatment, 1: 10 dilution of 0.1 per cent. collagenase (Sigma Chemical Company, Type III, Fraction A, 500 units/mg. solid) solution in 0.05 M-TES buffer (Tri-(hydroxymethyl) methyl 2-amino ethane sulfonate) with 0.36 mM-CaCl, at pH 7.5 for 3 h. at 37 “C. in a moist chamber. For trypsin treatment, sections were treated with trypsin in TES buffer with O-0115 M-CaCl, at pH 7.6 (9000 BAEE units/mg. powder/ml.) for 3 h. at 5 “C. in a moist chamber. Trypsin, bovine type XI (DCC treated) was obtained from Sigma Chemical Company, St Louis, Missouri, U.S.A.; (5) treating sections of horse tissues directly with FITC goat anti-rabbit Ig as a further control for nonspecific binding. Fluorescence microscopy andphotografihy. Stained preparations were examined with the aid of an immersion dark field condenser on a Leitz Wetzlar microscope using an Osram HBO 200 W light source, a KP 490 interference filter and a K 470 barrier filter. Black and white photographs and colour transparencies were taken on Tri-X panchromatic and high speed Ektachrome (ASA 400, 160) film using an Orthomat automatic camera. RESULTS
Liquid fixation 5 per cent. acetic
followed by paraffin acid in 95 per cent.
embedding, with the exception of methanol gave unsatisfactory results.
614
Fig.
S. A. KHALEEL
1. Equine staining
parotid salivary gland of interstitial connective
et Ul.
section stained for IgG(= + ,, + e), showing immunofluorescent tissue and cytoplasm of interstitial mononuclear cells. x 76.
Fig. 2. Equine parotid salivary gland section stained for IgG(T) showing localization of immunoglobulin in network of interstitial connective tissue spaces and in cytoplasm of a cluster of interstitial cells. X 76. Fig. 3. Equine discrete
parotid plasma
salivary gland cells randomly
stained for IgA. Immunofluorescence scattered through interstitial tissue.
localized x 284.
Fig. 4. Equine parotid salivary gland section stained for IgA showing immunoglobulin cytoplasm of epithelial cells of the duct and in duct contents. x 284.
in cytoplasm
of
localized
in
DISTRIBUTION
OF IMMUNOGLOBULINS
IN EQUINE
TISSUES
615
Histological quality was poor in the majority of such sections and even completely lost in a few sections. Furthermore, fluorescent material leached from its original site and floated under the coverslip. However, fixation with 5 per cent. acetic acid in 95 per cent. methanol provided sections with sharp histological features and stable localization of antigen. Similarly, cryostat sections fixed with 5 per cent. acetic acid in 95 per cent. methanol also gave satisfactory results. Paraffin embedding was preferred, however, since it helped prevent autofluorescence and material could be stored at 4 “C. in the paraffin block for further study.
Salivary Glands The immunohistochemical staining pattern for IgG produced by a polyvalent rabbit anti-horse (IgG,+IgG,+IgG,) reagent was consistent in all sections of horse parotid and submaxillary gland (Fig. 1). Fluorescence was present in the interstitial connective tissue, basement membrane and often within vascular lumina. Cytoplasmic staining for IgG was present in mononuclear cells scattered throughout the interstitium. Neither the duct lumina nor any part of the acinar cells showed specific immunofluorescence. Sections incubated with monospecific antisera against the heavy chain of equine IgG,, IgG,,, or IgG, gave a staining pattern identical to that obtained with polyvalent IgG,,,,,,,. Sections of salivary gland incubated with monospecific anti-IgG(T) gave a staining pattern (Fig. 2), similar to that obtained with anti-IgG reagents; however, IgG (T) immunofluorescence was more diffuse in the connective tissue. Clusters of interstitial cells showing cytoplasmic staining for IgG(T) were seen scattered irregularly throughout the interstitium. Absorption of antisera with powders of horse tendon, liver or fascia lata did not alter the staining pattern. Sections treated with trypsin before exposure to anti-horse IgG or IgG(T), lacked fluorescence in areas previously positive. Treatment with collagenase did not alter immunofluorescent staining. While IgM was found discretely localized in a small number of isolated, individual, oval shaped mononuclear cells (plasma cells) scattered in the interstitium, IgB was not demonstrable by immunofluorescence in any sections ofsalivary gland examined. Sections of salivary gland stained for IgA showed immunofluorescence in individual mononuclear cells (plasma cells) randomly scattered in the interstitium (Fig. 3), lumen of the glandular acini, ducts and epithelial cells (Fig. 4). IgA fluorescence was stronger in cells of the luminal layer in contact with ductular contents while cells of the basal layer were weakly positive.
Lymph .Nodes Sections of parotid lymph node stained for IgG,,,,,,, (Fig. 5) showed strong cytoplasmic fluorescence in cells of, the cortex, cells comprising the core of germinal centres and cells of medullary cords. A large amount of diffuse intercellular IgG fluorescence was also apparent in sections of lymph node. Staining for IgG(T) showed a pattern of distribution somewhat similar to IgG. While small numbers of cells showing cytoplasmic staining for IgG(T) were seen scattered irregularly throughout the cortex, intense IgG(T) staining
s. A. KHALEEL
et
ai.
Fig. 5. Equine parotid lymph node stained for IgG(,+ h + C). Immunofluorescence localized in cells of the germinal centre. x 76. Fig. 6. Equine parotid lymph node stained for IgG(T) showing immunofluorescent staining of cells at the periphery of a germinal centre and in the medullary cords. x 76. Fig. 7. Equine parotid lymph node stained for IgM showing the presence of fluorescent stained cells in the zone of large lymphocytes in the germinal centre and in medullary cords. x 76. Fig. 8. Equine parotid lymph node stained for IgA showing cytoplasmic staining of individual cells randomly scattered in the medullary cords. x 192.
DISTRIBUTION
OF
IMMUNOGLOBULINS
IN
EQUINE
TISSUES
617
was associated with cells located predominantly at the periphery of germinal centres and in the medullary cords (Fig. 6). Parotid lymph node stained for IgM showed the presence of immunofluorescent stained lymphoid cells in the zone of large lymphocytes of germinal
Fig.
9.
Fig.
10.
Fig.
Il.
Fig.
12.
Fig.
13.
Fig.
14.
showing immunofluorescent localization in the surface Equine duodenum stained for IgG,, mucin at the tips of villi. x 144. Equine duodenum stained for IgG, showing localization in lymphoid cells of the lamina prbpria and in the glands. x 57. Equine duodenum stained for IgG(T) showing cytoplasmic staining of individual lymphoid cells scattered in the lamina propria. x 144. Equine duodenum stained for IgM showing immunofluorescent localization in lymphoid cells of the lamina propria and in the apical portion of epithelial cells lining a crypt. x 144. Equine duodenum stained for IgA showing fluorescent staining of the cytoplasm of crypt. epithelium and plasma cells scattered throughout the lamina propria. x 144. Equine duodenum stained for IgA showing fluorescent staining of lymphoid cells at the periphery of a lymphoid nodule. x 144.
618
S. A. KHALEEL
et Ul.
centres as well as in medullary cords (Fig. 7). Although a few single, isolated cells staining for IgB were seen in the cortical areas of parotid and mesenteric lymph nodes, these cells were very few and rarely encountered in sections. Immunofluorescent staining of parotid lymph node for IgA, showed a distinctive pattern of distribution (Fig. 8). Cytoplasmic IgA fluorescence was confined to individual cells scattered in the cortex between and around germinal centres extending into the medullary cords, but was conspicuously absent from germinal centres. Mesenteric lymph node stained for IgG,,,b,,,, IgG(T), IgB, IgA and IgM gave distribution patterns comparable to those described for parotid lymph node.
Sections of horse duodenum stained with monospecific anti-IgG,, anti-IgG,, or anti-IgG, showed strong immunofluorescence associated with the mucin coating the luminal surface of villous epithelia (Fig. 9). Lymphoid cells with TABLE RELATIVE
AMOUNTS
OF IMMUNOGLOBULINS
Relative
Total
Horse no.
Tissue extract
1
Parotid gland Submaxillary gland Parotid gland Submaxillary gland Parotid gland Lymph node (parotid) Parotid gland Submaxillary gland Lymph node (mesenteric) Submaxillary gland Lymph node (mesenteric) Parotid gland Submaxillary gland Parotid gland Lymph node (mesenteric) Lacrimal gland
2 3 4
5 6 7
1
protein mg./ml.
57
~ IgCs
I.%
DETECTED
IN HORSE
concentration 4G
TISSUE
EXTRACTS
of immunoglobulin* Id
W(T)
--w
Zghi
0.031 0.063 0.03 1 0.008 0.016
0.016 0.03 1 0.03 1 0.008 0.031
0,016 0.016 0.016 0.006 0.031
0.500 0.125 0.125 0.250 0.016
0.007 0.007 0.060 0908 0.031
0.030 0.03 1 0,016
0.016 0.03 1 0.016
0.008 0.016 0.016
0.016 0.031 0.500
0.008 0.016 0.004
ND
DISTRIBUTION TISSUES
611
BY
OF IMMUNOGLOBULINS IN INDIRECT IMMUNOFLUORESCENCE
EQUINE
BY
S. A. KHALEEL University
and R. M.
of Pennsylvania
School of Veterinary Medicine, Kennett Square, Pa. 19348, U.S.A.
P. z.
KENNEY ,~eul Bolton Center,
ALLER'
Department of Microbiology, Division of Immunolopl. University of Rochester School of Medicine and Dentistry, Rochester, New York 14642, U.S.A.
INTRODUCTIOX Heterogeneity in the molecular forms and properties of equine immunoglobulins has been revealed by immunochemical studies on equine antibodies (Rockey, Klinman and Karush, 1964; Klinman, Rockey and Karush, 1965 ; Klinman, Rockey, Frauenberger and Karush, 1966; Klinman and Karush, 1967; Rockey, 1967 ; Raynaud and Iscaki, 1970; Rockey, Montgomery and Dorrington, 1970; Helms and Allen, 1970, 197 1; Allen and Johnson, 1972; Audibert and Sandor, 1972; Allen and Dalton, 1975). Although at least 10 antigenically distinct equine immunoglobulins have been recognized, only 7 equine immunoglobulins designated IgGa, IgGt,, IgG,, and IgM have been isolated and studied. I@, k-4 WV’) IgM appears to predominate among serum antibodies made early in the immune response of the horse against carbohydrate components of bacterial vaccines (Hill and Cebra, 1965; Kim and Karush, 1973). Prolonged or continued immunization with bacterial vaccines, however, leads to the production of relatively large amounts of IgB anti-carbohydrate (Zolla and Goodman, 1968; Sandor and Audibert, 1970; Lavergne and Raynaud, 1973). In contrast to this IgB response, hyperimmunization of horses with protein antigens usually leads to the prevalence of IgG(T) antibodies (Raynaud and Iscaki, 1970; Audibert and Sandor, 1972). and IgM in equine secretions was demonstrated The occurrence of IgG, IgG(T) by Genco, Yecies and Karush (1969), who described their isolation from equine parotid fluid and colostrum. Although evidence for a secretory IgA system was not obtained by these workers, the occurrence of IgA and secretory component (SC) in equine secretions has been established by several studies indicating the existence of an IgA secretory system in the horse analogous to that described for other species. Audibert and Sandor (1968) isolated an antigenically distinct immunoglobulin from equine colostrum and milk characterized as IgA on the basis of its electrophoretic mobility, behaviour with zinc ions, carbohydrate content and its selective Heremans and VanKerkhoven (1969), secretion by exocrine glands. Vaerman, Orlans and Feinstein (1971) and McGuire and Crawford (1972) identified horse serum IgA by demonstrating its immunological cross reaction with specific antihuman alpha chain reagents. Employing monospecific anti-horse IgA reagent, quantitative studies have shown IgA to be the major immunoglobulin present in equine milk, tears, saliva, nasal secretions and spermatic fluid, but a minor component of colostrum (Vaerman, Querinjean and Heremans, 197 1; McGuire and Crawford, 1972; Pahud and Mach, 1972). Comparable to findings in other species, the intestinal mucosa of the horse has also
612
s. A.
KHALEEL
et
al.
been identified as a site of local IgA synthesis (Vaerman, 1973). By means of immunofluorescent staining, IgA-containing plasma cells were identified in horse duodenum and proximal jejunum where they were found by Vaerman et al. (1971) to be more abundant than IgM-containing cells. Studies on the in vitro synthesis of immunoglobulins by salivary gland have identified the submandibular gland of the horse as another site of local IgA synthesis. Fluids from tissue cultures of the horse submandibular gland examined by radioimmunoelectrophoresis were found by Hurlim.ann and Darling (1971) to show the selective synthesis of a secretory IgA-like globulin when compared to lymph node cultures.
Immunofluorescent studies on the distribution of specific classes and subclassesof equine immunoglobulin in horse salivary glands and lymph nodes have not been previously reported. In the present investigation, monospecific rabbit antisera prepared against the heavy chains of equine IgG,, IgG,, IgG,, and IgM were used as reagents for the indirect immunok-4 IgB, W(T) fluorescent staining of tissue immunoglobulins. The identification of cells containing specific immunoglobulin and their distribution in horse parotid and submaxillary salivary glands was compared with that obtained for regional parotid and mesenteric lymph nodes as well as duodenum. In addition, tissue extracts prepared from horse salivary glands and lymph nodes were assayed by immunodiffusion to estimate the relative amount of the various immunoglobulins present. MATERIALS
AND
METHODS
Tissues. Parotid and submaxillary salivary glands; submandibular lymph nodes; mesenteric lymph nodes and duodenum were obtained from 7 horses within 5 min. after death. Slices 4 mm. thick were immediately quenched in isopentane chilled with liquid nitrogen or placed into one of several fixatives. Tissue extracts. Soluble tissue extracts were made from frozen tissues stored at -20 “C. Each gram of tissue was homogenized at 4 “C in a Servall tissue homogenizer, with 1 ml. of 0.1 M phosphate buffer pH 7.4, containing 0.9 per cent. sodium chloride. Homogenates were centrifuged at 10 000 g for 2 h. The clear supernatant fluid provided soluble tissue extract for immunodiffusion analysis. Protein concentration of clear extracts was determined by micro-Kjeldahl nitrogen analysis, assuming a nitrogen content of 16 per cent. for proteins. Antisera. The preparation and characterization of monospecific rabbit antisera against horse IgGa, IgG, IgGc, IgB, IgA, IgG(T), IgM and a polyvalent rabbit antiserum against horse (IgG, + Ig& + IgG,) h ave been previously described (Allen and Dalton, 1975 ; Allen and Johnson, 1972; Martineau and Allen, 1973). All antisera employed in immunofluorescent localization were absorbed with horse tissue powders of fascia lata, liver and tendon to remove nonspecific binding components. Tissue powders were prepared by the method of Mellors (1959). Goat antirabbit IgG globulin was obtained from a commercial source (Cappel Laboratories, Downingtown, Penna., U.S.A.). Fl uorescein conjugated goat anti-rabbit 7S-globulin was also purchased from a commercial source (Hyland Division, Travenol Laboratories, Inc., Costa Mesa, California, U.S.A.). Immunodi$usion analysis. Double diffusion in plates was carried out as previously described (Johnston and Allen, 1968). Double diffusion in microtubes by the method of Preer as described by Finger ( 197 I), was used to obtain a relative estimate of the amount of immunoglobulin present in tissue extracts. A single, commercial, pooled lot of normal horse serum (Hyland Laboratories, Los Angeles, California, U.S.A.) was used as a primary reference standard for all immunoglobulins. Serial, two-fold dilutions of reference serum standard or tissue extract were diffused through a 5 mm.
DISTRIBUTION
OF IMMUNOGLOBULINS
IN EQUINE
TISSUES
613
column of agar against 0.02 ml. of each monospecific anti-horse immunoglobulin reagent. The position of each precipitin band relative to the agar column height was measured daily for 5 days. The ratio of the dilutions of extract and serum standard producing a band at comparable positions was used to provide an estimate of the amount of immunoglobulin present in extracts relative to that of reference serum standard. IgM did not penetrate the intermediate agar column beyond 20 per cent. Difficulty was encountered in efforts to assay IgM by this method since the IgM band position did not show significant displacement on doubling dilution and thus quantitation of IgM was not carried out. Tissue extracts were qualitatively tested for the presence of IgM by both plate and Preer methods. Fixation. Post fixation: frozen material was sectioned (3 to 5 pm.) in a cryostat at - 15 “C. and each section was postfixed for 10 min. in one of the following solutions: acetone; 70 per cent. ethanol; 95 per cent. ethanol; 95 per cent. methanol; 95 per cent. methanol and 5 per cent. acetic acid. In addition, one or two sections from each tissue were stained with HE. Liquid fixation: tissues were embedded in paraffin and sectioned at 4 pm. after fixing the sections overnight at 4 “C. in one of the following fixatives: cold acetone; cold 70 per cent. ethanol; cold 95 per cent. ethanol; cold 10 per cent. neutral buffered saline; cold methanol; cold 95 per cent. methanol and 5 per cent. acetic acid and Bouin’s solution. Immunojkorescent staining procedure. Cryostat and paraffin embedded sections were incubated with unconjugated monospecific rabbit anti-equine immunoglobulin reagents in a moist chamber at 37 “C. for 40 min. They were individually rinsed in 0.01 M-phosphate buffered saline (PBS) pH 7.2 3 times (3 min. each wash) with continuous, gentle, agitation. Sections were air dried, incubated with fluorescein isothiocyanate (FITC) conjugated goat anti-rabbit serum in a moist chamber at 37 “C. for 40 min., then washed, air dried as described above and mounted in 90 per cent. glycerine in PBS. Controls. The specificity of staining reactions was controlled by: (1) the determination of the degree of blocking of the reaction with unconjugated goat anti-rabbit serum prior to incubation with conjugated goat anti-rabbit serum; (2) treating sections with normal rabbit serum before incubating with FITC-conjugated reagents; (3) treating sections with a heterologous unrelated antibody (guinea pig anti-hamster immunoglobulin obtained from Cappel Laboratories, Downingtown, Penna., U.S.A.) before staining with FITC-conjugates; (4) treating sections with trypsin or colsections were treated with a lagenase before staining. For collagenase treatment, 1: 10 dilution of 0.1 per cent. collagenase (Sigma Chemical Company, Type III, Fraction A, 500 units/mg. solid) solution in 0.05 M-TES buffer (Tri-(hydroxymethyl) methyl 2-amino ethane sulfonate) with 0.36 mM-CaCl, at pH 7.5 for 3 h. at 37 “C. in a moist chamber. For trypsin treatment, sections were treated with trypsin in TES buffer with O-0115 M-CaCl, at pH 7.6 (9000 BAEE units/mg. powder/ml.) for 3 h. at 5 “C. in a moist chamber. Trypsin, bovine type XI (DCC treated) was obtained from Sigma Chemical Company, St Louis, Missouri, U.S.A.; (5) treating sections of horse tissues directly with FITC goat anti-rabbit Ig as a further control for nonspecific binding. Fluorescence microscopy andphotografihy. Stained preparations were examined with the aid of an immersion dark field condenser on a Leitz Wetzlar microscope using an Osram HBO 200 W light source, a KP 490 interference filter and a K 470 barrier filter. Black and white photographs and colour transparencies were taken on Tri-X panchromatic and high speed Ektachrome (ASA 400, 160) film using an Orthomat automatic camera. RESULTS
Liquid fixation 5 per cent. acetic
followed by paraffin acid in 95 per cent.
embedding, with the exception of methanol gave unsatisfactory results.
614
Fig.
S. A. KHALEEL
1. Equine staining
parotid salivary gland of interstitial connective
et Ul.
section stained for IgG(= + ,, + e), showing immunofluorescent tissue and cytoplasm of interstitial mononuclear cells. x 76.
Fig. 2. Equine parotid salivary gland section stained for IgG(T) showing localization of immunoglobulin in network of interstitial connective tissue spaces and in cytoplasm of a cluster of interstitial cells. X 76. Fig. 3. Equine discrete
parotid plasma
salivary gland cells randomly
stained for IgA. Immunofluorescence scattered through interstitial tissue.
localized x 284.
Fig. 4. Equine parotid salivary gland section stained for IgA showing immunoglobulin cytoplasm of epithelial cells of the duct and in duct contents. x 284.
in cytoplasm
of
localized
in
DISTRIBUTION
OF IMMUNOGLOBULINS
IN EQUINE
TISSUES
615
Histological quality was poor in the majority of such sections and even completely lost in a few sections. Furthermore, fluorescent material leached from its original site and floated under the coverslip. However, fixation with 5 per cent. acetic acid in 95 per cent. methanol provided sections with sharp histological features and stable localization of antigen. Similarly, cryostat sections fixed with 5 per cent. acetic acid in 95 per cent. methanol also gave satisfactory results. Paraffin embedding was preferred, however, since it helped prevent autofluorescence and material could be stored at 4 “C. in the paraffin block for further study.
Salivary Glands The immunohistochemical staining pattern for IgG produced by a polyvalent rabbit anti-horse (IgG,+IgG,+IgG,) reagent was consistent in all sections of horse parotid and submaxillary gland (Fig. 1). Fluorescence was present in the interstitial connective tissue, basement membrane and often within vascular lumina. Cytoplasmic staining for IgG was present in mononuclear cells scattered throughout the interstitium. Neither the duct lumina nor any part of the acinar cells showed specific immunofluorescence. Sections incubated with monospecific antisera against the heavy chain of equine IgG,, IgG,,, or IgG, gave a staining pattern identical to that obtained with polyvalent IgG,,,,,,,. Sections of salivary gland incubated with monospecific anti-IgG(T) gave a staining pattern (Fig. 2), similar to that obtained with anti-IgG reagents; however, IgG (T) immunofluorescence was more diffuse in the connective tissue. Clusters of interstitial cells showing cytoplasmic staining for IgG(T) were seen scattered irregularly throughout the interstitium. Absorption of antisera with powders of horse tendon, liver or fascia lata did not alter the staining pattern. Sections treated with trypsin before exposure to anti-horse IgG or IgG(T), lacked fluorescence in areas previously positive. Treatment with collagenase did not alter immunofluorescent staining. While IgM was found discretely localized in a small number of isolated, individual, oval shaped mononuclear cells (plasma cells) scattered in the interstitium, IgB was not demonstrable by immunofluorescence in any sections ofsalivary gland examined. Sections of salivary gland stained for IgA showed immunofluorescence in individual mononuclear cells (plasma cells) randomly scattered in the interstitium (Fig. 3), lumen of the glandular acini, ducts and epithelial cells (Fig. 4). IgA fluorescence was stronger in cells of the luminal layer in contact with ductular contents while cells of the basal layer were weakly positive.
Lymph .Nodes Sections of parotid lymph node stained for IgG,,,,,,, (Fig. 5) showed strong cytoplasmic fluorescence in cells of, the cortex, cells comprising the core of germinal centres and cells of medullary cords. A large amount of diffuse intercellular IgG fluorescence was also apparent in sections of lymph node. Staining for IgG(T) showed a pattern of distribution somewhat similar to IgG. While small numbers of cells showing cytoplasmic staining for IgG(T) were seen scattered irregularly throughout the cortex, intense IgG(T) staining
s. A. KHALEEL
et
ai.
Fig. 5. Equine parotid lymph node stained for IgG(,+ h + C). Immunofluorescence localized in cells of the germinal centre. x 76. Fig. 6. Equine parotid lymph node stained for IgG(T) showing immunofluorescent staining of cells at the periphery of a germinal centre and in the medullary cords. x 76. Fig. 7. Equine parotid lymph node stained for IgM showing the presence of fluorescent stained cells in the zone of large lymphocytes in the germinal centre and in medullary cords. x 76. Fig. 8. Equine parotid lymph node stained for IgA showing cytoplasmic staining of individual cells randomly scattered in the medullary cords. x 192.
DISTRIBUTION
OF
IMMUNOGLOBULINS
IN
EQUINE
TISSUES
617
was associated with cells located predominantly at the periphery of germinal centres and in the medullary cords (Fig. 6). Parotid lymph node stained for IgM showed the presence of immunofluorescent stained lymphoid cells in the zone of large lymphocytes of germinal
Fig.
9.
Fig.
10.
Fig.
Il.
Fig.
12.
Fig.
13.
Fig.
14.
showing immunofluorescent localization in the surface Equine duodenum stained for IgG,, mucin at the tips of villi. x 144. Equine duodenum stained for IgG, showing localization in lymphoid cells of the lamina prbpria and in the glands. x 57. Equine duodenum stained for IgG(T) showing cytoplasmic staining of individual lymphoid cells scattered in the lamina propria. x 144. Equine duodenum stained for IgM showing immunofluorescent localization in lymphoid cells of the lamina propria and in the apical portion of epithelial cells lining a crypt. x 144. Equine duodenum stained for IgA showing fluorescent staining of the cytoplasm of crypt. epithelium and plasma cells scattered throughout the lamina propria. x 144. Equine duodenum stained for IgA showing fluorescent staining of lymphoid cells at the periphery of a lymphoid nodule. x 144.
618
S. A. KHALEEL
et Ul.
centres as well as in medullary cords (Fig. 7). Although a few single, isolated cells staining for IgB were seen in the cortical areas of parotid and mesenteric lymph nodes, these cells were very few and rarely encountered in sections. Immunofluorescent staining of parotid lymph node for IgA, showed a distinctive pattern of distribution (Fig. 8). Cytoplasmic IgA fluorescence was confined to individual cells scattered in the cortex between and around germinal centres extending into the medullary cords, but was conspicuously absent from germinal centres. Mesenteric lymph node stained for IgG,,,b,,,, IgG(T), IgB, IgA and IgM gave distribution patterns comparable to those described for parotid lymph node.
Sections of horse duodenum stained with monospecific anti-IgG,, anti-IgG,, or anti-IgG, showed strong immunofluorescence associated with the mucin coating the luminal surface of villous epithelia (Fig. 9). Lymphoid cells with TABLE RELATIVE
AMOUNTS
OF IMMUNOGLOBULINS
Relative
Total
Horse no.
Tissue extract
1
Parotid gland Submaxillary gland Parotid gland Submaxillary gland Parotid gland Lymph node (parotid) Parotid gland Submaxillary gland Lymph node (mesenteric) Submaxillary gland Lymph node (mesenteric) Parotid gland Submaxillary gland Parotid gland Lymph node (mesenteric) Lacrimal gland
2 3 4
5 6 7
1
protein mg./ml.
57
~ IgCs
I.%
DETECTED
IN HORSE
concentration 4G
TISSUE
EXTRACTS
of immunoglobulin* Id
W(T)
--w
Zghi
0.031 0.063 0.03 1 0.008 0.016
0.016 0.03 1 0.03 1 0.008 0.031
0,016 0.016 0.016 0.006 0.031
0.500 0.125 0.125 0.250 0.016
0.007 0.007 0.060 0908 0.031
0.030 0.03 1 0,016
0.016 0.03 1 0.016
0.008 0.016 0.016
0.016 0.031 0.500
0.008 0.016 0.004
ND
