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Differential enhancement and distribution of antigen-specific cells in various lymph nodes in response to locally inoculated bacterial antigens
Veterinary imtnunology and lmmunopathology, 29 ( 1991 )239-250
239
Elsevier Science Publishers B.V., Amsterdam
Differential enhancement and distribution of antigen-specific cells in various lymph nodes in response to locally inoculated bacterial antigens Mark J. Dobrzanski and Tsu-Ju (Thomas) Yang Department of Pathobiology, The University of Connecticut, Storrs, CT 06269-3089, USA (Accepted 9 October 1990)
ABSTRACT Dobrzanski, M.J. and Yang, T.J., 1991. Differential enhancement and distribution of antigen-specific cells in various lymph nodes in response to locally inoculated bacterial antigens. Vet. hnmunol. ImmunopathoL, 29: 239-250. The proliferation responses of antigen-specific lymphocytes from various anatomical sites were studied in dairy goats locally immunized with heat-killed Staphylococcus aureus (HKS). Animals were inoculated three times subcutaneously in the fight udder with HKS at l month intervals. One week following the last inoculation, prescapular, mesenteric and ipsilateral ~draining) and contralateral (non-draining) supramammary lymph nodes were collected and the cells assayed in 3- and 6-day cultures to determine the immune proliferative responses of antigen-specific lymphocytes to HKS and the polyclonal T cell mitogen phytohemagglutinin (PHA). The cells from draining and non-draining supramammary lymph nodes responded to HKS in 3-day cultures. Peripheral lymph nodes, such as the prescapular, showed similar responses. In contrast, mesenteric lymph nodes responded optimally in 6-day cultures, notably to lower concentrations of the antigen. Cells from all lymph nodes tested showed increased responses to PHA in immunized animals, although non-draining lymph nodes demonstrated a greater response to the T cell mitogen than those of draining lymph nodes. These results suggest that unilateral introduction of Staphylococcus cell antigens to the supramammary region can induce an anamnestic response in ipsilateral as well as contralateral supramammary lymph nodes and other distant peripheral lymphoid organs. Furthermore, these data indicate that cells from intestinal lymph nodes respond differently from those of peripheral lymph nodes, suggesting the presence of a unique gastrointestinal lymphoid cell circulation in goats. Concomitant peripheral responses may be attributed to memory ceil migration or to antigen leakage and relocation to distant sites from the inoculated region. Analysis with PHA suggests a difference in general responsiveness and perhaps, /mmunocompetence, by lymphocyte populations in various lymphoid tissues of immunized animals. ABBREVIATIONS
CPM, counts per minute; HBSS, Hanks' balanced salt solution; HKS, heat-killed Staphylococcus aureus; PHA, phytohemagglutinin.
0165-2427/91/$03.50 © 1991 Elsevier Science Publishers B.V. All fights reserved.
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M.J. DOBRZANSKI AND T.J. YANG
INTRODUCTION
Studies on the immune response in mammary tissues and the regional lymph nodes are important with respect to mucosal immunity in general and specific diseases like mastiffs. Several components of causative agents such as those of the Gram-positive bacteria, Staphylococcus aureus, have been shown to possess stimulating or suppressing properties in lymphocytes (Dziarski and Dziarski, 1979; Pryjma et al., 1986). Inoculation of mice with hea~-killed S. aureus (HKS) induces delayed-type hypersensitivity reactions (Easmon and Glynn, 1978); however, the effects of locally administered antigen on immune cell reaction and their migration to distant sites remains unclear (Husband, 1988). Lymphocytes have been shown to recirculate preferentially through either peripheral or mucosal tissues (Cahill et al., 1977; Hall et al., 1977; Chin and Hay, 1980) and thus, allow efficient mobilization of immune responses (Ford, 1975). Many investigators have observed antigen-mediated recruitment of both B and T cells from the circulation into lymphoid tissues draining the site of antigen injection (Sprent and Miller, 1976; Baine et al., 1981 ). Persisting antigen in lymph nodes has been thought to mediate a greater accumulation of antigen-specific B cells where local interaction with T cells may assist in causing memory cell proliferation and differentiation (Ponzio et al., 1977). Cells and antigens released subsequently by primed lymph nodes during immune responses have been shown to influence immune reactions at sites remote from the stimulated lymph node (Smith et al., 1970). Thus, evaluation a f anti~an-~n~cific,,__ -r ~I v m - - nr -h-a-c~v t a.~ . r e. ~.p n. n.~ i v a n e ~
a t ~ite~ p r o x i m a l
and distal to
local antigen challenge is critical to understanding the role of the immune response to local infection by microbes such as S. aureus. The present study was designed to assess the antigen-specific and phytohemagglutinin (PHA) proliferative responses in 3- and 6-day cultures of resident lymph node cells from various anatomical sites of dairy goats locally immunized in the right supramammary region with HKS. MATERIAL AND METHODS
A,~imals
Twenty-three (13 female, 10 male) Alpine and Saanen goats and their crosses, ranging in ages from 4 months to 3 years, were used in this study. They were bred and maintained at the University of C o i ~ t i c a t Spring H.ill Animal Research Facility according to standard animal care guidelines. Bacterial antigen Staphylococcus aureus Cowan I was cultured in trypticase soy broth (Difco
Laboratories, Detroit, MI) at 37°C for 18 h on a rotary shaker. The cells were
DISTRIBUTION AND Ag SPECIFIC REACTIVITY OF LYMPHOID CELLS
241
collected by centrifugation at 2000×g for l0 rain, washed twice in Hanks' balanced salt solution (HBSS), resuspended, submerged in an 80°C waterbath for 15 rain, and then cooled. Stock cell suspensions of the HKS ( l × l 0 s colony forming units/ml) for immunization and in vitro mitogenic assays were adjusted spectrophotometrically (Spec 20-Bausch and Lomb) at an optical density of 550 nm and stored at - 2 0 ° C (Goding, 1978 ).
Immunization Nineteen goats ( 11 female, 8 male) were inoculated subcutaneously into the right mammary region 10-15 mm lateral to the teat with 8-8.5 × l 0 s colony forming units of HKS in l ml of HBSS. Injections were made three times at monthly intervals and tissues were harvested 7 days after the final injection. Four normal non-immunized goats (two female, two male) served as controls.
Lymph node cells Following euthanasia (T-61 National Laboratories Corp., Somerville, NJ) and exsanguination by cardiac puncture, prescapular, mesenteric, right (ipsilateral) and left (contralateral) supramammary lymph nodes were collected, weighed and minced in HBSS (pH 7.2). After three washes in HBSS, lymphoid cells were resuspended in RPMI 1640 (GIBCO, Grand Island, NY ), supplemented with 2 mM glutamine, l mM pyruvatz~ ! 00 units/ml penicillin, 100/zg/ml streptomycin, 10 mM HEPES, and 10% heat-inactivated fetal calf serum (Hy-Clone Sterile System, Inc., Logan, UT) to attain a cell concentration of I X ! 06 viable cells/m_!:
Spleen cells Following extirpation, spleens were weighed and minced in HBSS (pH 7.2). The cells were washed in HBSS and erythrocytes lysed by hypotonic shock (Tris-buffered NH4CI (0.16 M) ) for l rain. Remaining cells were subjected to Ficoll-Diatrizoate gradient centdfugation (d= 1.084 g / c c , Ficoll type 400, Sigma Chemical Company, St. Louis, MO; Diatrizoate-sodium, Stealing Drug Company, New York, NY) as described by Yang and Rabinovsky (1987). The mononuclear cells in the interphase were recovered, washed three times with HBSS, and resuspended in RPMI 1640 containing 10% fetal calf serum to attain a cell concentration of I × 106 viable cells/ml.
Lymphocyte proliferation assay Mononuclear cells ( 1 × 106 cells/ml) were cultured in 96-well flat-bottomed microtiter plates (Coming Laboratory Science Co., Coming, NY). Triplicate cultures were run using 0. l ml of cell suspension plus 0. l ml of medium, or medium containing serial dilutions of HKS or phytohemagglutinin-P (PHA-P) (Difco). 'The HKS and PHA-P doses used ranged from 0.4
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M.J. DOBRZANSKI AND T.J. YANG
to 100 ng/ml and 5.3 to 340 g/ml, respectively. Incubation with HKS was for 3 days and 6 days at 37°C in a humidified atmosphere at 5% CO2 to determine the immune responses of antigen-primed cells (Rose et al., 1986). Eighteen hours before harvest, each well received 1 #Ci oftritiated thymidine (specific activity 20-40 Ci/mmol; ICN Radiochemicals, Inc., Irvine, CA) in 50/d of RPMI 1640 medium. Cultures were harvested with a Multiple Automated Sample Harvester (Brandel Inc., Gaithersburg, MD). The glass fiber filter strips were air dried and individual disks were placed in scintillation vials with 5 ml of toluene scintillation fluid containing PPO and POPOP. The samples were counted in a Beckman LS3801 liquid scintillation spectrometer (Beckman Instruments Inc., Fullerton, CA) and results expressed as the mean_+ standard error of triplicate cultures.
Tissue sections Portions of lymph nodes and supramammary tissues were fixed in 10% formalin-buffered saline, sectioned, and stained with hematoxylin and eosin.
Statistical analyses The difference in counts per minute (Delta CPM) was calculated by subtracting the mean CPM ofunstimulated cultures from the mean CPM of HKS or PHA stimulated cultures. For statistical anal)sis the one-tailed Student's ttest w a s used. RESULTS
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Lymph node cells from 19 immunized ( 11 female, 8 male) and four control (two female, two male) goats were cultured with HKS for 3 days and 6 days to assess secondary antigen responsiveness. Three day cultures with PHA served as a monitor of general responsiveness and culture conditions. Cells from draining (right) and non-draining (left) supramammary lymph nodes of antigen-injected animals showed a greater response in 3-day than in 6-day cultures, at all concentrations of HKS antigen used (Fig. 1 ). As shown in Fig. 2, cells from ipsilateral lymph nodes exhibited higher reactivity than those of contralateral lymph nodes at nearly all doses of HKS after 3 days in culture. Optimal responses for both lymph nodes were evident at antigen concentrations of 12.5, 25 and 50 ng/ml. Analogous lymph nodes from non-immunized control goats showed negligible responses to antigen exposure in both 3- and 6-day cultures (Figs. 1 and 2). Figure 3 shows proliferative responses of ipsilateral and contralateral supramammary lymph node cells to the polyclonal T cell mitogen, PHA. Although dose-response profiles were similar, cells from contralateral lymph
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nodes responded better to graded concentrations of the mitogen than those from ipsilateral lymph nodes in 3-day cultures. Supramammary lymph nodes from the control group showed less response to PHA in 3-day cultures. Draining lymph nodes were hyperplastic and weighed four times the amount of non-draining nodes (6.08 _+ 1.41 versus 1.46 +_0.21 g, P<0.002) and the ab-
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solute cell counts of draining lymph nodes were seven times more than those of non-draining lymph nodes (4.3 X 10 9 versus 6.4 X 10 s cells).
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Proliferation responses of distant lymphoid organs to HKS and PHA Spleen, prescapular and mesentedc lymph nodes from immunized and control goats were assayed as described above. As shown in Fig. 4, prescapular lymph node cells responded better to HKS in 3-day than in 6-day cultures
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with peak responses of the former nearly seven-fold greater and at higher antigen concentrations (25 and 50 ng/ml). In contrast, mesenteric lymph node cells demonstrated peak responses for all cultures at lower antigen concentrations of 1.6 ng/ml HKS with nearly a five-fold greater activity in 6-day than
DISTRIBUTION AND Ag SPECIFIC REACTIVITY OF LYMPHOID CELLS
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in 3-day responses (Fig. 5 ). Cells from both mesenteric and prescapular lymph nodes of non-immunized animals exhibited negligible proliferative activity in both 3- and 6-day cultures (Figs. 4 and 5 ). Spleen cells from both immunized and control goats responded to HKS antigen in 6-day and 3-day cultures, however, the magnitude of activity in the immunized group was significantly higher than that of controls in respective cultures (Fig. 6 ). Figure 7 shows that mesenteric lymph nodes from immunized goats had an enhanced responsiveness to PHA in 3-day cultures. There were two- to threefold increases in activity over that of control animals at PHA concentrations of 10.5-340 gg/ml. Similarly, the PHA responses of spleen and prescapular lymph node cells demonstrated greater activity at 3 days when compared with analogous cells from control goats (data not shown). DISCUSSION
This study was designed to evaluate the induction and/or recruitment of specific memory cells to sites proximal and distal to local injection of antigen. Subcutaneous inoculation of HKS antigen into the fight udder of dairy goats induced specific memory cell responses in both ipsilateral and contralateral supramammary lymph nodes. Although cells from both supramammary lymph nodes exhibited secondary immune responses to HKS, the intensity of the responses from draining nodes was greater. The data demonstrated this in two ways: ( 1 ) hyperplasia of the draining node as evidenced by increases in organ weight and cell number; and (2) heightened proliferative activity of these cells to the HKS antigen. The higher responsiveness in draining nodes may be because of the presence of persisting antigen which has been shown to m d~aiuiu~ induce immunologic memory to resident and c]rcmaung -" . . . . . . -" ccm __,1_-_ lymph nodes (Ponzio et al., 1977). In contrast, the responsiveness of cells from contralateral nodes may be attributed to memory cell migration from ipsilateral nodes as suggested by other investigators (Smith et al., 1970; Sprent et al., 1971; Sprent, 1973; Strober and Dilly, 1973) or by antigen leakage and relocation to distant sites where specific cell responses could be induced in situ. Memory cell activity was clearly present in the periphery as demonstrated by the responsiveness of spleen and prescapular and contra!ateral supramammary lymph nodes to HKS in 3-day cultures. All peripheral lymphoid tissues demonstrated increased responses with peak activity at or near 25 ng/ml of HKS antigen in culture. In contrast, cells from mesenteric lymph nodes showed heightened responses to HKS at lower concentrations of antigen challenge ( 1.6 ng/ml) in 6-day and to a lesser degree in 3-day cultures. This finding suggests that antigen spill-over into the circulation and the number of memory cells migrating from the inoculated area to the visceral lymph nodes is rather limited, requiting increased time in culture for adequate manifestation
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M.J. DOBRZANSKI AND T.J. YANG
of activity. Although we are not in a position either to establish or dispute the existence of a gastrointestinal-peripheral (mammary gland) recirculating axis in response to local antigen challenge (Hanson et al., 1979; Jeurissen et al., 1985), our findings on the limited memory cell homing from the supramammary region to the mesenteric lymph nodes suggests the presence of functionally separate peripheral and intestinal lymphoid cell circulations (Cahill et al., 1977; Chin and Hay, 1980). Analysis of lymph node cell responses to the polyclonal T cell mitogen, PHA, shows that cells from both peripheral and mesenteric lymph nodes of immunized animals had increased reactivity to PHA than those from analogous nodes in non-immunized animals, suggesting that T cells in general are hyperresponsive as a result of the HKS inoculation. However, cells from the draining supramammary lymph nodes were less responsive to PHA when compared with those of contralateral lymph nodes. This lower response may be caused by suppressor effects by antigen activated cells (Easmon and Glynn, 1978; Yamamoto et al., 1985; Cox and Kennell, 1988) or to inhibition of previously activated cells by PHA in culture (Yang, 1975; Farkas et al., 1986). Alternatively, it is possible that in response to local infection, select lymphocyte populations are attracted (Wood and Neff, 1978; Baine et al., 1981 ) or proliferate (Reichert et al., 1983) causing changes in distribution patterns of T and B lymphocytes in lymph nodes draining these sites and in the periphery (Yang et al., 1980, 1988), suggesting that quantitative and/or qualitative differences in responding lymphocyte populations, as indicated here by stimulation with PHA, may influence immune cell responses to HKS at sites proximal and distal to local antigen deposition. in conclusion, these studies demonstrate that unilateral introduction of S. a u r e u s cell antigens to the supramammary region can induce an antigen-specific anamnestic response accompanied by a heightened general responsiveness in ipsilateral as well as contralateral supramammary lymph nodes and other distant peripheral lymphoid organs. In contrast, lymphocytes from mesenteric lymph nodes responded differently from those of peripheral lymph nodes suggesting the presence of a unique gastrointestinal lymphoid cell circulation (Reynolds et al., 1988 ). Experiments are currently underway to define whether this differential enhancement and distribution of antigen-specific reactivity functions in a microbiostatic and/or prophylactic capacity to such bacterial components. ACKNOWLEDGMENTS
This work was supported by the United States Department of Agriculture Animal Health Grant No. 86-CRSR-2-2895 and the Storrs (Connecticut) Agricultural Experiment Station Project CONS 00625, and is submitted as Sci-
DISTRIBUTION AND Ag SPECIFIC REACTIVITY OF LYMPHOID CELLS
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entific Contribution No. 1260, Storrs Agricultural Experiment Station, University of Connecticut, Storrs, CT 06269.
REFERENCES Baine, Y., Ponzio, N.M. and Thorbecke, S.J., 1981. Transfer of memory cells into antigen-pretreated hosts. II. Influence of localized antigen on the migration of specific memory B cells. Eur. J. Immunol., 11: 990-996. Cahill, R.N.P., Poskitt, D.C., Fr~,zt, H. and Trnka, Z., 1977. Two distinct pools of recirculating T lymphocytes: Migratory characteristics of nodal and intestinal T lymphocytes. J. Exp. Med., 145: 420-429. Chin, W. and Hay, J.B., 1980. A comparison of lymphocyte migration through intestinal lymph nodes, subcutaneous lymph nodes, and chronic inflammatory sites of sheep. Gastroenterology, 79: 1231-1242. Cox, R.A. and Kennell, W., 1988. Suppression of T-lymphocyte response by Coccidioides immitis antigen. Infect. Immun., 56: 1423-1429. Dziarski, R. and Dziarski, A., 1979. Mitogenic activity of staphylococcal peptidoglycan. Infect. Immun., 23: 706-710. Easmon, C.S.F. and Glynn, A.A., 1978. Role of Staphylococcus aureus cell wall antigens in the stimulation of delayed type hypersensitivity after Staphylococcus infection. Infect. Immun., 19:341-342. Farkas, R., Manor, Y. and Klajman, A., 1986. Generation of B suppressor cells by phytohaemagglutinin. Immunology, 57: 395-401. Ford, W.L., 1975. Lymphocyte migration and immune responses. Prog. Allergy, 19: 1-59. Goding, J.W., 1978. Use of staphylococcal protein A as an immunological reagent. J. Immunol. Meths, 20:241-253. Ha!!~JoG, Hopkins, J. and Orlans, E., 1977. Studies on the lymphocytes of sheep. Ill. Destination of lymph-borne immunoblasts in relation to their tissue of origin. Eur. J. Immunol., 7: 30-37. Hanson, L.A., Carlsson, B., Cruz, J.R., Garcia, B., Holmgren, J., Kahn, S.R., Lindblad, B.S., Svennerholm, A.M., Svennerholm, B. and Urrutia, J., 1979. Immune response in the mammary gland. In: P.L. Ogra and D.H. Dayton (Editors), Immunology of Breast Milk. Raven Press, New York, NY, pp. 145-157. Husband, A.J., 1988. Migration and Homing of Lymphoid Cells. Vols. I and II. CRC Press, Boca Raton, FL. Jeurissen, S.H.M., Claassen, E., van Rooijen, N. and Kraal, G., 1985. Intraintestinal priming leads to antigen-specific IgA memory cells in peripheral lymphoid organs. Immunology, 56: 417-423. Ponzio, N.M., Chapman-Alexander, J.M. and Thorbecke, G.J., 1977. Transfer of memory cells into antigen-pretreated hosts. I. Functional detection of migration sites for antigen-specific B cells. Cell. Immunol., 34: 79-92. Pryjma, J., Pituch-Noworolska, A., Flad, H.D., Ulmer, A.J. and Ernst, M., 1986 Suppression of Staphylococcus aureus Cowan I-induced immunoglobulin synthesis in vitro: discrimination between the presence of suppressor T cell precursors and effectors. Clin. Exp. Immunol., 66: 348-357. Reichert, R.A., Gallatin, W.M., Weissman, I.L. and Butcher, E.C., 1983. Germinal center B cells lacking homing receptors necessary for normal lymphocyte recirculation. J. Exp. Med., 157:813-827.
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Reynolds, J.D., Chin, W. and Shmoorkoff, J., 1988. T and B cells have similar recirculation kinetics in sheep. Eur. J. Immunol., 18: 835-840. Rose, N.R., Friedman, H. and Fahey, J.L., 1986. Manual of Clinical Laboratory Immunology. American Society for Microbiology, Washington, D.C., pp. 260-273. Smith, J.B., Cunningham, A.J., Lafferty, K.J. and Morris, B., 1970. The role of the lymphatic system and lymphoid cells in the establishment of immunological memory. Aust. J. Exp. Med. Sci., 48: 57-70. Sprent, J., 1973. Circulating T and B lymphocytes of the mouse. I. Migratory properties. Ceil. lmmunol., 7. 10-39. Sprent, J. and Miller, J.F.A.P., 1976. Effects of antigen priming on adoptive immune responses. Ill. Antigen-induced selective recruitment of subsets of circulating lymphocytes reactive to H-2 determinants. J. Exp. Med., 143: 585-600. Sprent, J., Miller, J.F.A. and Mitchell, G.F., 1971. Antigen-induced selective recruitment of circulating lymphocytes. Cell. Immunol., 2:171-181. Strober, S. and Dilly, J., 1973. Maturation of B lymphocytes in rats. I. Migration pattern, tissue distribution, and turnover rate of unprimed and primed B lymphocytes involved in the adoptive antidinitrophenyl response. J. Exp. Med., 138:1331-1344. Wood, G.W. and Neff, J.R., 1978. A re-evaluation of B lymphocyte levels in peripheral blood from cancer patients. J. Natl. Cancer Inst., 61: 715-718. Yamamoto, K., Kato, K. and Kimur, T., 1985. Killed Listeria-induced suppressor T cells involved in suppression of delayed-type hypersensitivity and protection against Listeria infection. Immunology, 55:609-619. Yang, T.J., 1975. Inhibitory effects ofphytohemagglutinin on growth of leukemia lymphoblasts L5178Y in vitro and in vivo. J. Natl. Cancer Inst., 55: 323-327. Yang, T.J. and Rabinovsky, E.D., 1987. Separation and identification of bovine lymphocyte populations. Vet. Immunol. Immunopathol., 14: 77-84. Yang, T.J., Jantzen Abo, P.A. and Williams, L.F., 1980. Depression of B-lymphocyte levels in the peripheral blood of cows with mastitis. Infect. Immun., 27: 90-93. Yang, T.J., Mather, J.F. and Rabinovsky, E.D., 1988. Changes in sub-populations of lymphocytes in peripheral blood, supramammary and prescapular lymph nodes of..~ows with mastitis and normal cows. Vet. Immunol. Immunopathol.~ 18: 279-285.
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Elsevier Science Publishers B.V., Amsterdam
Differential enhancement and distribution of antigen-specific cells in various lymph nodes in response to locally inoculated bacterial antigens Mark J. Dobrzanski and Tsu-Ju (Thomas) Yang Department of Pathobiology, The University of Connecticut, Storrs, CT 06269-3089, USA (Accepted 9 October 1990)
ABSTRACT Dobrzanski, M.J. and Yang, T.J., 1991. Differential enhancement and distribution of antigen-specific cells in various lymph nodes in response to locally inoculated bacterial antigens. Vet. hnmunol. ImmunopathoL, 29: 239-250. The proliferation responses of antigen-specific lymphocytes from various anatomical sites were studied in dairy goats locally immunized with heat-killed Staphylococcus aureus (HKS). Animals were inoculated three times subcutaneously in the fight udder with HKS at l month intervals. One week following the last inoculation, prescapular, mesenteric and ipsilateral ~draining) and contralateral (non-draining) supramammary lymph nodes were collected and the cells assayed in 3- and 6-day cultures to determine the immune proliferative responses of antigen-specific lymphocytes to HKS and the polyclonal T cell mitogen phytohemagglutinin (PHA). The cells from draining and non-draining supramammary lymph nodes responded to HKS in 3-day cultures. Peripheral lymph nodes, such as the prescapular, showed similar responses. In contrast, mesenteric lymph nodes responded optimally in 6-day cultures, notably to lower concentrations of the antigen. Cells from all lymph nodes tested showed increased responses to PHA in immunized animals, although non-draining lymph nodes demonstrated a greater response to the T cell mitogen than those of draining lymph nodes. These results suggest that unilateral introduction of Staphylococcus cell antigens to the supramammary region can induce an anamnestic response in ipsilateral as well as contralateral supramammary lymph nodes and other distant peripheral lymphoid organs. Furthermore, these data indicate that cells from intestinal lymph nodes respond differently from those of peripheral lymph nodes, suggesting the presence of a unique gastrointestinal lymphoid cell circulation in goats. Concomitant peripheral responses may be attributed to memory ceil migration or to antigen leakage and relocation to distant sites from the inoculated region. Analysis with PHA suggests a difference in general responsiveness and perhaps, /mmunocompetence, by lymphocyte populations in various lymphoid tissues of immunized animals. ABBREVIATIONS
CPM, counts per minute; HBSS, Hanks' balanced salt solution; HKS, heat-killed Staphylococcus aureus; PHA, phytohemagglutinin.
0165-2427/91/$03.50 © 1991 Elsevier Science Publishers B.V. All fights reserved.
240
M.J. DOBRZANSKI AND T.J. YANG
INTRODUCTION
Studies on the immune response in mammary tissues and the regional lymph nodes are important with respect to mucosal immunity in general and specific diseases like mastiffs. Several components of causative agents such as those of the Gram-positive bacteria, Staphylococcus aureus, have been shown to possess stimulating or suppressing properties in lymphocytes (Dziarski and Dziarski, 1979; Pryjma et al., 1986). Inoculation of mice with hea~-killed S. aureus (HKS) induces delayed-type hypersensitivity reactions (Easmon and Glynn, 1978); however, the effects of locally administered antigen on immune cell reaction and their migration to distant sites remains unclear (Husband, 1988). Lymphocytes have been shown to recirculate preferentially through either peripheral or mucosal tissues (Cahill et al., 1977; Hall et al., 1977; Chin and Hay, 1980) and thus, allow efficient mobilization of immune responses (Ford, 1975). Many investigators have observed antigen-mediated recruitment of both B and T cells from the circulation into lymphoid tissues draining the site of antigen injection (Sprent and Miller, 1976; Baine et al., 1981 ). Persisting antigen in lymph nodes has been thought to mediate a greater accumulation of antigen-specific B cells where local interaction with T cells may assist in causing memory cell proliferation and differentiation (Ponzio et al., 1977). Cells and antigens released subsequently by primed lymph nodes during immune responses have been shown to influence immune reactions at sites remote from the stimulated lymph node (Smith et al., 1970). Thus, evaluation a f anti~an-~n~cific,,__ -r ~I v m - - nr -h-a-c~v t a.~ . r e. ~.p n. n.~ i v a n e ~
a t ~ite~ p r o x i m a l
and distal to
local antigen challenge is critical to understanding the role of the immune response to local infection by microbes such as S. aureus. The present study was designed to assess the antigen-specific and phytohemagglutinin (PHA) proliferative responses in 3- and 6-day cultures of resident lymph node cells from various anatomical sites of dairy goats locally immunized in the right supramammary region with HKS. MATERIAL AND METHODS
A,~imals
Twenty-three (13 female, 10 male) Alpine and Saanen goats and their crosses, ranging in ages from 4 months to 3 years, were used in this study. They were bred and maintained at the University of C o i ~ t i c a t Spring H.ill Animal Research Facility according to standard animal care guidelines. Bacterial antigen Staphylococcus aureus Cowan I was cultured in trypticase soy broth (Difco
Laboratories, Detroit, MI) at 37°C for 18 h on a rotary shaker. The cells were
DISTRIBUTION AND Ag SPECIFIC REACTIVITY OF LYMPHOID CELLS
241
collected by centrifugation at 2000×g for l0 rain, washed twice in Hanks' balanced salt solution (HBSS), resuspended, submerged in an 80°C waterbath for 15 rain, and then cooled. Stock cell suspensions of the HKS ( l × l 0 s colony forming units/ml) for immunization and in vitro mitogenic assays were adjusted spectrophotometrically (Spec 20-Bausch and Lomb) at an optical density of 550 nm and stored at - 2 0 ° C (Goding, 1978 ).
Immunization Nineteen goats ( 11 female, 8 male) were inoculated subcutaneously into the right mammary region 10-15 mm lateral to the teat with 8-8.5 × l 0 s colony forming units of HKS in l ml of HBSS. Injections were made three times at monthly intervals and tissues were harvested 7 days after the final injection. Four normal non-immunized goats (two female, two male) served as controls.
Lymph node cells Following euthanasia (T-61 National Laboratories Corp., Somerville, NJ) and exsanguination by cardiac puncture, prescapular, mesenteric, right (ipsilateral) and left (contralateral) supramammary lymph nodes were collected, weighed and minced in HBSS (pH 7.2). After three washes in HBSS, lymphoid cells were resuspended in RPMI 1640 (GIBCO, Grand Island, NY ), supplemented with 2 mM glutamine, l mM pyruvatz~ ! 00 units/ml penicillin, 100/zg/ml streptomycin, 10 mM HEPES, and 10% heat-inactivated fetal calf serum (Hy-Clone Sterile System, Inc., Logan, UT) to attain a cell concentration of I X ! 06 viable cells/m_!:
Spleen cells Following extirpation, spleens were weighed and minced in HBSS (pH 7.2). The cells were washed in HBSS and erythrocytes lysed by hypotonic shock (Tris-buffered NH4CI (0.16 M) ) for l rain. Remaining cells were subjected to Ficoll-Diatrizoate gradient centdfugation (d= 1.084 g / c c , Ficoll type 400, Sigma Chemical Company, St. Louis, MO; Diatrizoate-sodium, Stealing Drug Company, New York, NY) as described by Yang and Rabinovsky (1987). The mononuclear cells in the interphase were recovered, washed three times with HBSS, and resuspended in RPMI 1640 containing 10% fetal calf serum to attain a cell concentration of I × 106 viable cells/ml.
Lymphocyte proliferation assay Mononuclear cells ( 1 × 106 cells/ml) were cultured in 96-well flat-bottomed microtiter plates (Coming Laboratory Science Co., Coming, NY). Triplicate cultures were run using 0. l ml of cell suspension plus 0. l ml of medium, or medium containing serial dilutions of HKS or phytohemagglutinin-P (PHA-P) (Difco). 'The HKS and PHA-P doses used ranged from 0.4
242
M.J. DOBRZANSKI AND T.J. YANG
to 100 ng/ml and 5.3 to 340 g/ml, respectively. Incubation with HKS was for 3 days and 6 days at 37°C in a humidified atmosphere at 5% CO2 to determine the immune responses of antigen-primed cells (Rose et al., 1986). Eighteen hours before harvest, each well received 1 #Ci oftritiated thymidine (specific activity 20-40 Ci/mmol; ICN Radiochemicals, Inc., Irvine, CA) in 50/d of RPMI 1640 medium. Cultures were harvested with a Multiple Automated Sample Harvester (Brandel Inc., Gaithersburg, MD). The glass fiber filter strips were air dried and individual disks were placed in scintillation vials with 5 ml of toluene scintillation fluid containing PPO and POPOP. The samples were counted in a Beckman LS3801 liquid scintillation spectrometer (Beckman Instruments Inc., Fullerton, CA) and results expressed as the mean_+ standard error of triplicate cultures.
Tissue sections Portions of lymph nodes and supramammary tissues were fixed in 10% formalin-buffered saline, sectioned, and stained with hematoxylin and eosin.
Statistical analyses The difference in counts per minute (Delta CPM) was calculated by subtracting the mean CPM ofunstimulated cultures from the mean CPM of HKS or PHA stimulated cultures. For statistical anal)sis the one-tailed Student's ttest w a s used. RESULTS
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Lymph node cells from 19 immunized ( 11 female, 8 male) and four control (two female, two male) goats were cultured with HKS for 3 days and 6 days to assess secondary antigen responsiveness. Three day cultures with PHA served as a monitor of general responsiveness and culture conditions. Cells from draining (right) and non-draining (left) supramammary lymph nodes of antigen-injected animals showed a greater response in 3-day than in 6-day cultures, at all concentrations of HKS antigen used (Fig. 1 ). As shown in Fig. 2, cells from ipsilateral lymph nodes exhibited higher reactivity than those of contralateral lymph nodes at nearly all doses of HKS after 3 days in culture. Optimal responses for both lymph nodes were evident at antigen concentrations of 12.5, 25 and 50 ng/ml. Analogous lymph nodes from non-immunized control goats showed negligible responses to antigen exposure in both 3- and 6-day cultures (Figs. 1 and 2). Figure 3 shows proliferative responses of ipsilateral and contralateral supramammary lymph node cells to the polyclonal T cell mitogen, PHA. Although dose-response profiles were similar, cells from contralateral lymph
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nodes responded better to graded concentrations of the mitogen than those from ipsilateral lymph nodes in 3-day cultures. Supramammary lymph nodes from the control group showed less response to PHA in 3-day cultures. Draining lymph nodes were hyperplastic and weighed four times the amount of non-draining nodes (6.08 _+ 1.41 versus 1.46 +_0.21 g, P<0.002) and the ab-
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solute cell counts of draining lymph nodes were seven times more than those of non-draining lymph nodes (4.3 X 10 9 versus 6.4 X 10 s cells).
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Proliferation responses of distant lymphoid organs to HKS and PHA Spleen, prescapular and mesentedc lymph nodes from immunized and control goats were assayed as described above. As shown in Fig. 4, prescapular lymph node cells responded better to HKS in 3-day than in 6-day cultures
246
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with peak responses of the former nearly seven-fold greater and at higher antigen concentrations (25 and 50 ng/ml). In contrast, mesenteric lymph node cells demonstrated peak responses for all cultures at lower antigen concentrations of 1.6 ng/ml HKS with nearly a five-fold greater activity in 6-day than
DISTRIBUTION AND Ag SPECIFIC REACTIVITY OF LYMPHOID CELLS
L.,'),~-7,
in 3-day responses (Fig. 5 ). Cells from both mesenteric and prescapular lymph nodes of non-immunized animals exhibited negligible proliferative activity in both 3- and 6-day cultures (Figs. 4 and 5 ). Spleen cells from both immunized and control goats responded to HKS antigen in 6-day and 3-day cultures, however, the magnitude of activity in the immunized group was significantly higher than that of controls in respective cultures (Fig. 6 ). Figure 7 shows that mesenteric lymph nodes from immunized goats had an enhanced responsiveness to PHA in 3-day cultures. There were two- to threefold increases in activity over that of control animals at PHA concentrations of 10.5-340 gg/ml. Similarly, the PHA responses of spleen and prescapular lymph node cells demonstrated greater activity at 3 days when compared with analogous cells from control goats (data not shown). DISCUSSION
This study was designed to evaluate the induction and/or recruitment of specific memory cells to sites proximal and distal to local injection of antigen. Subcutaneous inoculation of HKS antigen into the fight udder of dairy goats induced specific memory cell responses in both ipsilateral and contralateral supramammary lymph nodes. Although cells from both supramammary lymph nodes exhibited secondary immune responses to HKS, the intensity of the responses from draining nodes was greater. The data demonstrated this in two ways: ( 1 ) hyperplasia of the draining node as evidenced by increases in organ weight and cell number; and (2) heightened proliferative activity of these cells to the HKS antigen. The higher responsiveness in draining nodes may be because of the presence of persisting antigen which has been shown to m d~aiuiu~ induce immunologic memory to resident and c]rcmaung -" . . . . . . -" ccm __,1_-_ lymph nodes (Ponzio et al., 1977). In contrast, the responsiveness of cells from contralateral nodes may be attributed to memory cell migration from ipsilateral nodes as suggested by other investigators (Smith et al., 1970; Sprent et al., 1971; Sprent, 1973; Strober and Dilly, 1973) or by antigen leakage and relocation to distant sites where specific cell responses could be induced in situ. Memory cell activity was clearly present in the periphery as demonstrated by the responsiveness of spleen and prescapular and contra!ateral supramammary lymph nodes to HKS in 3-day cultures. All peripheral lymphoid tissues demonstrated increased responses with peak activity at or near 25 ng/ml of HKS antigen in culture. In contrast, cells from mesenteric lymph nodes showed heightened responses to HKS at lower concentrations of antigen challenge ( 1.6 ng/ml) in 6-day and to a lesser degree in 3-day cultures. This finding suggests that antigen spill-over into the circulation and the number of memory cells migrating from the inoculated area to the visceral lymph nodes is rather limited, requiting increased time in culture for adequate manifestation
248
M.J. DOBRZANSKI AND T.J. YANG
of activity. Although we are not in a position either to establish or dispute the existence of a gastrointestinal-peripheral (mammary gland) recirculating axis in response to local antigen challenge (Hanson et al., 1979; Jeurissen et al., 1985), our findings on the limited memory cell homing from the supramammary region to the mesenteric lymph nodes suggests the presence of functionally separate peripheral and intestinal lymphoid cell circulations (Cahill et al., 1977; Chin and Hay, 1980). Analysis of lymph node cell responses to the polyclonal T cell mitogen, PHA, shows that cells from both peripheral and mesenteric lymph nodes of immunized animals had increased reactivity to PHA than those from analogous nodes in non-immunized animals, suggesting that T cells in general are hyperresponsive as a result of the HKS inoculation. However, cells from the draining supramammary lymph nodes were less responsive to PHA when compared with those of contralateral lymph nodes. This lower response may be caused by suppressor effects by antigen activated cells (Easmon and Glynn, 1978; Yamamoto et al., 1985; Cox and Kennell, 1988) or to inhibition of previously activated cells by PHA in culture (Yang, 1975; Farkas et al., 1986). Alternatively, it is possible that in response to local infection, select lymphocyte populations are attracted (Wood and Neff, 1978; Baine et al., 1981 ) or proliferate (Reichert et al., 1983) causing changes in distribution patterns of T and B lymphocytes in lymph nodes draining these sites and in the periphery (Yang et al., 1980, 1988), suggesting that quantitative and/or qualitative differences in responding lymphocyte populations, as indicated here by stimulation with PHA, may influence immune cell responses to HKS at sites proximal and distal to local antigen deposition. in conclusion, these studies demonstrate that unilateral introduction of S. a u r e u s cell antigens to the supramammary region can induce an antigen-specific anamnestic response accompanied by a heightened general responsiveness in ipsilateral as well as contralateral supramammary lymph nodes and other distant peripheral lymphoid organs. In contrast, lymphocytes from mesenteric lymph nodes responded differently from those of peripheral lymph nodes suggesting the presence of a unique gastrointestinal lymphoid cell circulation (Reynolds et al., 1988 ). Experiments are currently underway to define whether this differential enhancement and distribution of antigen-specific reactivity functions in a microbiostatic and/or prophylactic capacity to such bacterial components. ACKNOWLEDGMENTS
This work was supported by the United States Department of Agriculture Animal Health Grant No. 86-CRSR-2-2895 and the Storrs (Connecticut) Agricultural Experiment Station Project CONS 00625, and is submitted as Sci-
DISTRIBUTION AND Ag SPECIFIC REACTIVITY OF LYMPHOID CELLS
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entific Contribution No. 1260, Storrs Agricultural Experiment Station, University of Connecticut, Storrs, CT 06269.
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