Cellular Sites of Immunologic Unresponsiveness*

Transcription

1 Proceedings of the National Academy of Sciences Vol. 65, No. 3, pp , March 1970 Cellular Sites of Immunologic Unresponsiveness* Jacques M. Chiller,t Gail S. Habicht, and William 0. Weiglet DEPARTMENT OF EXPERIMENTAL PATHOLOGY, SCRIPPS CLINIC AND RESEARCH FOUNDATION, LA JOLLA, CALIFORNIA Communicated by Karl Habel, December 22, 1969 Abstract. The reconstitution of the immune response of lethally irradiated mice to human 7-globulin is dependent on the synergistic action of bone marrow with thymus cells. Immunologic unresponsiveness appears to involve a functional defect at each of these cellular levels, inasmuch as neither bone marrow nor thymus cells from unresponsive donors are capable of demonstrating synergism in combination with their normal counterpart. There is an accumulation of evidence supporting the requirement of two or more cell types in the initiation of the primary immune response.1 Cells have been separated on the basis of physical property, that is, either adherence to plastic,2 or anatomic localization in the thymus, bone marrow, or thoracic duct lymph.3 4 The function of each cell type in the over-all reaction has not been clearly defined. There is suggestive evidence that one may act in the processing5 or localization6 of antigen, another in specifically recognizing antigen, undergoing proliferation without forming antibody,7 and still a third cell type involved in the actual synthesis of specific antibody.8 Theoretical relationships between such cells have been proposed.' Studies in mice of synergism between two or more cells as a prerequisite for an antibody response have predominantly employed sheep red blood cells as the antigen. To delineate the specificity of each cell type and more particularly the site of cellular lesions in immunologic unresponsiveness, it is important to demonstrate synergism in a system employing an antigen to which an animal can be made totally unresponsive. In mice, human 7-globulin in its deaggregated form is such an antigen.9 The present report demonstrates synergism between mouse thymus and bone marrow cells in the immune response to human y- globulin. Furthermore, combinations of cells obtained from normal and tolerant animals suggest that immunologic unresponsiveness is a function of both cell types. Materials and Methods. Animals: BALB/c and A/J male mice were obtained from the Jackson Laboratory, Bar Harbor, Maine. Preparation of cell suspensions: Mice were sacrificed by exsanguination. The thymus was excised from the thoracic cavity and the bone marrow was expressed from both femurs and tibiae by hydrostatic pressure using a syringe and attached 25 gauge needle containing cold balanced salt solution.'0 This solution containing 100 jug streptomycin and 100 units penicillin/ml was used for suspending and washing cells throughout these studies. Thymic cell suspensions were prepared using a pool of donor thymi. Single cell 551

2 5>52 MICROBIOLOGY: CHILLER ET AL. PROC. N. A. S. suspensions were made by manipulating the thymi stainless steel gauze followed by filtration through nylon netting. The cells were washed three times, resuspended, filtered 'again through nylon netting and adjusted to a final concentration of approximately 240 X 106 cells/ml. Bone marrow cell suspensions were prepared by flushing the bone marrow-balanced salt solution mixture repeatedly through a syringe without needle and then through a 19 gauge needle. This mixture was washed and filtered as described for thynmus cells and adjusted to Ia final concentration of approximately 150 X 10 cells/ml. Recipient mice were subjected to 900 r whole body irradiation and within 3 to 4 hr were injected intravenously with thymus and/or bone marrow cells. Antigen injections were given intravenously at the time of cell transfer. Subsequent antigen injections were intraperitoneal. X-irradiation: Mice used as recipients were given 900 r whole body irradiation. The animals were housed in a leucite container placed between opposing 220 kv Picker machines which were 160 cm apart. Operation took place at 210 kv and 15 ma with an inherent filtration equivalent to 0.25 cm Cu and 1 mm Al. The dose rate was 51 r/min. Induction of unresponsiveness: Mice were made unresponsive by a single intraperitoneal injection of 2.5 mg deaggregated, DEAE-fractionated human 7-globulin in 1 ml 0.15 M NaCl. The protein was deaggregated by high speed centrifugation (100,000 X g) for 150 min using an SW 50.1 rotor (Beckman Instruments). Only the human y-globulin contained in the top third of each tube was used to render mice unresponsive. The animals were used as donors of bone marrow and thymus days following this treatment. Antigens: Burro and goat blood in Alsever's solution were obtained from Colorado Serum Company, Denver. Before use the cells were washed four times in phosphate buffered saline, ph 7.2, and suspended in balanced salt solution to the appropriate concentration. Human 7-globulin (Cohn fraction II) was obtained through the courtesy of the American Red Cross and prepared as Cohn Fraction II by Squibb (batch no. 2123). It was further purified by chromatography on a DEAE cellulose column equilibrated with 0.01 Mphosphate buffer ph 8.0. Only such DEAE-purified human y-globulin was used for either deaggregation or aggregation. Heat aggregation was performed essentially as by Gamble."' Hemolytic plaque assay: Immunocompetence of recipient animals was assayed by enumeration of antibody-forming cells using a modification of the Jerne plaque technique, where protein antigens are covalently coupled to red blood cells.'0' 12 Goat red blood cells and rabbit red blood cells conjugated with human y-globulin were used as indicator cells. Of these, the rabbit cells provided the advantage of a low background number of plaque-forming cells in mouse spleen suspensions. Indirect plaque-forming cells were developed with squirrel monkey anti-mouse y globulin serum at a final concentration of 1:100. Results. An initial experiment was performed to test whether synergism was obtainable with a cellular antigen, goat red blood cells. Male BALB/c mice eight weeks of age were used as donors and recipients. Lethally-irradiated recipients were injected with various combinations of normal bone marrow (31 X 106 cells) and thymus (121 X 106) along with antigen (4 X 10' goat red blood cells) on day 0. Seven days later the mice were sacrificed and the spleens were pooled within each group for assay of both direct and indirect plaqueforming cells. Striking synergism was demonstrated (Table 1), in that recipients which received both bone marrow and thymus cells had 56 times more direct plaque-forming cells per spleen and 68 times more indirect plaque-forming cells per spleen than could be accounted for by taking the sum of these cells in recipients of bone marrow and in recipients of thymus.

3 VOL. 65, 1970 MICROBIOLOGY: CHILLER ET AL. 553 TABLE 1. Anti-goat red blood cells plaque-forming cells in spleens of lethally irradiated BALB/c mice immunologically reconstituted with normal bone marrow and thymus cells. Direct Plaque-Forming No. of Cells Indirect Plaque-Forming Cells Treatment mice No./106 No./spleen No./106 No./spleen BM* + Tt + Ag$ ,000 BM + T BM + Ag BM T + Ag * 30.9 X 106 bone marrow cells. t X 106 thymus cells. t 4.1 X 108 goat red blood cells. I Recipients assayed for plaque-forming cells. In order to show the synergistic effect of bone marrow and thymus cells in response to human -y-globulin, the experimental protocol had to be modified so that the recipient animals received two injections of the antigen. This was necessary since y-globulin plaque-forming cells could not be detected in irradiated recipients reconstituted with bone marrow and thymus and given a single dose of human y-globulin. Male A/J mice eight weeks of age were used as donors and recipients. Lethally irradiated recipients received various combinations of 22.5 X 106 bone marrow cells, 120 X 106 thymus cells and 0.4 mg On day 10 those groups which had heat-aggregated human y-globulin on day 0. received antigen on day 0 were given a second injection of 0.4 mg human y-globulin. All groups were sacrificed on day 15 and the spleens within each group were pooled and assayed. The results shown in Table 2 represent an average of five separate experiments and clearly demonstrate synergism between thymus and bone marrow cells in the response to human -y-globulin. No human y-globulin plaque-forming cells were found in animals which received only bone marrow and antigen or bone marrow, thymus and no antigen. No animals which received only thymus cells and antigen survived the 15 days from irradiation to time of assay. However, animals surviving until the eleventh day showed no plaqueforming cells. It is unlikely that normal thymus cells would produce 3860 plaque-forming cells per spleen should an animal survive until day 15. If this was the case, it would be expected that a similar number of such cells should be seen in animals protected from the radiation effects by unresponsive bone marrow and given normal thymus cells. TABLE 2. Anti-human y-globulin plaque-forming cells in spleens of lethally irradiated A/J mice immunologically reconstituted with normal bone marrow and thymus cells. No. of Indirect Plaque-Forming Cells Treatment mice No./106 No./spleen BM* + Tt +Ag BM+T BM + Ag * 22.5 X 106 bone marrow cells. t 120 X 106 thymus cells. t 0.4 mg heat-aggregated human y-globulin. Recipients assayed for plaque-forming cells.

4 554 MICROBIOLOGY: CHILLER ET AL. PROC. N. A. S. A subsequent experiment was performed using normal thymus and bone marrow cells in combination with marrow and thymus cells taken from unresponsive mice. Lethally irradiated recipients were injected with various combinations of 42 X 106 unresponsive bone marrow cells, 99 X 106 unresponsive thymus cells, 46 X 106 normal bone marrow cells and 46 X 106 normal thymus cells. Each cell injected group, along with groups of mice from the pool of unresponsive donors and normal donors, received 0.4 mg heat-aggregated human a-globulin on days 0 and 10. The spleens were assayed on day 15 after cell transfer. Neither bone marrow nor thymus cells from unresponsive animals were able to react with normal cells to produce human 7y-globulin plaque-forming cells (Table 3). Similar results were obtained using combinations of 120 X 106 thymus cells and 30 X 106 bone marrow cells. It is also clear that the toleranceinducing regimen was fully effective in rendering mice unresponsive to human,y-globulin (Table 3). Recipient mice which received various combinations of normal or unresponsive bone marrow and thymus cells all responded equally well to an injection of burro red blood cells. Hence the unresponsiveness of the thymus and bone marrow cells was specific to human 7-globulin. TABLE 3. Anti-human y-globulin plaque-forming cells in spleens of lethally irradiated A/J mice immunologically reconstituted with combinations of normal or unresponsive bone marrow and thymus cells. No. of Indirect Plaque-Forming Cells Treatment mice No./ 106 No./spleen NT + NBM + Ag* ,250 NT + TBM + Ag TT + NBM + Ag TT+TBM+Ag Normalt + Ag ,000 Tolerantt + Ag X 106 tolerant thymus cells. TT: NBM: 46 X 106 normal bone marrow cells. NT: 46 X 106 normal thymus cells. TBM: 42 X 106 tolerant bone marrow cells. * 0.4 mg heat-aggregated human e-globulin. t Normal animals from normal donor group. $ Tolerant animals from tolerant donor group. Recipients assayed for plaque-forming cells. To measure the amount of human 7-globulin that may be carried over in the cell transfer, mice were rendered tolerant using 2.5 mg deaggregated human 7- globulin labeled with I3'l. Fifteen days later these animals were used as donors of unresponsive bone marrow and thymus. At that time a pool of washed thymus or bone marrow cells from sixteen animals had no detectable counts. A pool of washed spleen cells from the same donors contained 1.04 X 10-5 mg of antigen. Since recipient animals were each injected with a fraction of the collected bone marrow or thymus cells, each would have received less than 1 X 10-6 mg of human 7-globulin. Discussion. The present data demonstrate that in the mouse neither unresponsive bone marrow nor thymus cells participate synergistically with normal thymus or bone marrow in the immunologic reconstitution of lethally irradiated mice. This implies that there exist in each of these organs populations of cells

5 VOL. 65, 1970 MICROBIOLOGY: CHILLER ET AL. 555 which carry antigen-specific receptors, and that the functional defect in immunologic tolerance to human 7-globulin is present at each of these cellular levels. Previous reports have implicated an involvement of the thymus in unresponsiveness to bovine serum albumin,13 bovine y-globulin,'4 and sheep red blood cells.'5 On the other hand, Playfair'6 concluded that cells from bone marrow, not thymus, were responsible for unresponsiveness in a state of tolerance to sheep red blood cells. The discrepancy between the former results and those presently reported may well be a function of the degree of unresponsiveness obtained to the varying antigens used. In the present study it has been observed that total unresponsiveness of donor animals leads to the demonstration of tolerance at the level of both thymus and bone marrow. However, when cells are obtained from hyporesponsive donors, i.e., animals per cent tolerant, those from thymus are ineffective in synergism, while those from bone marrow demonstrate synergism with normal thymus cells, albeit at a level much lower than the normal-normal combination.'8 Furthermore, the procedure used to achieve a state of tolerance may well influence the type of cells which are specifically rendered unresponsive. Thus, cyclophosphamide-induced tolerance may be restricted to antigen reactive cells, i.e., thymus cells,'5 if these are the only cells specifically triggered to a cyclophosphamide-sensitive stage by antigen. Taylor's experiments'3 in which only thymus, not bone marrow, cells were found unresponsive when obtained from donors given a tolerogenic dose of bovine serum albumin 24 hours before sacrifice, may reflect a difference in the rate of induction of tolerance in thymus and bone marrow cell populations. The kinetics of this induction in each cell population is currently being investigated. Although the experiments reported here suggest that thymus and bone marrow cell populations carry antigen-specific receptors active in the induction of unresponsiveness, they do not distinguish between the possibilities that the observed defects involve cell inactivation, cell death or the migration of specific cells from these tissues to other anatomical sites. In rabbits there appears to be a specific depletion of bone marrow cells following contact with antigen in ViVo.17 If a similar mechanism was applicable in explaining the present observations, it would be limited to a migration induced by a tolerogenic form of the antigen, inasmuch as donors injected with an immunogenic form possess bone marrow and thymus cell populations, each of which is fully competent in demonstrating synergism with its normal counterpart.'8 It is unlikely that in the present experiments normal cells were rendered unresponsive by antigen transferred with cells from tolerant donors, since if antigen is present on such unresponsive cells it is in extremely low quantity, i.e., less than 1 X 10-6 mg. Tolerance is not induced in mice with such low doses of human 7-globulin. 1' The existence of two different cell populations each exhibiting specificity toward a single protein antigen leads to the intriguing problem of the function of each population in immunocompetence. Assuming that only one population (bone marrow?) has the capacity to specifically synthesize antibody, the basic question then revolves around the role which another antigen-reactive population (thymus?) plays in the process of production of specific immunoglobulins.

6 556 MICROBIOLOGY: CHILLER ET AL. PROC. N. A. S. * Publication No. 379 from the Department of Experimental Pathology, Scripps Clinic and Research Foundation. The work was supported by U.S. Public Health Service grant AI and Atomic Energy Commission Contract AT (04-3)410. t Supported by U.S. Public Health Service Training grant GM Research Career Awardee of the U.S. Public Health Service (5-K6-GM-6936). 'Talmage, D. W., J. Radovich, and H. Hemingsen, J. Allergy, 43, 323 (1969). 2 Mosier, D. E., Science, 158, 1573 (1967). 3 Claman, H. N., E. A. Chaperon, and R. F. Triplett, Proc. Soc. Exptl. Biol. Med., 122, 1167 (1966). 4Miller, J. F. A. P., and G. F. Mitchell, Nature, 216, 659 (1967). 6 Fishman, M., J. Exp. Med., 114, 837 (1961). 6 Unanue, E. R., and B. A. Askonas, J. Exptl. Med., 127, 915 (1968). 7Davies, A. J. S., E. Leuchars, V. Wallis, and P. C. Koller, Transplantation, 4, 488 (1966). 8 Mitchell, G. F., and J. F. A. P. Miller, J. Exptl. Med., 128, 821 (1968). 9 Golub, E. S., and W. 0. Weigle, J. Immunol., 99, 624 (1967). 10 Golub, E. S., R. I. Mishell, W. 0. Weigle, and R. W. Dutton, J. Immunol., 100, 133 (1968). 11 Gamble, C. N., Int. Arch. Allergy Appl. Immunol., 30, 446 (1966). 12 Daniels, J. C., and W. 0. Weigle, J. Immunol., 101, 1223 (1968). 18 Taylor, R. B., Nature, 220, 611 (1968). 14 Isakovic, K., S. B. Smith, and B. H. Waksman, J. Exptl. Med., 122, 1103 (1965). '5 Miller, J. F. A. P.,-in Immunological Tolerance (New York: Academic Press, 1969) p Playfair, J. H. L., Nature, 222, 882 (1969). 17 Abdou, N. I., and M. Richter, Int. Arch. Allergy Appl. Immunol., 35, 335 (1969). 18 Chiller, J. M., G. S. Habicht, and W. 0. Weigle, in preparation. 19 Golub, E. S., and W. 0. Weigle, J. Immunol., 102, 389 (1969).