CELLULAR
IMMUNOLOGY
The Enrichment
43,176- 184 (1979)
of Thymocytes Cortisone
Bearing C3 Receptors Involution
following
A. S. WALIA’, B. ANDERSSON, E. W. FUSON, AND E. W. LAMON~ Veterans Administration Hospital, Birmingham, Alabama 35233; Department of Surgery and Cancer Research and Training Center, University of Alabama in Birmingham, School of Medicine, Birmingham, Alabama 35294; and the Department of Tumor Biology, Karolinska Institute. Stockholm, Sweden Received
September 25, 1978
Young CBA mice were injected with 2.5 mg of cortisone acetate, following which their spleen cells, thymocytes, and lymph node cells were tested for receptors for the third component of complement (C3) over a 20-day period. An erythrocyte-antibody-complement (EAC) rosette assay was used to detect C3 receptors. Cells bearing C3 receptors in the thymus emerged as early as 2 days after cortisone injection and peaked to a level of 18% at Day 7. This was followed by a decline to control levels by Day 14. There was no significant change in the percentage of C3 receptor-bearing cells in lymph node and spleen of the cortisone injected animals compared to appropriately matched uninjected animals. Evidence has been presented that cortisone-resistant thymocytes may bind EAC, and that these cells are surface Ig negative, contain no or very few macrophages, and bear thy-l antigen. Complement receptor lymphocytes (CRLs) were separated from the nonrosetting thymus cells by sedimentation in an Isopaque-Ficoll gradient. These enriched C3 receptor-bearing cells were found to possess thy-l antigens by indirect immunofluorescence. Specificity controls using antisera with thy-l. 1 and thy-l.2 specificity and donor animals with thy-l. 1 or thy-l.2 phenotypic expression indicated that C3 receptor-bearing cells appearing in the thymi of these respective donors following cortisone involution possessed the appropriate thy- 1 phenotype.
INTRODUCTION It is well established that lymphoid cells possess receptors for antigen-antibody-complement complexes (l-3). It has been shown that receptors exist on lymphocytes, macrophages, granulocytes, and platelets for the third component of complement (C3) (1, 4-8) and are present in different proportions in various lymphoid organs (9). In mice, complement receptor lymphocytes (CRL’s) comprise 30-40% of the cells of the spleen, lo-25% of the lymph node, lo-20% of the thoracic duct, and about 5% of the mononuclear cells of the bone marrow (1, 10). Membrane receptors for complement have been detected through the binding of sheep erythrocytes sensitized with antibody and complement leading to the formation of rosettes which can be microscopically enumerated (1, 11). Lymphocytes bearing complement receptors have been generally considered to r Recipient of a Fellowship Award from the National Cancer Institute. * Recipient of a Research Career Development Award from the National Cancer Institute. 176
0008-8749/79/030176-09$02.00/O Copyright All rights
0 1979 by Academic Press, of reproduction in any form
Inc. reserved.
C3 RECEPTORS
ON THYMOCYTES
177
be part of the immunoglobulin (Ig)-bearing (B) lymphocyte population (1, 12). Bianco and Nussenzweig’s (13) studies on mice using selective depletion of 8-, bearing (T) cells and of the CRL-rosetting population by means of cytotoxicity and sedimentation velocity, respectively, followed by statistical analysis, suggested that f&bearing cells and CRL were nonoverlapping populations and that CRL and Ig-bearing cells were coincidental (13). Specific isolation of a small fraction (IS-30%) of the CRL was obtained with about 95% purity (14); in this recovered population, no cells were o-bearing (T) cells and the majority were Ig-positive (B) cells. It was also shown that a number of CRL can be generated in lymph nodes from the bone marrow in the absence of the thymus (12) and that CRL localize preferentially in the lymph node and spleen B-cell areas (15, 16). Ross et al. (17), however, demonstrated a subpopulation of lymphocytes which had complement receptors and was surface Ig negative in mouse spleen (lo-20%). By using a double method of indirect immunofluorescence and rosetting, evidence has also been presented that mouse spleen and lymph node contained T cells which possess complement receptors (18, 19). These studies provided estimates that 20% of spleen T cells and 10% of lymph node T cells are CRL. Previous studies (20-22) have shown that the murine thymus consists of 95% small cortical lymphocytes which are sensitive to cortisone and are immunologically inert. The remaining 5% medullary lymphocytes are relatively resistant to cortisone and immunologically reactive as measured by the graft-vs-host assay (20). In this paper, we present evidence that a subpopulation of cortisone-resistant thymocytes possess C3 receptors. MATERIALS
AND METHODS
Animals. Young CBA mice 6-12 weeks old were used as sources of lymphocytes. Each experiment was performed with 3-4 experimental animals and 1-2 control animals matched by age. Young AKR mice 6- 12 weeks old were also used for specificity control experiments. Serum from A/J mice (C5) deficient was used as a source of complement. Outbred rabbits were used to raise IgM anti-sheep red blood cell (SRBC) antibodies. Antisera. A primary antiserum to SRBC was raised in rabbits by injecting 0.5 ml of a 50% suspension of SRBC via the ear vein. These rabbits were bled after five days and the serum was removed, heat inactivated, and precipitated with ammonium sulfate. The y-globulin was dialyzed against water to obtain the euglobulin fraction. The euglobulin precipitate was dissolved in PBS and fractionated on Sephadex G-200. The first half of the leading protein peak (19 S) was pooled and concentrated to 1 mg/ml. The purified IgM gave a single precipitation line in the IgM region when analyzed by immunoelectrophoresis. A/J mice were bled from the retro-orbital plexus and the fresh serum diluted 1:5 was used as the source of complement. Rabbit anti-thy-l and mouse anti-thy-l serum was kindly provided by Dr. Robert K. Zwemer, Department of Microbiology, University of Alabama in Birmingham. Rabbit anti-thy-l was raised in rabbits against purified thy-l preparations from the T-cell lymphoma B WS147. This antiserum has been demonstrated to be specific for murine thy-l by multiple criteria (23).
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ET AL.
Antiserum specific for thy-l.2 was raised by injecting AKWcum thymocytes (thy- 1.2) into AKR/Jax (thy- 1.1) mice (23). Specific antiserum for thy- 1.1 was raised by reversing the above injection scheme. Rabbit anti-mouse immunoglobulin (fluorescein conjugated) was obtained from Cappel Laboratories, Downingtown, Pa. Medium. Eagle’s minimal essential medium (MEM with 100&ml of gentamicin was used. Lymphoid cells. Spleens, lymph nodes, and thymi from cortisone-treated and control mice were dissected out under sterile conditions and prepared separately as follows: The organs were pressed through fine nylon mesh into cold MEM without FCS, mixed with a Pasteur pipet, and passed over a fine nylon mesh filter. The cells were pelleted by centrifugation and spleen cells were resuspended in tris(hydroxymethyl)aminomethane-buffered, 0.75% ammonium chloride, pH 7.2, and incubated for 10 min at 37°C to lyse the erythrocytes. This step was omitted with thymus and lymph node cells when erythrocyte contamination was minimal. The cells were washed and resuspended in MEM without FCS. Removal of macrophages. Cells prepared as above in 5 ml of MEM were mixed with iron powder and incubated for 30 min at 37°C with agitation every 5- 10 min. Iron and phagocytic cells were removed by passing the suspension through a series of five sterile tubes using a magnet to retain the iron in the discarded tube. The cells were then washed twice with MEM before use. The cells were counted in a hemacytometer, and appropriate dilutions were used in rosette assays. Thymocyte fractionation. The approach taken was to pharmacologically fractionate the thymocytes in vivo by injecting cortisone acetate. Six- to eight-week-old CBA mice were injected with 2.5 mg of cortisone acetate (125 mg/kg body weight). The thymi were harvested from such animals at 2-day intervals for 20 days. From Days 4 to 8, however, experiments were done each day. At Day 2, after injection of cortisone, the thymus is reduced to approximately 5% of its original size and the remaining cortisone-resistant cells are immunologically competent (20). Fourteen days after cortisone injection, the thymus is repopulated to near normal size (22). Preparation of EAC. The technique of Bianco, et al. (1) with modifications was used. The sensitization of erythrocyte with antibody and mouse complement was performed as follows: A 2% suspension of SRBC was washed twice with MEM and the SRBC were incubated in ice for 1 hr with rabbit IgM anti-SRBC diluted 1:200. A relatively low IgM concentration was used to avoid detection of IgM Fc receptors (24). The essentially negative rosette count in the absence of complement was due to the low concentration of IgM used. However, with higher concentrations of IgM, EA rosettes were seen (24). After the incubation, the pellet was washed with MEM three times and divided into two parts, complement (A/J serum) diluted I:5 was added to one part, and the cells were again incubated for 30 min at 37°C. After three more washings with MEM a final suspension of 2% EAC was made in MEM. Assay for CRL. The 50 ~1 of the EAC suspension was added to wells of No. 3040 Falcon Microtiter Plates (Falcon Plastics, Div. of BioQuest, Oxnard, Calif.). To these wells 50 ~1 of lymphocytes from the spleen, thymus, or lymph node from control or cortisone-treated animals at a concentration of 10’ cells/ml was added. As
C3 RECEPTORS ON THYMOCYTES
179
controls, suspensions of E and EA were used in separate wells. The percent rosettes were counted after a 3-hr incubation at 4°C. Lymphocytes with two or more firmly attached SRBC were counted as CRL. Enrichment of EAC-RFC’s and immunojluorescence. One milliliter of 2% EAC and 1 ml of 1 x 10’ thymocytes obtained from animals 7 days after cortisone injection were mixed together and centrifuged. The pellet was incubated at 37°C for 30 min. After the incubation 2 ml of MEM was added to bring the volume back to 2 ml. These cells were layered over 1.5 ml of 14% Isopaque-Ficoll and centrifuged at 400g for 20 min. Enriched EAC-RFCs were collected from the pellet and washed two times. These cells were examined for thy-l antigens using rabbit anti-thy-l or mouse antisera followed by FITC-labeled goat anti-rabbit Ig, or rabbit anti-mouse Ig respectively. Controls included cells incubated first with normal rabbit or mouse serum. Similarly EAC-RFCs obtained from both CBA and AKR mice were examined for thy-l. 1 and thy-l.2 antigens using mouse antisera with thy-l, 1 and thy- 1.2 specificity. Membrane immunofluorescence was detected using a fluorescence microscope. The percentage of surface Ig-positive cells in spleen, thymus, and lymph node from both normal and cortisone-treated mice was determined by direct anti-Ig immunofluorescence. RESULTS Figure 1 shows the enrichment of CRL in the thymus after cortisone treatment. Two days after cortisone treatment, the level of rosette-forming cells increased to 4%. The percentage of CRL continued to increase with time reaching a peak of 18% at 7 days after cortisone injection. Thereafter, the percentage of CRL declined sharply to control levels by Day 14. To evaluate whether differences in the percentages of CRL appeared in spleen and lymph node as well as thymus following cortisone injection we compared the splenic and lymph node CRL from control and cortisone treated animals. Table 1 compares complement receptor-bearing cells in spleens, thymi, and lymph 201
0
2
4
6 8 IO I2 14 16 18 20 Days After Cortisone
FIG. 1. Percentage of EAC-RFC in the thymus after cortisone treatment from Days 2 to 20. Each point represents at least three experiments. The vertical bars represent the standard error of the mean.
180
WALIA ET AL. TABLE 1 EAC Receptor-Bearing Cells in Spleens, Thymi, and Lymph Nodes from Normal and Cortisone-Treated Animals EAC-RFC (%) Organ
ControP
Thymus Spleen Lymph node
0.6 ” 0.7 39 2 13 18 k 10
Experimental*
P valuec 0.05).
nodes from normal and cortisone-treated animals. The data represented in the table are the mean &SD of CRLs from cortisone-treated animals from Days 2 to 7 when high numbers of CRL appeared in the thymus compared to the mean + SD of CRLs from control animals tested. Spleens and lymph node, exhibited no significant difference in CRLs between the normal and cortisone-injected animals. However, there was a significant difference between the percentage of CRL’s in cortisone-resistant and normal thymuses. Table 2 shows the percentage of surface Ig-positive cells for spleens, thymuses, and lymph node obtained from normal and cortisone-treated animals from Day 2 to 20. With both normal and cortisone-resistant thymocytes, less than 1% of the cells were surface Ig-positive (B cells). Spleens and lymph node contained the usual percentage of B cells from normal and cortisone-treated hosts. To evaluate whether the increase in CRL in the thymus was due to macrophages entering the organ following cortisone involution, we tested for CRL before and after treatment of the cell suspension with iron powder and magnetism. Table 3 TABLE 2 Percentage of Surface Ig”-Positive Cells from Spleen, Thymus, and Lymph Node Day after cortisone
SP normal
LN normal
42 40 44 42 49 44 39
2 4 6 7 8 11 20 All days”
SP expt
39 f 9
43 2 3
LN expt
Thymus normal
1 0.5 0 1.0 0 1 1
14 14 11 14 10 11 15 14 + 4
13 f 2
Thymus expt
0.7 Ik 0.4
a Fluorescein-conjugated goat anti-mouse Ig was used for surface Ig studies. * Shows mean k SD for all the days (2 to 20).
0.7 2 0.5
181
C3 RECEPTORS ON THYMOCYTES TABLE 3
Percentage of CRL before and after Removal of Macrophages by Iron Powder and Magnetism EAC-RFC after treatment with iron powder and magnet (%)
EAC-RFC before treatment with iron powder (%) Organ
Control
Experimental
Control
Spleen Thymus Lymph node
25 k 8 1.5 k 0.7 15 2 3
24 f 6 16 k 4 14 2 4
23 + 6 1.0 k 0 17 + 5
Experimental 26 f 96 182 1 17 2 4
D Spleens, thymuses, and lymph nodes were used from normal and experimental animals which were injected with cortisone 7 days before. * Represents mean f SD of percentage of CRLs.
shows the differences before and after iron-powder and magnetism treatment in the number of CRL 7 days after cortisone injection. Cortisone-resistant thymocytes exhibited no decrease in the number of CRL after iron powder and magnet fractionation. Also, with spleen and lymph node cells from both normal and cortisone-treated donors there was no significant difference in the number of CRL before and after iron-powder treatment. (Our lymphocyte preparations contain few, if any, adherent macrophages since the cells are prepared using unsiliconized glass and plastic and passed twice through fine nylon mesh.) To evaluate the antigens on CRL in the thymus in a more direct way we performed immunofluorescence studies on rosette-forming cells in the thymus. The thymocytes forming EAC-RFC were enriched on an Isopaque-Ficoll gradient from 15 to 50% and the immunofluorescence was performed on the enriched population. Table 4 shows the percentage of EAC-RFC which were stained by rabbit anti-thy-l and mouse anti-thy-l. Also, by this procedure, we directly TABLE 4 Indirect Immunofluorescence on EAC-RFCs”
Antiserab
Positive fluorescence (%I
Rabbit anti-thy-l (1:5) NRSd Mouse anti-thy- 1 ( 1:5) NMS
99 + 0.6 0.5 2 0.7 97 2 4 455
0 Thymocytes were obtained from experimental animals which have been injected with cortisone 7 days before. The number of EAC-RFCs were 14% which were increased to 50% after sedimentation on 14% Ficoll-Isopaque. * Fluorescein-conjugated goat anti-rabbit and anti-mouse-Ig antisera were used for the second layer in indirect immunofluorescence. c Percentage positive fluorescence represents mean t- SD of four experiments. d NRS stands for normal rabbit serum and NMS stands for normal mouse serum.
182
WALIA ET AL.
visualized thy- 1 positive cells binding EAC. To further confirm the immunofluorescence specificity, we tested cortisone-resistant thymocytes obtained from AKR and CBA mice with mouse anti-thy-l. 1 and thy-l .2. EAC-RFCs were enriched to 43% in cortisone-resistant AKR thymocytes and to 46% with CBA thymocytes following sedimentation with EACs in an Isopaque-Ficoll gradient. Table 5 shows the percentage of positive fluorescing cells after treatment with thy-l. 1 and thy-l.2 on the C3 receptor-enriched population. As shown the CBA thymocytes enriched to 46%. C3 receptor-bearing cells were 100% thy- 1.2 positive and completely negative with thy-l. 1 antiserum. Likewise the AKR thymocytes enriched to 43%. EAC-RFCs were 98% thy-l.1 positive and only 5% stained with anti-thy- 1.2. DISCUSSION We have thus presented evidence that a subpopulation of mouse thymocytes possess C3 receptors following cortisone involution. Previously, C3 receptors have been considered to be a B-cell marker (1, 11). However, there have been some conflicting reports regarding C3 receptors on T cells. Using heterologous antisera specific for immunoglobulin and T cells, Arnaiz-Villena et al. (19) have presented evidence that 30% of CRL obtained from mouse spleen and lymph node were T cells. These results were in conflict with previous reports (13) that e-bearing (T) cells and CRL were nonoverlapping populations. Parish et al. (25) and Pepys et al. (26) were unable to confirm the results of Amaiz-Villena, that T cells have C3 receptors. From cortisone-involuted mouse thymus, we have detected 1820% CRL and shown that these cells possess thy-l antigens and are not B cells or macrophages. Han and Minowada (27) have previously demonstrated C3 receptors on a human leukemia cell which also bound SRBC (a T-cell characteristic). Pretreatment of this cell line with neuraminidase enhanced both E- and EAC-rosette formation. In their hands normal human T lymphocytes (PHA-induced lymphoblasts or thymus cells) possessed 3 to 6% EAC-rosettes which did not increase with neuraminidase treatment (27). It has been shown by previous studies that a minority of the lymphocytes in the mouse thymus are immunocompetent (20). These cells are relatively cortisone
TABLE 5 Indirect Immunofluorescence on EAC-RFCs Obtained from Cortisone-Treated AKR and CBA Mice Antiserum” Donor
Thy 1.2
Thy 1.1
CBA AKR
100 5
0 98
EAC-RFCs m 46 43
n Fluorescein-conjugated rabbit anti-mouse-Ig antisera were used for the second layer in indirect immunofluorescence.
C3 RECEPTORS
ON THYMOCYTES
183
resistant (20, 21) and are located in the medullary areas of the organ (20). The majority of the cells which are located in the thymic cortex, constituting approximately 95% of the total thymus cell population, does not seem to exhibit any type of immunological reactivity. It is important to point out that absolute numbers of EAC-binding cells after treatment with cortisone may not increase. The number of EAC-RFCs in the normal thymus is about 0.6% and since only 5% of the thymus remains after cortisone treatment (20) this would indicate that the absolute number of C3-bearing cells in the thymus increases only slightly. Since the thymus is completely involuted by 2 days after cortisone treatment (22) the absolute numbers of C3 receptor-bearing cells may actually decrease during the first 2 days. Why 7 days is required for the peak of C3 receptor-bearing cells to appear is unknown. At 8 days after cortisone treatment the thymus regenerates its cortical cells rapidly (22) which corresponds to the rapid decrease in C3 receptor-bearing cells. The cortisone-resistant thymocytes binding EAC were characterized by the following criteria: Surface Ig as assessed by anti-mouse Ig immunofluorescence was present on less than 1% of the cells indicating that the thymocytes were not contaminated with B cells. A suspension of cortisone-resistant thymocytes were treated with iron powder and magnetism. This treatment removes macrophages (29). No decrease in the number of complement rosettes following this treatment indicates that thymus-derived CRL were not macrophages. Using rabbit and murine anti-thy-l serum in an indirect immunofluorescence assay we visualized the presence of thy-l on the EAC-RFC of the CR thymocytes. Table 5 further confirms that EAC-RFCs, indeed, possess thy-l antigen as EAC-RFCs from CBA mice which had been treated with cortisone acetate were not stained with anti-thy-l. 1 but were stained with anti-thy- 1.2. EAC-RFCs from AKR mice treated with cortisone showed similar results. Only 5% of the cells were stained with anti-thy- 1.2 but 98% were stained with anti-thy- 1.1 antiserum. These experiments clearly show that EAC-RFCs obtained from cortisone-treated animals possess the appropriate thy-l antigen on their surface. The present experiments give no information as to whether peripheralT cells also possess C3 receptors. This question is currently under investigation in our laboratories. The function of C3 receptors on thymocytes or on peripheral T cells (if they exist) is not clear. Bianco, Patrick, and Nussenzweig (1) suggest that the binding of activated C3 to the C3 receptor on murine B cells provides a necessary “second signal” for T-cell-independent B-cell triggering (30). Feldmann and Pepys (31) suggested that C3 may be involved in T-cell-B-cell cooperation. Klaus and Humphrey (32) have suggested that the development of B-memory cells involves the formation of antigen-antibody-C3 complexes on dendritic cells in lymphoid follicles. They showed that Cobra Venom Factor treatment of T-cell-depleted mice following priming with DNP-KLH abrogates the development of B-cell memory. They also showed that the localization of DNP-KLH in lymphoid follicles is both C3 and antibody dependent (32). It is conceivable that antigen-antibody-complement complexes may bind B cells, T cells, and macrophages together via their C3 receptors to provide optimal cellular cooperation in thymus-dependent immune responses. Note added in proof. Chiao, J. W., Pantic, V. S., and Good, R. A., (C/in. Exp. Immun&. 18, 483, 1974) have also described a subpopulation of human T cells possessing C3 receptors.
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ACKNOWLEDGMENTS Supported by Grant CA-17273-04 from the National Cancer Institute, National Institutes of Health. The project was supported by funds provided in part by the International Cancer Research Data Bank Programme of the National Cancer Institute, National Institutes of Health (United States), under contract NOI-CO-65341 with the International Union against Cancer. Credit is given to National Research Service Award I-T32CA09128 from the National Cancer Institute and Project 5132-02of the Birmingham Veterans Administration Hospital. Also supported by a grant from the Swedish Cancer Society. Technical assistance of Barbara Williams, Lena Hamrin, Gail Ray, and Joyce Woods is greatly appreciated.
REFERENCES 1. 2. 3. 4. 5. 6.
Bianco, C., Patrick, R., and Nussenzweig, V., J. Exp. Med. 132, 702, 1970. Basten, A., Miller, J. F. A. P., Sprent, J., and Pye, J., J. Exp. Med. 135, 610, 1972. Basten, A., Warner, N. L., and Mandel, T., J. Exp. Med. 135, 627, 1972. Uhr, J. W., Proc. Nat. Acad. Sci. 54, 1599, 1965. Uhr, J. W., and Phillips, Y. M., Ann. N. Y. Acad. Sci. 129, 793, 1966. Huber, H., Polley, M. J., Linscott, W. D., Fudenberg, H. H., and Muller-Eberhard, H. J., Science 162, 1281, 1968. 7. Henson, P. M., Immunology 16, 107, 1969. 8. Eden, A., Bianco, C., and Nussenzweig, V., Cell. Zmmunol. 7, 459, 1973. 9. Ross, G. D., Polley, M. J., Rabellino, E. M., and Grey, H. M., J. Exp. Med. 138, 798, 1973. 10. Michlmayr, G., and Huber, H., J. Zmmunol. 105, 670, 1970. 11. Lay, W. H., and Nussenzweig, V., .Z. Exp. Med. 128, 991, 1968. 12. Nussenzweig, V., Bianco, C., Dukor, P., and Eden, A., In “Progress in Immunology” (B. Amos, Ed.), p. 73, Academic Press, New York, 1971. 13. Bianco, C., and Nussenzweig, V., Science 173, 154, 1971. 14. Eden, A., Bianco, C., and Nussenzweig, V., Cell. Zmmunol. 2, 658, 1971. 15. Dukor, P., Bianco, C., and Nussenzweig, V., Proc. Nat. Acad. Sci. 67, 991, 1970. 16. Silveira, N. P. A., Mendes, M. F., and Tolnai, M., J. Zmmunol. 108, 1456, 1972. 17. Ross, G. D., Rabellino, E. M., Polley, M. J., and Grey, H. M., J. Clin. Invest. 52, 377, 1973. 18. Gyongyossy, M. I. C., and Playfair, J. H. L., Clin. Exp. Zmmunol. 18, 169, 1974. 19. Amaiz-Villena, A., Gyongyossy, M. I. C., andplayfair, J. H. L., Clin. Exp. Zmmunol. 18,177,1974. 20. Blomgren, H., and Andersson, B., Exp. Cell Res., 57, 185, 1969. 21. Andersson, B., and Blomgren, H., Cell. Zmmunol. 1, 362, 1970. 22. Blomgren, H., and Andersson, B., Cell. Zmmunol. 1, 545, 1970. 23. Zwemer, R. K., and Acton, R. T., J. Exp. Med. 142, 378, 1975. 24. Lamon, E. W., Andersson, B., Whitten, H. D., Hurst, M. M., and Ghanta, V., J. Zmmunol. 116, 1199, 1976. 25. Parish, C. R., and Chilcott, A. B., Cell Zmmunol. 20, 290, 1975. 26. Pepys, M. B., Bell, A. J., and Rowe, I. F. In “Immunoadsorbents in Protein Purification” (E. Ruoslahti, Ed.), J. Zmmunol. Suppl. 3, 79. University Park Press, Baltimore 1976. 27. Han, T., and Minowada, J., J. Zmmunol. Methods 12, 253, 1976. 28. Ishidate, J., Jr., and Metcalf, D., J. Exp. Biol. 41, 637, 1963. 29. Lamon, E. W., Wigzell, H., Klein, E., Andersson, B., and Skurzak, H. M.,J. Exp. Med. 137, 1472, 1973. 30. Dukor, P., and Hartmann, K. U., Cell Zmmunol. 7, 349, 1973. 31. Feldmann, M., and Pepys, M. B., Nature (London) 249, 159, 1974. 32. Klaus, G. G. B., and Humphrey, J. H., Immunology 33, 31, 1977.
IMMUNOLOGY
The Enrichment
43,176- 184 (1979)
of Thymocytes Cortisone
Bearing C3 Receptors Involution
following
A. S. WALIA’, B. ANDERSSON, E. W. FUSON, AND E. W. LAMON~ Veterans Administration Hospital, Birmingham, Alabama 35233; Department of Surgery and Cancer Research and Training Center, University of Alabama in Birmingham, School of Medicine, Birmingham, Alabama 35294; and the Department of Tumor Biology, Karolinska Institute. Stockholm, Sweden Received
September 25, 1978
Young CBA mice were injected with 2.5 mg of cortisone acetate, following which their spleen cells, thymocytes, and lymph node cells were tested for receptors for the third component of complement (C3) over a 20-day period. An erythrocyte-antibody-complement (EAC) rosette assay was used to detect C3 receptors. Cells bearing C3 receptors in the thymus emerged as early as 2 days after cortisone injection and peaked to a level of 18% at Day 7. This was followed by a decline to control levels by Day 14. There was no significant change in the percentage of C3 receptor-bearing cells in lymph node and spleen of the cortisone injected animals compared to appropriately matched uninjected animals. Evidence has been presented that cortisone-resistant thymocytes may bind EAC, and that these cells are surface Ig negative, contain no or very few macrophages, and bear thy-l antigen. Complement receptor lymphocytes (CRLs) were separated from the nonrosetting thymus cells by sedimentation in an Isopaque-Ficoll gradient. These enriched C3 receptor-bearing cells were found to possess thy-l antigens by indirect immunofluorescence. Specificity controls using antisera with thy-l. 1 and thy-l.2 specificity and donor animals with thy-l. 1 or thy-l.2 phenotypic expression indicated that C3 receptor-bearing cells appearing in the thymi of these respective donors following cortisone involution possessed the appropriate thy- 1 phenotype.
INTRODUCTION It is well established that lymphoid cells possess receptors for antigen-antibody-complement complexes (l-3). It has been shown that receptors exist on lymphocytes, macrophages, granulocytes, and platelets for the third component of complement (C3) (1, 4-8) and are present in different proportions in various lymphoid organs (9). In mice, complement receptor lymphocytes (CRL’s) comprise 30-40% of the cells of the spleen, lo-25% of the lymph node, lo-20% of the thoracic duct, and about 5% of the mononuclear cells of the bone marrow (1, 10). Membrane receptors for complement have been detected through the binding of sheep erythrocytes sensitized with antibody and complement leading to the formation of rosettes which can be microscopically enumerated (1, 11). Lymphocytes bearing complement receptors have been generally considered to r Recipient of a Fellowship Award from the National Cancer Institute. * Recipient of a Research Career Development Award from the National Cancer Institute. 176
0008-8749/79/030176-09$02.00/O Copyright All rights
0 1979 by Academic Press, of reproduction in any form
Inc. reserved.
C3 RECEPTORS
ON THYMOCYTES
177
be part of the immunoglobulin (Ig)-bearing (B) lymphocyte population (1, 12). Bianco and Nussenzweig’s (13) studies on mice using selective depletion of 8-, bearing (T) cells and of the CRL-rosetting population by means of cytotoxicity and sedimentation velocity, respectively, followed by statistical analysis, suggested that f&bearing cells and CRL were nonoverlapping populations and that CRL and Ig-bearing cells were coincidental (13). Specific isolation of a small fraction (IS-30%) of the CRL was obtained with about 95% purity (14); in this recovered population, no cells were o-bearing (T) cells and the majority were Ig-positive (B) cells. It was also shown that a number of CRL can be generated in lymph nodes from the bone marrow in the absence of the thymus (12) and that CRL localize preferentially in the lymph node and spleen B-cell areas (15, 16). Ross et al. (17), however, demonstrated a subpopulation of lymphocytes which had complement receptors and was surface Ig negative in mouse spleen (lo-20%). By using a double method of indirect immunofluorescence and rosetting, evidence has also been presented that mouse spleen and lymph node contained T cells which possess complement receptors (18, 19). These studies provided estimates that 20% of spleen T cells and 10% of lymph node T cells are CRL. Previous studies (20-22) have shown that the murine thymus consists of 95% small cortical lymphocytes which are sensitive to cortisone and are immunologically inert. The remaining 5% medullary lymphocytes are relatively resistant to cortisone and immunologically reactive as measured by the graft-vs-host assay (20). In this paper, we present evidence that a subpopulation of cortisone-resistant thymocytes possess C3 receptors. MATERIALS
AND METHODS
Animals. Young CBA mice 6-12 weeks old were used as sources of lymphocytes. Each experiment was performed with 3-4 experimental animals and 1-2 control animals matched by age. Young AKR mice 6- 12 weeks old were also used for specificity control experiments. Serum from A/J mice (C5) deficient was used as a source of complement. Outbred rabbits were used to raise IgM anti-sheep red blood cell (SRBC) antibodies. Antisera. A primary antiserum to SRBC was raised in rabbits by injecting 0.5 ml of a 50% suspension of SRBC via the ear vein. These rabbits were bled after five days and the serum was removed, heat inactivated, and precipitated with ammonium sulfate. The y-globulin was dialyzed against water to obtain the euglobulin fraction. The euglobulin precipitate was dissolved in PBS and fractionated on Sephadex G-200. The first half of the leading protein peak (19 S) was pooled and concentrated to 1 mg/ml. The purified IgM gave a single precipitation line in the IgM region when analyzed by immunoelectrophoresis. A/J mice were bled from the retro-orbital plexus and the fresh serum diluted 1:5 was used as the source of complement. Rabbit anti-thy-l and mouse anti-thy-l serum was kindly provided by Dr. Robert K. Zwemer, Department of Microbiology, University of Alabama in Birmingham. Rabbit anti-thy-l was raised in rabbits against purified thy-l preparations from the T-cell lymphoma B WS147. This antiserum has been demonstrated to be specific for murine thy-l by multiple criteria (23).
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Antiserum specific for thy-l.2 was raised by injecting AKWcum thymocytes (thy- 1.2) into AKR/Jax (thy- 1.1) mice (23). Specific antiserum for thy- 1.1 was raised by reversing the above injection scheme. Rabbit anti-mouse immunoglobulin (fluorescein conjugated) was obtained from Cappel Laboratories, Downingtown, Pa. Medium. Eagle’s minimal essential medium (MEM with 100&ml of gentamicin was used. Lymphoid cells. Spleens, lymph nodes, and thymi from cortisone-treated and control mice were dissected out under sterile conditions and prepared separately as follows: The organs were pressed through fine nylon mesh into cold MEM without FCS, mixed with a Pasteur pipet, and passed over a fine nylon mesh filter. The cells were pelleted by centrifugation and spleen cells were resuspended in tris(hydroxymethyl)aminomethane-buffered, 0.75% ammonium chloride, pH 7.2, and incubated for 10 min at 37°C to lyse the erythrocytes. This step was omitted with thymus and lymph node cells when erythrocyte contamination was minimal. The cells were washed and resuspended in MEM without FCS. Removal of macrophages. Cells prepared as above in 5 ml of MEM were mixed with iron powder and incubated for 30 min at 37°C with agitation every 5- 10 min. Iron and phagocytic cells were removed by passing the suspension through a series of five sterile tubes using a magnet to retain the iron in the discarded tube. The cells were then washed twice with MEM before use. The cells were counted in a hemacytometer, and appropriate dilutions were used in rosette assays. Thymocyte fractionation. The approach taken was to pharmacologically fractionate the thymocytes in vivo by injecting cortisone acetate. Six- to eight-week-old CBA mice were injected with 2.5 mg of cortisone acetate (125 mg/kg body weight). The thymi were harvested from such animals at 2-day intervals for 20 days. From Days 4 to 8, however, experiments were done each day. At Day 2, after injection of cortisone, the thymus is reduced to approximately 5% of its original size and the remaining cortisone-resistant cells are immunologically competent (20). Fourteen days after cortisone injection, the thymus is repopulated to near normal size (22). Preparation of EAC. The technique of Bianco, et al. (1) with modifications was used. The sensitization of erythrocyte with antibody and mouse complement was performed as follows: A 2% suspension of SRBC was washed twice with MEM and the SRBC were incubated in ice for 1 hr with rabbit IgM anti-SRBC diluted 1:200. A relatively low IgM concentration was used to avoid detection of IgM Fc receptors (24). The essentially negative rosette count in the absence of complement was due to the low concentration of IgM used. However, with higher concentrations of IgM, EA rosettes were seen (24). After the incubation, the pellet was washed with MEM three times and divided into two parts, complement (A/J serum) diluted I:5 was added to one part, and the cells were again incubated for 30 min at 37°C. After three more washings with MEM a final suspension of 2% EAC was made in MEM. Assay for CRL. The 50 ~1 of the EAC suspension was added to wells of No. 3040 Falcon Microtiter Plates (Falcon Plastics, Div. of BioQuest, Oxnard, Calif.). To these wells 50 ~1 of lymphocytes from the spleen, thymus, or lymph node from control or cortisone-treated animals at a concentration of 10’ cells/ml was added. As
C3 RECEPTORS ON THYMOCYTES
179
controls, suspensions of E and EA were used in separate wells. The percent rosettes were counted after a 3-hr incubation at 4°C. Lymphocytes with two or more firmly attached SRBC were counted as CRL. Enrichment of EAC-RFC’s and immunojluorescence. One milliliter of 2% EAC and 1 ml of 1 x 10’ thymocytes obtained from animals 7 days after cortisone injection were mixed together and centrifuged. The pellet was incubated at 37°C for 30 min. After the incubation 2 ml of MEM was added to bring the volume back to 2 ml. These cells were layered over 1.5 ml of 14% Isopaque-Ficoll and centrifuged at 400g for 20 min. Enriched EAC-RFCs were collected from the pellet and washed two times. These cells were examined for thy-l antigens using rabbit anti-thy-l or mouse antisera followed by FITC-labeled goat anti-rabbit Ig, or rabbit anti-mouse Ig respectively. Controls included cells incubated first with normal rabbit or mouse serum. Similarly EAC-RFCs obtained from both CBA and AKR mice were examined for thy-l. 1 and thy-l.2 antigens using mouse antisera with thy-l, 1 and thy- 1.2 specificity. Membrane immunofluorescence was detected using a fluorescence microscope. The percentage of surface Ig-positive cells in spleen, thymus, and lymph node from both normal and cortisone-treated mice was determined by direct anti-Ig immunofluorescence. RESULTS Figure 1 shows the enrichment of CRL in the thymus after cortisone treatment. Two days after cortisone treatment, the level of rosette-forming cells increased to 4%. The percentage of CRL continued to increase with time reaching a peak of 18% at 7 days after cortisone injection. Thereafter, the percentage of CRL declined sharply to control levels by Day 14. To evaluate whether differences in the percentages of CRL appeared in spleen and lymph node as well as thymus following cortisone injection we compared the splenic and lymph node CRL from control and cortisone treated animals. Table 1 compares complement receptor-bearing cells in spleens, thymi, and lymph 201
0
2
4
6 8 IO I2 14 16 18 20 Days After Cortisone
FIG. 1. Percentage of EAC-RFC in the thymus after cortisone treatment from Days 2 to 20. Each point represents at least three experiments. The vertical bars represent the standard error of the mean.
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WALIA ET AL. TABLE 1 EAC Receptor-Bearing Cells in Spleens, Thymi, and Lymph Nodes from Normal and Cortisone-Treated Animals EAC-RFC (%) Organ
ControP
Thymus Spleen Lymph node
0.6 ” 0.7 39 2 13 18 k 10
Experimental*
P valuec 0.05).
nodes from normal and cortisone-treated animals. The data represented in the table are the mean &SD of CRLs from cortisone-treated animals from Days 2 to 7 when high numbers of CRL appeared in the thymus compared to the mean + SD of CRLs from control animals tested. Spleens and lymph node, exhibited no significant difference in CRLs between the normal and cortisone-injected animals. However, there was a significant difference between the percentage of CRL’s in cortisone-resistant and normal thymuses. Table 2 shows the percentage of surface Ig-positive cells for spleens, thymuses, and lymph node obtained from normal and cortisone-treated animals from Day 2 to 20. With both normal and cortisone-resistant thymocytes, less than 1% of the cells were surface Ig-positive (B cells). Spleens and lymph node contained the usual percentage of B cells from normal and cortisone-treated hosts. To evaluate whether the increase in CRL in the thymus was due to macrophages entering the organ following cortisone involution, we tested for CRL before and after treatment of the cell suspension with iron powder and magnetism. Table 3 TABLE 2 Percentage of Surface Ig”-Positive Cells from Spleen, Thymus, and Lymph Node Day after cortisone
SP normal
LN normal
42 40 44 42 49 44 39
2 4 6 7 8 11 20 All days”
SP expt
39 f 9
43 2 3
LN expt
Thymus normal
1 0.5 0 1.0 0 1 1
14 14 11 14 10 11 15 14 + 4
13 f 2
Thymus expt
0.7 Ik 0.4
a Fluorescein-conjugated goat anti-mouse Ig was used for surface Ig studies. * Shows mean k SD for all the days (2 to 20).
0.7 2 0.5
181
C3 RECEPTORS ON THYMOCYTES TABLE 3
Percentage of CRL before and after Removal of Macrophages by Iron Powder and Magnetism EAC-RFC after treatment with iron powder and magnet (%)
EAC-RFC before treatment with iron powder (%) Organ
Control
Experimental
Control
Spleen Thymus Lymph node
25 k 8 1.5 k 0.7 15 2 3
24 f 6 16 k 4 14 2 4
23 + 6 1.0 k 0 17 + 5
Experimental 26 f 96 182 1 17 2 4
D Spleens, thymuses, and lymph nodes were used from normal and experimental animals which were injected with cortisone 7 days before. * Represents mean f SD of percentage of CRLs.
shows the differences before and after iron-powder and magnetism treatment in the number of CRL 7 days after cortisone injection. Cortisone-resistant thymocytes exhibited no decrease in the number of CRL after iron powder and magnet fractionation. Also, with spleen and lymph node cells from both normal and cortisone-treated donors there was no significant difference in the number of CRL before and after iron-powder treatment. (Our lymphocyte preparations contain few, if any, adherent macrophages since the cells are prepared using unsiliconized glass and plastic and passed twice through fine nylon mesh.) To evaluate the antigens on CRL in the thymus in a more direct way we performed immunofluorescence studies on rosette-forming cells in the thymus. The thymocytes forming EAC-RFC were enriched on an Isopaque-Ficoll gradient from 15 to 50% and the immunofluorescence was performed on the enriched population. Table 4 shows the percentage of EAC-RFC which were stained by rabbit anti-thy-l and mouse anti-thy-l. Also, by this procedure, we directly TABLE 4 Indirect Immunofluorescence on EAC-RFCs”
Antiserab
Positive fluorescence (%I
Rabbit anti-thy-l (1:5) NRSd Mouse anti-thy- 1 ( 1:5) NMS
99 + 0.6 0.5 2 0.7 97 2 4 455
0 Thymocytes were obtained from experimental animals which have been injected with cortisone 7 days before. The number of EAC-RFCs were 14% which were increased to 50% after sedimentation on 14% Ficoll-Isopaque. * Fluorescein-conjugated goat anti-rabbit and anti-mouse-Ig antisera were used for the second layer in indirect immunofluorescence. c Percentage positive fluorescence represents mean t- SD of four experiments. d NRS stands for normal rabbit serum and NMS stands for normal mouse serum.
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WALIA ET AL.
visualized thy- 1 positive cells binding EAC. To further confirm the immunofluorescence specificity, we tested cortisone-resistant thymocytes obtained from AKR and CBA mice with mouse anti-thy-l. 1 and thy-l .2. EAC-RFCs were enriched to 43% in cortisone-resistant AKR thymocytes and to 46% with CBA thymocytes following sedimentation with EACs in an Isopaque-Ficoll gradient. Table 5 shows the percentage of positive fluorescing cells after treatment with thy-l. 1 and thy-l.2 on the C3 receptor-enriched population. As shown the CBA thymocytes enriched to 46%. C3 receptor-bearing cells were 100% thy- 1.2 positive and completely negative with thy-l. 1 antiserum. Likewise the AKR thymocytes enriched to 43%. EAC-RFCs were 98% thy-l.1 positive and only 5% stained with anti-thy- 1.2. DISCUSSION We have thus presented evidence that a subpopulation of mouse thymocytes possess C3 receptors following cortisone involution. Previously, C3 receptors have been considered to be a B-cell marker (1, 11). However, there have been some conflicting reports regarding C3 receptors on T cells. Using heterologous antisera specific for immunoglobulin and T cells, Arnaiz-Villena et al. (19) have presented evidence that 30% of CRL obtained from mouse spleen and lymph node were T cells. These results were in conflict with previous reports (13) that e-bearing (T) cells and CRL were nonoverlapping populations. Parish et al. (25) and Pepys et al. (26) were unable to confirm the results of Amaiz-Villena, that T cells have C3 receptors. From cortisone-involuted mouse thymus, we have detected 1820% CRL and shown that these cells possess thy-l antigens and are not B cells or macrophages. Han and Minowada (27) have previously demonstrated C3 receptors on a human leukemia cell which also bound SRBC (a T-cell characteristic). Pretreatment of this cell line with neuraminidase enhanced both E- and EAC-rosette formation. In their hands normal human T lymphocytes (PHA-induced lymphoblasts or thymus cells) possessed 3 to 6% EAC-rosettes which did not increase with neuraminidase treatment (27). It has been shown by previous studies that a minority of the lymphocytes in the mouse thymus are immunocompetent (20). These cells are relatively cortisone
TABLE 5 Indirect Immunofluorescence on EAC-RFCs Obtained from Cortisone-Treated AKR and CBA Mice Antiserum” Donor
Thy 1.2
Thy 1.1
CBA AKR
100 5
0 98
EAC-RFCs m 46 43
n Fluorescein-conjugated rabbit anti-mouse-Ig antisera were used for the second layer in indirect immunofluorescence.
C3 RECEPTORS
ON THYMOCYTES
183
resistant (20, 21) and are located in the medullary areas of the organ (20). The majority of the cells which are located in the thymic cortex, constituting approximately 95% of the total thymus cell population, does not seem to exhibit any type of immunological reactivity. It is important to point out that absolute numbers of EAC-binding cells after treatment with cortisone may not increase. The number of EAC-RFCs in the normal thymus is about 0.6% and since only 5% of the thymus remains after cortisone treatment (20) this would indicate that the absolute number of C3-bearing cells in the thymus increases only slightly. Since the thymus is completely involuted by 2 days after cortisone treatment (22) the absolute numbers of C3 receptor-bearing cells may actually decrease during the first 2 days. Why 7 days is required for the peak of C3 receptor-bearing cells to appear is unknown. At 8 days after cortisone treatment the thymus regenerates its cortical cells rapidly (22) which corresponds to the rapid decrease in C3 receptor-bearing cells. The cortisone-resistant thymocytes binding EAC were characterized by the following criteria: Surface Ig as assessed by anti-mouse Ig immunofluorescence was present on less than 1% of the cells indicating that the thymocytes were not contaminated with B cells. A suspension of cortisone-resistant thymocytes were treated with iron powder and magnetism. This treatment removes macrophages (29). No decrease in the number of complement rosettes following this treatment indicates that thymus-derived CRL were not macrophages. Using rabbit and murine anti-thy-l serum in an indirect immunofluorescence assay we visualized the presence of thy-l on the EAC-RFC of the CR thymocytes. Table 5 further confirms that EAC-RFCs, indeed, possess thy-l antigen as EAC-RFCs from CBA mice which had been treated with cortisone acetate were not stained with anti-thy-l. 1 but were stained with anti-thy- 1.2. EAC-RFCs from AKR mice treated with cortisone showed similar results. Only 5% of the cells were stained with anti-thy- 1.2 but 98% were stained with anti-thy- 1.1 antiserum. These experiments clearly show that EAC-RFCs obtained from cortisone-treated animals possess the appropriate thy-l antigen on their surface. The present experiments give no information as to whether peripheralT cells also possess C3 receptors. This question is currently under investigation in our laboratories. The function of C3 receptors on thymocytes or on peripheral T cells (if they exist) is not clear. Bianco, Patrick, and Nussenzweig (1) suggest that the binding of activated C3 to the C3 receptor on murine B cells provides a necessary “second signal” for T-cell-independent B-cell triggering (30). Feldmann and Pepys (31) suggested that C3 may be involved in T-cell-B-cell cooperation. Klaus and Humphrey (32) have suggested that the development of B-memory cells involves the formation of antigen-antibody-C3 complexes on dendritic cells in lymphoid follicles. They showed that Cobra Venom Factor treatment of T-cell-depleted mice following priming with DNP-KLH abrogates the development of B-cell memory. They also showed that the localization of DNP-KLH in lymphoid follicles is both C3 and antibody dependent (32). It is conceivable that antigen-antibody-complement complexes may bind B cells, T cells, and macrophages together via their C3 receptors to provide optimal cellular cooperation in thymus-dependent immune responses. Note added in proof. Chiao, J. W., Pantic, V. S., and Good, R. A., (C/in. Exp. Immun&. 18, 483, 1974) have also described a subpopulation of human T cells possessing C3 receptors.
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ACKNOWLEDGMENTS Supported by Grant CA-17273-04 from the National Cancer Institute, National Institutes of Health. The project was supported by funds provided in part by the International Cancer Research Data Bank Programme of the National Cancer Institute, National Institutes of Health (United States), under contract NOI-CO-65341 with the International Union against Cancer. Credit is given to National Research Service Award I-T32CA09128 from the National Cancer Institute and Project 5132-02of the Birmingham Veterans Administration Hospital. Also supported by a grant from the Swedish Cancer Society. Technical assistance of Barbara Williams, Lena Hamrin, Gail Ray, and Joyce Woods is greatly appreciated.
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