CL.INI 0.1). The lymphotoxin titers were not changed by the preincubation. Mixing equal numbers of SFL and PBL from the same donor does not significantly decrease @ > 0.3) the PHA-induced 3HTdR incorporation or lymphotoxin
RA
THE
SUMMARY
LYMPHOCYTE
OF .THE
SYNOVIAL
FLUID
237
IN VITRO
RESPONSES
TABLE 2 1,~ VITRO RESPONSES OF PERIPHERAL (SFL) LYMPHOCYTES FROM RHEUMATOID
(PBL) AND
BLOOD
AKTHRITIS
Lymphocytes stimulated by Nonstimulated PBLISFL” PBLISFU’ Lt titer PBL SFL
0.8 10.1 (19) -
PHA
Con A
PWM
11.4 k 3.1 (19) 31.1 + 11.9 (19)
6.3 + 1.7 (9) 25.2 + 8.7 (9)
7.0 + 2.3 (10) 35.1 + 13.5(10)
66.0 2 21.1’ 8.2 + 1.7
26.8 f 6.7 3.3 t 1.2
25.5 k 0.3 12.1 + 6.5
U Ratio of peripheral bloodkynovial fluid lymphocyte response to mitogen stimulation measured by the incorporation $HTdR; mean 2 SE; number of patients in sample given in parentheses. h Ratio of peripheral blood/synovial fluid lymphocyte response to mitogen stimulation measured by incorporation of 3HTdR and corrected by subtraction of the nonstimulated (basal) response; mean t SE; number of patients in sample given in parentheses. c Lymphotoxin production induced by stimulation with mitogen, reciprocal of the dilution of supernatant resulting in 10% specific release of “‘Cr from prelabeled L cells: mean 2 SEM.
production compared to the responses of the same number of PBL cultured alone (Table 4). In fact, in one case the addition of SFL significantly increased (p < 0.01) the 3HTdR incorporation (patient No. 2, Table 4). Altering the concentration of mitogen with a constant number of lymphocytes (Fig. 1) or increasing the number of lymphocytes with a constant concentration of mitogen (Fig. 2) did not affect TABLE EFFECTS
3
OF PREINCUBATION IN AUTOLOGOUS SYNOVIAL FLUID (ASF) RESPONSES OF LYMPHOCYTES FROM RHEUMATOID ARTHRITIS
Peripheral blood”
ON PHA-SrrMuL.1rro PArrENTs Synovial fluid”
Mediab
ASF*
Mediab
ASF”
MH-3HTdR incorporation Nonstimulated PHA Lt titer
1.0 + 0.4’ 20.1 2 0.8 100”
3.0 2 0.2 27.0 k 1.6 100
0.9 2 0.1 3.8 2 1.1 2
2.6 t 0.3 5.2 f 2.5 3
LD-:‘HTdR incorporation Nonstimulated PHA L,t titer
6.5 2 1.9 31.8 c 2.3 5
8.1 -t 1.4 27.4 5 2.0 5
6.3 t 1.8 15.1 + 3.2 1
8.4 + 2.2 5.6 2 0.5 1
JE-“HTdR incorporation Nonstimulated PHA Lt titer
1.2 + 0.1 5.7 + 0.3 104
4.6 + 0.4 6.3 xt 1.0 107
n.t.’
n.t.
n.t.
n.t.
I’ Lymphocyte source. !’ Preincubation. c Counts per minute x 103: mean 2 SE. ‘I Lymphotoxin production induced by stimulation with mitogen, reciprocal of the dilution of supernatant resulting in 10% specific release of “Cr from prelabeled L cells. I’ n.t. = not tested.
238
STRATTON
MB-“HTdR incorporation Nonstimulated PHA Lt titer BS-:lHTdR incorporation Nonstimulated PHA Lt titer LD-“HTdR incorporation Nonstimulated PHA Lt titer LH-2HTdR incorporation Nonstimulated PHA Lt titer
AND
PE’I‘ER
PBL”
PBL A SFL”
SFL”
2.1 i 0.w 7.7 t 0.8 80”
0.9 -+ 0.1 10.3 + 3.6 100
1.1 t 0.3 2.3 t 0.7 14
1.8 i 0.2 10.1 ir 0.9 100
2.9 t- 1.0 19.8 -t 2.5 100
1.8 f 0.7 5.6 k 0.3 4
4.6 + 1.5 6.6 t 2.2 13
4.8 t 1.3 9.5 2 3.1 45
6.4 t 1.8 7.7 + 3.1 1
0.9 f 0.1 42.9 + 4.8 100
0.9 f 0.4 58.0 -+ 9.7 92
0.4 + 0.1 I.1 -t 0.1 I
I’ Lymphocytes: 5 x 10” peripheral blood lymphocytes (PBL) or synovial fluid lymphocytes (SFL): PBL + SFL consisted of 5 x lOti PBL plus 5 x 10fi SFL. ’ “HTdR incorporation (counts per minute x IO”): mean 2 SEM. c Lymphotoxin titer, reciprocal of dilution of mitogen-stimulated supernatant resulting in 10% specific release of :’ Cr from prelabeled L cells.
the ratio of the PBL to SFL response; in addition, neither the ‘HTdR tion nor the lymphotoxin production was affected.
incorpora-
DISCUSSION
We have been unable to demonstrate enhanced 3HTdR incorporation in RA synovial fluid lymphocytes after stimulation with several mitogens including phytohemagglutinin, concanavalin A, and pokeweed mitogen. This contrasts with a 3- to loo-fold increase in 3HTdR incorporation of paired RA-PBL and confirms the impaired SFL blastogenic response observed by other workers (2, 15-21). On the other hand, previous work shows that lymphocytes isolated from synovial fluid are as competent as PBL in certain cell-mediated immune responses, such as cytotoxicity on human fibroblasts (22, 23) and on Chang liver cells (24). The latter observations do not support the notion that the decreased mitogen responsiveness is due to effects of certain drugs, e.g., salicylates (25, 26). In addition some of our samples (e.g., DG, Table 1) were from patients who were not taking any drugs. That the SFL are viable and immunocompetent is also substantiated by our data (Tables 1 and 2) which demonstrate that the cells are capable of producing lymphotoxin in response to mitogen stimulation. A possible explanation of decreased SFL reactivity is suggested by the work of Robbins and Levis (27) who demonstrated that the peak of 3HTdR incorporation could be delayed if either suboptimal concentrations of mitogen or lympho-
RA
LYMPHOCYTE
RESPONSES
IN VITRO
239
. can A --
pgm
MITOGEN
0 PHA 0 PWM PBL SFL
/ ml
Flo. I. Response of mononuclear cells isolated from peripheral blood (PBL; solid line) and synovial fluid (SFL; dashed line) of rheumatoid arthritis patient to increasing concentrations of mitogen in tissue culture. The blastogenic response (incorporation of ‘HTdR) and the lymphotoxin production were assayed after stimulation with 0.5, 1.0. and 3.0 pg of PHA (0): 0.5. 15.0, and 30.0 p.g of PWM (0): and 5.0. 10.0, and 25.0 yg of Con A (0).
cytes were present. The delayed peak could be the result of a delay in response of all the cells or a gradual increase in a small number of responding rapidly dividing cells (28). This possibility was evaluated but the 3HTdR incorporation of RA-SFL was not altered by changing either the mitogen concentration or the cell numbers (cf. Figs. 1 and 2). However, we were able to substantiate Takasugi and Hollingworth’s (29) finding that the unstimulated incorporation of 3HTdR is greater in SFL than in PBL of RA patients. These data suggest that compared to RAPBL, more RA-SFL are entering the S phase after 3 days in unstimulated culture but that the SFL as a group are poorly responsive to mitogens. The possibility that SFL are coated with immunoglobulin and thus unable to respond is unlikely since the addition of one-tenth rabbit complement to either SFL or PBL (5 x lo6 lymphocytes in 1 ml of RPMI-1640, 5% FBS) did not kill any lymphocytes (trypan blue exclusion at either 1 or 72 hr). RA-PBL, so treated, were able to respond normally to PHA. In addition, incubation of RA-PBL in autologous SF did not impair the PHA response (Table 3). Rosenstreich et ~1. (30) found that lymphocytes from peritoneal exudates
240
--
lJ-rT4-3 NUMBER
2 OF LYMPHOCYTES
. Con A 0 PHA PEL SFL
4
(I lO-6)
Frc. 2. Response of increasing number of mononuclear cells isolated from peripheral blood (PBL; solid line) and synovial fluid (SFL: dashed line) of rheumatoid arthritis patient to a stimulation with either 1.O pg of PHA (0) or 10.0 pg of Con A (0). The blastogenic response (incorporation of 3HTdR) and Iymphotoxin production were assayed on 0.5, 1.0. 2.5, and 5.0 x 10’~ stimulated mononuclear cells.
responded normally to both antigen and PHA. whereas lymphocytes from draining lymph nodes had a markedly decreased response to both. They suggested that lymphocytes sequestered into inflammatory sites represent a population selected on the basis of some physiological property, perhaps surface membrane characteristics since these cells do not adhere to nylon fiber or glass beads (30). The unresponsiveness does not appear to be due to the presence of suppressor cells (31), since the addition of SFL to PBL does not inhibit the mitogen responses (c.f. Table 4). Alternatively the decreased reactivity of these lymphocytes may be explained by the possibility that they are already “terminal,” that is they are already committed and cannot be further stimulated (32-35). Schellenkens and Eijsoovogel(35) showed there was a summation of antigen stimulation to a ceiling and if the response to one antigen was already at this ceiling there is no further stimulation. Also, pretreatment of lymphocytes with one antigen decreased the ability of the lymphocytes to respond with DNA synthesis to later additions of the same antigen, unrelated antigen, or nonspecific mitogens (33). The most likely explanation of this phenomenon is an inhibition of immunocompetent cells due to direct contact with antigen-activated cells, since no soluble inhibitor was found (34).
RA
LYMPHOCYTE
RESPONSES
IN
VITRO
241
After contact with the antigen, the antigen-responsive lymphocytes in murine thymus migrate to the spleen where they proliferate and eventually recirculate (36). The “activated” thymocytes are immunocompetent and highly specific for the activating antigen, and since they have already divided many times, they are incapable of further extensive division. That this type of cell may be present in the synovial fluid of rheumatoid arthritis patients is supported by the work of Herman et cl/. (37), who were unable to elicit a specific antibody response to tetanus toxoid even though the synovial lymphoid infiltrate produces five to nine times more immunoglobulin than PBL. In summary, we have demonstrated that lymphocytes isolated from the synovial fluid of patients with rheumatoid arthritis are immunocompetent (they produce lymphotoxin in response to mitogen stimulation) but are incapable of being stimulated by mitogens to incorporate 3HTdR. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. I I.
Williams. R. C., Emmons, J. D.. and Astorga, G. P., Arthritis Rheum. 14, 697. 1971. Astorga. G. A., and Williams. R. C., Jr., Arthritis Rheum. 12, 547. 1969. Hedburg, H.. Kallen, B., Low, B., et al.. C/in. Bp. Immunol. 9, 201, 1971. Silverman. H. A., Johnson, J. S., Vaughan, J. H., er al., Arthritis Rheum. 19, 509, 1976. Rawson, A. J., and Huang, T. C., Arfhritis Rhcurn. 19, 720, 1976. Maclaurin, B. P., Larzcer 1, 1070, 1971. Stastny. P., Arthritis Rheum. 18, 428, 1975. Stastny. P., J. Clin. Znvrst. 57, 1148, 1976. Perper. R. J.. Zee. T. W., and Michkelson, M. M.. J. Lab. C/in. Med. 72, 842. 1968. Peter, J. B.. Stratton, J. A., Stemple. K. B.. et al., J. Immunol. 111, 770. 1973. Yu. D. T. Y., Peter, J. B., Stratton, J. A.. Paulus. H. E., and Machleder, H. I., C/in. Immunol.
12. 13. 14. IS. 16.
Granger, G. A., and Williams, W., Progr. Immunol. 1, 438, 1971. Spofford, B. T.. Daynes, R. A., and Granger, G. A., J. Immunol. 112, 211 I, 1974. Stratton, J. A.. and Byfield. P. E., Cell. I,nmuno/. 28, 1, 1977. Stratton. J. A.. and Peter, J. B., Arthritis Rheum. 15, 457. 1972. Reynolds, M. D.. and Abdou, N. I.. J. C/in. Invest. 52, 1627, 1973. Ivanyi, L., Lehner. T., and Burry, H. C., Immunology 25, 905, 1973. Sheldon. P. J., Papamichaeil, M., and Holborrow, E. J., Ann. Rheum. Di.?. 33, 509, 1974. Griffiths, M. M., and Williams, R. C., Arthritis Rheum. 17, 111, 1974. Hepburn, B., McDuffie, F. C., and Ritts, R. E., J. Rheum. 3, 118, 1976. Panayi. G. S.. Lower 2, 512. 1973. Hedburg. H., and Kallen. B., Acttr Pathol. Microbial. Stand. 62, 177, 1964. Subernik, R., Hanin, A., and Mosolov. A., Clitz. hp. Imrttunol. 3, 171, 1968. Maclennan, I. C. M., and Loewi, G., C/in. Exp. Immunol. 6, 713, 1970. Ambrose. C. T., J. Esp. Med. 124, 461, 1966. Opelz, G.. Terasaki, P. I., and Hirata, A. A., Loncet 2, 478, 1973. Robbins. J. H., and Levis, W. R., fnt. Arch. A//erg)’ Appl. Immu~w(. 39, 580, 1970. Douglas. S. D., Kamin, R. M.. and Fudenberg, H. H., J. Immwwl. 103, 1185, 1969. Takasugi, K.. and Hollingworth, J. W.. Arthritis Rheum. 11, 44, 1968. Rosenstreich. D. L.. Blake, J. T., and Rosenthal, A. S., J. Exp. Med. 134, 1170. 1971. Gershon. R. K., Cohen, P., Hencin, R., et a/., J. Immurwl. 108, 586. 1972. Moller, G., Irn~t~rzolog~ 19, 583, 1970. Moller, G.. and Kashiwagi, N., I~~rmunology 22, 441. 1972. Sjoberg. 0.. Imu7urzolog~ 21, 351, 1971. Schellekens. P. Th. A.. and Eijsvoogel, V. P., Cli17. Erp. Immunol. 8, 187, 1971. Sprent, J.. and Miller, J. F. A. P.. Nnture New, Biol. 234, 195. 1971. Herman, J. H.. Bradley, J., Ziff. M., et nl., J. C/in. Invest. 50, 266. 1971.
17.
18. 19. 20. II. 21. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37.
RA
THE
SUMMARY
LYMPHOCYTE
OF .THE
SYNOVIAL
FLUID
237
IN VITRO
RESPONSES
TABLE 2 1,~ VITRO RESPONSES OF PERIPHERAL (SFL) LYMPHOCYTES FROM RHEUMATOID
(PBL) AND
BLOOD
AKTHRITIS
Lymphocytes stimulated by Nonstimulated PBLISFL” PBLISFU’ Lt titer PBL SFL
0.8 10.1 (19) -
PHA
Con A
PWM
11.4 k 3.1 (19) 31.1 + 11.9 (19)
6.3 + 1.7 (9) 25.2 + 8.7 (9)
7.0 + 2.3 (10) 35.1 + 13.5(10)
66.0 2 21.1’ 8.2 + 1.7
26.8 f 6.7 3.3 t 1.2
25.5 k 0.3 12.1 + 6.5
U Ratio of peripheral bloodkynovial fluid lymphocyte response to mitogen stimulation measured by the incorporation $HTdR; mean 2 SE; number of patients in sample given in parentheses. h Ratio of peripheral blood/synovial fluid lymphocyte response to mitogen stimulation measured by incorporation of 3HTdR and corrected by subtraction of the nonstimulated (basal) response; mean t SE; number of patients in sample given in parentheses. c Lymphotoxin production induced by stimulation with mitogen, reciprocal of the dilution of supernatant resulting in 10% specific release of “‘Cr from prelabeled L cells: mean 2 SEM.
production compared to the responses of the same number of PBL cultured alone (Table 4). In fact, in one case the addition of SFL significantly increased (p < 0.01) the 3HTdR incorporation (patient No. 2, Table 4). Altering the concentration of mitogen with a constant number of lymphocytes (Fig. 1) or increasing the number of lymphocytes with a constant concentration of mitogen (Fig. 2) did not affect TABLE EFFECTS
3
OF PREINCUBATION IN AUTOLOGOUS SYNOVIAL FLUID (ASF) RESPONSES OF LYMPHOCYTES FROM RHEUMATOID ARTHRITIS
Peripheral blood”
ON PHA-SrrMuL.1rro PArrENTs Synovial fluid”
Mediab
ASF*
Mediab
ASF”
MH-3HTdR incorporation Nonstimulated PHA Lt titer
1.0 + 0.4’ 20.1 2 0.8 100”
3.0 2 0.2 27.0 k 1.6 100
0.9 2 0.1 3.8 2 1.1 2
2.6 t 0.3 5.2 f 2.5 3
LD-:‘HTdR incorporation Nonstimulated PHA L,t titer
6.5 2 1.9 31.8 c 2.3 5
8.1 -t 1.4 27.4 5 2.0 5
6.3 t 1.8 15.1 + 3.2 1
8.4 + 2.2 5.6 2 0.5 1
JE-“HTdR incorporation Nonstimulated PHA Lt titer
1.2 + 0.1 5.7 + 0.3 104
4.6 + 0.4 6.3 xt 1.0 107
n.t.’
n.t.
n.t.
n.t.
I’ Lymphocyte source. !’ Preincubation. c Counts per minute x 103: mean 2 SE. ‘I Lymphotoxin production induced by stimulation with mitogen, reciprocal of the dilution of supernatant resulting in 10% specific release of “Cr from prelabeled L cells. I’ n.t. = not tested.
238
STRATTON
MB-“HTdR incorporation Nonstimulated PHA Lt titer BS-:lHTdR incorporation Nonstimulated PHA Lt titer LD-“HTdR incorporation Nonstimulated PHA Lt titer LH-2HTdR incorporation Nonstimulated PHA Lt titer
AND
PE’I‘ER
PBL”
PBL A SFL”
SFL”
2.1 i 0.w 7.7 t 0.8 80”
0.9 -+ 0.1 10.3 + 3.6 100
1.1 t 0.3 2.3 t 0.7 14
1.8 i 0.2 10.1 ir 0.9 100
2.9 t- 1.0 19.8 -t 2.5 100
1.8 f 0.7 5.6 k 0.3 4
4.6 + 1.5 6.6 t 2.2 13
4.8 t 1.3 9.5 2 3.1 45
6.4 t 1.8 7.7 + 3.1 1
0.9 f 0.1 42.9 + 4.8 100
0.9 f 0.4 58.0 -+ 9.7 92
0.4 + 0.1 I.1 -t 0.1 I
I’ Lymphocytes: 5 x 10” peripheral blood lymphocytes (PBL) or synovial fluid lymphocytes (SFL): PBL + SFL consisted of 5 x lOti PBL plus 5 x 10fi SFL. ’ “HTdR incorporation (counts per minute x IO”): mean 2 SEM. c Lymphotoxin titer, reciprocal of dilution of mitogen-stimulated supernatant resulting in 10% specific release of :’ Cr from prelabeled L cells.
the ratio of the PBL to SFL response; in addition, neither the ‘HTdR tion nor the lymphotoxin production was affected.
incorpora-
DISCUSSION
We have been unable to demonstrate enhanced 3HTdR incorporation in RA synovial fluid lymphocytes after stimulation with several mitogens including phytohemagglutinin, concanavalin A, and pokeweed mitogen. This contrasts with a 3- to loo-fold increase in 3HTdR incorporation of paired RA-PBL and confirms the impaired SFL blastogenic response observed by other workers (2, 15-21). On the other hand, previous work shows that lymphocytes isolated from synovial fluid are as competent as PBL in certain cell-mediated immune responses, such as cytotoxicity on human fibroblasts (22, 23) and on Chang liver cells (24). The latter observations do not support the notion that the decreased mitogen responsiveness is due to effects of certain drugs, e.g., salicylates (25, 26). In addition some of our samples (e.g., DG, Table 1) were from patients who were not taking any drugs. That the SFL are viable and immunocompetent is also substantiated by our data (Tables 1 and 2) which demonstrate that the cells are capable of producing lymphotoxin in response to mitogen stimulation. A possible explanation of decreased SFL reactivity is suggested by the work of Robbins and Levis (27) who demonstrated that the peak of 3HTdR incorporation could be delayed if either suboptimal concentrations of mitogen or lympho-
RA
LYMPHOCYTE
RESPONSES
IN VITRO
239
. can A --
pgm
MITOGEN
0 PHA 0 PWM PBL SFL
/ ml
Flo. I. Response of mononuclear cells isolated from peripheral blood (PBL; solid line) and synovial fluid (SFL; dashed line) of rheumatoid arthritis patient to increasing concentrations of mitogen in tissue culture. The blastogenic response (incorporation of ‘HTdR) and the lymphotoxin production were assayed after stimulation with 0.5, 1.0. and 3.0 pg of PHA (0): 0.5. 15.0, and 30.0 p.g of PWM (0): and 5.0. 10.0, and 25.0 yg of Con A (0).
cytes were present. The delayed peak could be the result of a delay in response of all the cells or a gradual increase in a small number of responding rapidly dividing cells (28). This possibility was evaluated but the 3HTdR incorporation of RA-SFL was not altered by changing either the mitogen concentration or the cell numbers (cf. Figs. 1 and 2). However, we were able to substantiate Takasugi and Hollingworth’s (29) finding that the unstimulated incorporation of 3HTdR is greater in SFL than in PBL of RA patients. These data suggest that compared to RAPBL, more RA-SFL are entering the S phase after 3 days in unstimulated culture but that the SFL as a group are poorly responsive to mitogens. The possibility that SFL are coated with immunoglobulin and thus unable to respond is unlikely since the addition of one-tenth rabbit complement to either SFL or PBL (5 x lo6 lymphocytes in 1 ml of RPMI-1640, 5% FBS) did not kill any lymphocytes (trypan blue exclusion at either 1 or 72 hr). RA-PBL, so treated, were able to respond normally to PHA. In addition, incubation of RA-PBL in autologous SF did not impair the PHA response (Table 3). Rosenstreich et ~1. (30) found that lymphocytes from peritoneal exudates
240
--
lJ-rT4-3 NUMBER
2 OF LYMPHOCYTES
. Con A 0 PHA PEL SFL
4
(I lO-6)
Frc. 2. Response of increasing number of mononuclear cells isolated from peripheral blood (PBL; solid line) and synovial fluid (SFL: dashed line) of rheumatoid arthritis patient to a stimulation with either 1.O pg of PHA (0) or 10.0 pg of Con A (0). The blastogenic response (incorporation of 3HTdR) and Iymphotoxin production were assayed on 0.5, 1.0. 2.5, and 5.0 x 10’~ stimulated mononuclear cells.
responded normally to both antigen and PHA. whereas lymphocytes from draining lymph nodes had a markedly decreased response to both. They suggested that lymphocytes sequestered into inflammatory sites represent a population selected on the basis of some physiological property, perhaps surface membrane characteristics since these cells do not adhere to nylon fiber or glass beads (30). The unresponsiveness does not appear to be due to the presence of suppressor cells (31), since the addition of SFL to PBL does not inhibit the mitogen responses (c.f. Table 4). Alternatively the decreased reactivity of these lymphocytes may be explained by the possibility that they are already “terminal,” that is they are already committed and cannot be further stimulated (32-35). Schellenkens and Eijsoovogel(35) showed there was a summation of antigen stimulation to a ceiling and if the response to one antigen was already at this ceiling there is no further stimulation. Also, pretreatment of lymphocytes with one antigen decreased the ability of the lymphocytes to respond with DNA synthesis to later additions of the same antigen, unrelated antigen, or nonspecific mitogens (33). The most likely explanation of this phenomenon is an inhibition of immunocompetent cells due to direct contact with antigen-activated cells, since no soluble inhibitor was found (34).
RA
LYMPHOCYTE
RESPONSES
IN
VITRO
241
After contact with the antigen, the antigen-responsive lymphocytes in murine thymus migrate to the spleen where they proliferate and eventually recirculate (36). The “activated” thymocytes are immunocompetent and highly specific for the activating antigen, and since they have already divided many times, they are incapable of further extensive division. That this type of cell may be present in the synovial fluid of rheumatoid arthritis patients is supported by the work of Herman et cl/. (37), who were unable to elicit a specific antibody response to tetanus toxoid even though the synovial lymphoid infiltrate produces five to nine times more immunoglobulin than PBL. In summary, we have demonstrated that lymphocytes isolated from the synovial fluid of patients with rheumatoid arthritis are immunocompetent (they produce lymphotoxin in response to mitogen stimulation) but are incapable of being stimulated by mitogens to incorporate 3HTdR. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. I I.
Williams. R. C., Emmons, J. D.. and Astorga, G. P., Arthritis Rheum. 14, 697. 1971. Astorga. G. A., and Williams. R. C., Jr., Arthritis Rheum. 12, 547. 1969. Hedburg, H.. Kallen, B., Low, B., et al.. C/in. Bp. Immunol. 9, 201, 1971. Silverman. H. A., Johnson, J. S., Vaughan, J. H., er al., Arthritis Rheum. 19, 509, 1976. Rawson, A. J., and Huang, T. C., Arfhritis Rhcurn. 19, 720, 1976. Maclaurin, B. P., Larzcer 1, 1070, 1971. Stastny. P., Arthritis Rheum. 18, 428, 1975. Stastny. P., J. Clin. Znvrst. 57, 1148, 1976. Perper. R. J.. Zee. T. W., and Michkelson, M. M.. J. Lab. C/in. Med. 72, 842. 1968. Peter, J. B.. Stratton, J. A., Stemple. K. B.. et al., J. Immunol. 111, 770. 1973. Yu. D. T. Y., Peter, J. B., Stratton, J. A.. Paulus. H. E., and Machleder, H. I., C/in. Immunol.
12. 13. 14. IS. 16.
Granger, G. A., and Williams, W., Progr. Immunol. 1, 438, 1971. Spofford, B. T.. Daynes, R. A., and Granger, G. A., J. Immunol. 112, 211 I, 1974. Stratton, J. A.. and Byfield. P. E., Cell. I,nmuno/. 28, 1, 1977. Stratton. J. A.. and Peter, J. B., Arthritis Rheum. 15, 457. 1972. Reynolds, M. D.. and Abdou, N. I.. J. C/in. Invest. 52, 1627, 1973. Ivanyi, L., Lehner. T., and Burry, H. C., Immunology 25, 905, 1973. Sheldon. P. J., Papamichaeil, M., and Holborrow, E. J., Ann. Rheum. Di.?. 33, 509, 1974. Griffiths, M. M., and Williams, R. C., Arthritis Rheum. 17, 111, 1974. Hepburn, B., McDuffie, F. C., and Ritts, R. E., J. Rheum. 3, 118, 1976. Panayi. G. S.. Lower 2, 512. 1973. Hedburg. H., and Kallen. B., Acttr Pathol. Microbial. Stand. 62, 177, 1964. Subernik, R., Hanin, A., and Mosolov. A., Clitz. hp. Imrttunol. 3, 171, 1968. Maclennan, I. C. M., and Loewi, G., C/in. Exp. Immunol. 6, 713, 1970. Ambrose. C. T., J. Esp. Med. 124, 461, 1966. Opelz, G.. Terasaki, P. I., and Hirata, A. A., Loncet 2, 478, 1973. Robbins. J. H., and Levis, W. R., fnt. Arch. A//erg)’ Appl. Immu~w(. 39, 580, 1970. Douglas. S. D., Kamin, R. M.. and Fudenberg, H. H., J. Immwwl. 103, 1185, 1969. Takasugi, K.. and Hollingworth, J. W.. Arthritis Rheum. 11, 44, 1968. Rosenstreich. D. L.. Blake, J. T., and Rosenthal, A. S., J. Exp. Med. 134, 1170. 1971. Gershon. R. K., Cohen, P., Hencin, R., et a/., J. Immurwl. 108, 586. 1972. Moller, G., Irn~t~rzolog~ 19, 583, 1970. Moller, G.. and Kashiwagi, N., I~~rmunology 22, 441. 1972. Sjoberg. 0.. Imu7urzolog~ 21, 351, 1971. Schellekens. P. Th. A.. and Eijsvoogel, V. P., Cli17. Erp. Immunol. 8, 187, 1971. Sprent, J.. and Miller, J. F. A. P.. Nnture New, Biol. 234, 195. 1971. Herman, J. H.. Bradley, J., Ziff. M., et nl., J. C/in. Invest. 50, 266. 1971.
17.
18. 19. 20. II. 21. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37.
