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Mixed connective tissue disease: A disorder of immune regulation
Mixed
Connective Tissue Disease: Immune Regulation
A Disorder
of
Donato Alar&n-Segovia
INCE mixed connective tissue disease (MCTD) was first recognized in 1972 by Sharp and his coworkers’ as a disease with features that were previously considered characteristic of various other connective tissue, or collagen-vascular, diseases, there has been much debate as to whether it is a distinct disease entity or a subset of one of the other collagen diseases.’ We approached this question by systematically studying the immunoregulatory T cell circuits in the various connective tissue diseases, our main thesis is that different disturbances leading to autoimmunity occur in each of disease. Should the disturbance found in MCTD be different from those occurring in the diseases it mimicks, the question of its entity could be solved by showing that it has distinct pathogenetic mechanisms. These studies have been done only of patients who were receiving no treatment, either because their disease had become quiescent or because they were studied before treatment was started. We believe this fact to be particularly important because medications such as corticosteroids, immunosuppressors, or even nonsteroidal antiinflammatory agents obviously would affect T cells. In addition, normal controls have been matched for sex and age of all patients who entered into these studies. In the process of acquiring this information, we have added new methods and systems as they become available. Therefore, the studies were done in different groups of patients who were not receiving concomitant treatment when a particular study was underway.
S
From the Department of Immunology and Rheumatology Institute National de la Nutricibn “‘Salvador Zubiran.” D. Alar&m-Segovia, MD, MS, FACP: Professor of Rheumatology and Immunology, Universidad National Auibnoma de Mexico. and Chairman, Department of Immunology and Rheumatology, lnstituto National de la Nutricion “Salvador Zubiran,” Mexico City, Mexico. Address reprint requests to Dr. D. Alar&n-Segovia, Instituto National de la Nutricibn ‘Salvador Zubiran. ” Vasco de Quiroga No. 15, Delegacibn Tlalpon. Mexico. D. F., 14000, Mexico. o I983 by Grune & Stratton. Inc. 0049-0172/83/1301~006$ I .00/O
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The studies we performed can be divided as follows: Studies on T cell subpopuiations; studies of suppressor T cell functions; studies of functions of postthymic precursor cells; autologous mixed lymphocyte reactions, studies on interleukins- I and 2, natural killer cell function, and studies on the role of autoantibodies in immune regulation. The roles of these various cell subpopulations, functions and factors in T cell circuits are depicted in Fig. 1. Results that are available thus far in five of the consecutive tissue diseases, including MCTD, are summarized in Fig. 2.
STUDIES
ON T CELL SUBPOPULATIONS
As do patients with SLE,3 patients with MCTD have decreased numbers of T cells with surface receptors for the Fc portion of IgG (Ty cells),4 whereas their T cells with receptors for the Fc portion of IgM are normal, as opposed to what is found in scleroderma where these cells are decreased.5,6 We have previously shown7 that the autologous rosette-forming T cell (T,,, cell) behaves as a postthymic precursor cell capable of generating either help or suppression under the stimulus of the serum thymic facto? (Fig. 1). This cell is also the main cell that produces the T cell growth factor interleukin-2 (IL-2)9 and is an important responding cell in the autologous mixed lymphocyte reaction (AMLR).‘” T,* cell are decreased in SLE” and in Sjligren’s syndrome (SS)‘* but are increased in MCTD, the only connective tissue disease where this occurst3 The use of monoclonal antibodies has permitted the differentiation of several T cell subpopulations.14 T3 cells represent most, but not all, circulating T cells and probably exclude precisely the Ty cells; T4 cells are cells with a helper/inducer function-a function that is also probably exerted by Tp cells; and T8 cells are known to have an important role in suppressor and cytotoxic functions’ although this is also not an absolute since Ty cells are important, too. Indeed, there are many indications that Tr are suppressor ceIls,‘5~‘6 and a subpopulation of T4 cells may also have the suppressor function.
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Fig. 1. lmmunoregulatory circuits in humans. Four main areas are identified: the thymus (T) derived cells with a first group of postthymic precursor (T-1 cells. Tti cells encompass a peanut agglutinin (+I and a peanut agglutinin negative (-1 subpopulation, as well as T8+ and T4+ Tti cell subpopulations. Within this area there are also groups of helper (H) and suppressor (8) subpopulations that are generated from TM cells. H cells include T4 and Tp cells, whereas S cells include Ty and T8 cells. as well as a subpopulation of T4 cells with suppressor capacity. Tp cells send messages to Tti cells in a feedback inhibition system that generates suppression through Ty cells. In this area various factors also participate: FTS, serum thymic factor that exerts it effects on Tti cells and to a lesser extent on Ty cells; IL-2 that is mainly produced by the T4 + Ta cell and acts primarily on a non-T& T8 + cell, and IL-l, coming from other areas, that of monocytes (M&L which acts primarily on the T&T8 + cell. As a result of the balance between Hand S cells. a net regulation is obtained to act on 8 cells in another (stippled) area. 8 cells proliferate to produce surface Ig-bearing 8 cells and plasma cells that produce autoantibodies which, in turn. have immunoregulatory potential. 8 cells and Mr$ participate as stimulator cells in AMLR which, in turn. generates R-2, suppression. and natural cytotoxicity (not shown).
Nevertheless, T3, T4, and T8 cells have been found to have normal proportions in the blood of patients with MCTD when compared to their age/sex-matched controls.‘7 In this regard, MCTD is also different from all the other connective tissue diseases that present with various
MCTD m
among
Tar :t
T,
T4
T8
these T cell subpopulations”
STUDIES ON SUPPRESSOR CELL FUNCTIONS
Patients with SLE’* and patients with MCTD19 have decreased suppressor T cell func-
CCCD~~CI
j----/
Ta %I
imbalances (Fig. 2).
mmCmC
;UJ~FEE&-EGE, INklIE.
HELP
AMLR BLOW-=9 HEARLY
IL-2
IL-2 FTS
IL-I PROD
IL-I RESP
Fig. 2. lmmunoregulatory patterns in the connective tissue diseases. To each of five connective tissue diseases there corresponds an horizontal line of rectangles. The black rectangles represent abnormalities: the empty ones, normalcy: the hatched ones, a peculiar abnormality that is explained below, and those with a line are functions that have not yet been studied. The half black/half empty rectangle of IL-2 production in MCTD represents a normal production with AMLR stimulus and a decreased production with mitogen stimulation.
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DONATO ALAR&N-SEGOVIA
tions on B cells, either when this function is elicited by simply culturing the cells before adding them in a coculture to autologous fresh cells that are stimulated with pokeweed mitogen (spontaneous suppression), or when the T cells are precultured with the T cell mitogen concanavalin-A (Concanavalin-A-induced suppression). There are two main differences between SLE and MCTD patients in regard to this loss of suppressor T cell function. On the one hand, this function is more markedly reduced in SLE than in MCTD patients; on the other hand, patients with MCTD have a more homogeneous reduction of both these functions” than do patients with SLE who may have one or the other that is more markedly reduced.‘* We have also shown that the anti-RNP antibody that is the serologic marker, when present in high titers of MCTD, may cause abrogation of the suppressor function of normal T cells.*’ This abrogation of T suppressor cell functions caused by anti-RNP antibody has been shown to be due to its penetration into Tr cells and is less marked on T cells from MCTD patients than in normal T cells, presumably because of the prior (in vivo) effect of the anti-RNP antibody on MCTD patients’ cells. STUDIES ON FUNCTIONS OF POSTTHYMIC
cells because of a defect in thymic factors, but these cells otherwise are functionally intact in feedback inhibition. Similar findings were discovered in another system, generation of suppression,’ where T, cells, not themselves suppressors, generate the suppressor function in culture. In this system, again, MCTD Tti cells were found to be functionally normal in aliogeneic combinations of cells and the defective generation of suppression was found to be in mononuclear cells that were depleted of T, cells.13 AUTOLOGOUS MIXED LYMPHOCYTE REACTION (AMLR)
T cells cocultured with autologous non-T cells that are rendered inactive by radiation or mitomycin treatment respond by proliferating after seven days of coculture.*’ This reaction has been found to have both memory and specificity** and probably reflects in vitro the complex interactions of the immunoregulatory T cell circuits. Indeed, T& cells have been determined by ourselves and others to behave as important responding cells in AMLR.” This system may, therefore, give much insight into the differences and similarities of the immunoregulatory T cell circuits in the connective tissue diseases. Reports on the response at seven
PRECURSOR CELLS
We have determined that the human Tti cells behave as postthymic precursor cells in a manner similar to that of the Ly l+, 2+, 3+ cell of the mouse.’ This includes the participation in a system of feedback inhibition where, driven by PWM, that T, cells receive influences of T/1 cells to exert suppression (Fig. 1). Both patients with SLE” and patients with MCTD13 were found to have decreased feedback inhibition function, which in the case of MCTD was surprising since they have increased rather than decreased Tti cells.‘3 Conversely, patients with primary Sjiigren’s syndrome (SS) were found to have a normal feedback inhibition function despite having low Tee cells.‘* In coculture studies with normal and MCTD cells (Fig. 3) we were able to detect that as opposed to SLE where the T, cells are themselves defective, in MCTD the abnormality resides in the messages provided by Tp cells rather than in the Ta cells. Patients with SS seem to have decreased Tti
TO -
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E
3
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a
Em 2 t
20-
8
IO oIO20-
Q’
YCTO
NC10
Tar
UC10
NORMAL
NORYAL
NORMAL
UC10
NORYAL
Fig. 3. Feedback inhibition with diverse combinations of normal or MCTP Tp or T- cells. Two experiments (A) and (B) are shown.
IMMUNOREGULATION
IN MCTD
117
days have shown it to be decreased in both SLE23-26 and SS2’ We decided to study the kinetics of this response, rather than only the proliferation on day 7, which provided interesting findings.28 MCTD patients were found to have a peculiar pattern of response (Fig. 4), in that they had a proliferative response on the third day of culture and low normal mean proliferative response on day 7. This was unlike any of the kinetic curves that were discovered in other connective tissue diseases, except perhaps scleroderma, where a peak proliferative response occurred on the fourth day. This early response in AMLR suggests a prior autosensitization that took place in vivo, with behavior in vitro akin to that of an anamnestic, or secondary response. Indeed, if the cells are left in culture for six more days, a second peak of response occurs. This is also related to what happens in the experimental secondary AMLR.29 Recent observations have suggested that the AMLR may actually represent a proliferative response to xenoantigens present in sheep red blood cells and/or in fetal calf serum3’ but neither we3’ nor others32 have found this to be a primary factor in AMLR.
“-I “*
I-
? I
/c,
I
2
3
4 DAYS
5
d
r-
r
Fig. 4. Characteristic pattern of kinetics of AMLR throughout seven days with cells from an MCTD patient.
INTERLEUKINS
Interleukin-2 (IL-2) is a soluble product of T lymphocytes that supports the immunoregulatory functions of other T lymphocytes.33*34 Deviations from normal in the production of and/or the response to IL-2 may both reflect and contribute to the aberration of immune regulation in the connective tissue diseases. We have initially reported that both the production of and the response to IL-2 are deficient in SLE.35 Further study of the production of IL-2 in response to two
different stimuli, namely, activation with phytohemagglutinin (PHA) and activation in AMLR and in the response to purified IL-2 yielded interesting differences among the various connective tissue diseases.36 Thus, as opposed to SLE patients, those with scleroderma had normal production of and response to IL-2. Patients with MCTD had decreased production of IL-2 upon PHA stimulation but had normal production with AMLR stimulus. Response of their T cells to IL-2 was moderately diminished as compared to their age/sex-matched controls. Conversely, patients with RA showed moderately decreased production of IL-2 with both stimuli, but normal response to the soluble factor. Patients with primary SS behaved similarly to those with SLE in that both the production of and the response to IL-2 were diminished, although both significantly less so than in SLE. Patients with dermatopolymyositis showed decreased production of IL-2 by their T cells in AMLR but showed normal production upon PHA stimulation as well as normal response to IL-2. Thus, when studying both the production of and the response to IL-2, we found different patterns that were peculiar to each connective tissue disease. Interleukin-1 is a monocyte product with diverse amplifying effects on immune cell reactions by acting on T cells. Monocytes from SLE patients were found to produce significantly less IL- 1 than those of the controls. Response to IL- 1 by T cells from SLE patients was also found to have decreased as compared to those of normal controls.3 In preliminary studies, we have found that the monocyte production of and T lymphocyte response to IL-l are both normal in scleroderma, whereas the production is decreased but the response is normal in MCTD (J. Alcocer-Varela and D. Alarcon-Segovia, unpublished observations). NATURAL KILLER CELL FUNCTION
A subpopulation of T lymphocytes has an innate capacity to kill tumor target cells without the intervention of either complement or antibodies. This function has been found to be diminished in both SLE and Sjogren’s syndrome,38*39 but was normal in MCTD.40 Natural killer function, however, increases with 11-2, but in MCTD it was found to increase considerably less than it
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would in normal subjects, probably reflecting the abnormal response to IL-2 that was found with 3H-thymidine incorporation.36 STUDIES ON THE ROLE OF AUTOANTIBODIES IN IMMUNE
REGULATION
Autoantibodies may contribute to immune regulation, or dysregulation, by various mechanisms that have recently been reviewed.4’ Antilymphocytic antibodies are found in about three quarters of the patients with MCTD.42 More important, however, is the possible role of the marker anti-RNP antibody that is present in MCTD at such high titers. This antibody as well as other autoantibodies have been found to be capable of penetrating into Tr cells through their Fc receptor43 and, in so doing, abrogate their suppressor cell function, ultimately causing their T-y cells from MCTD death.*’ Circulating patients have been determined to have intranuclear IgG44 and the suppressor cell function of circulating T cells in MCTD patients is mitigated a little further by incubation in anti-RNP antibody (Fig. 5). SUMMARY
The findings resulting from these studies indicate significant differences between MCTD and
SPONTANEOUS
60
-60
CON-A-
lND”CE0
ANTIRNP
-
-
-NORMAL
ANTI-
NORMAL
IP6
RNP
106
Fig. 5. Effect of anti-RNP IgG or control normal IgG on spontaneously expanded or concanavalin-A (Con-A) induced suppressor functions on mononuclear cells from either MCTD patients or of normal controls (stippled bars). The bars represent the mean values of eight experiments with different individuals.
the other connective tissue diseases, so that there is a distinct disease entity that mimicks other diseases. In MCTD, the marked diminution of Ty cells may explain the uniformly diminished function of suppressor T cells on B cells in the absence of a decrease of the other recognized suppressor T cell subpopulation, the T8 cells, and in the absence of decreased or functionally incompetent suppressor-generating T,, cells. Thus, T,, cells from MCTD patients were determined to behave normally in feedback inhibition and generation of suppression assays where a defect at the level of T/1 cells and perhaps other T cells was discovered. Indeed, Tne cells were found to be increased in MCTD, indicating both a normal priming by serum thymic factor* as well as possibly a feedback mechanism in an attempt to replenish the Ty cells that they generate.’ Tti cells are also important responding cells in AMLR.‘0.45,46 Besides the possibility that the early AMLR may be due to in vivo self-recognition, there are the possibilities that increased proportions of T,. cells may have contributed to early AMLR or that the loss of supressor Tr cells may have caused it. The normal production of IL-2 upon AMLR stimulus is also indicative of the normalcy of TO+cells in MCTD, because they are both responding cells in this system as well as in the main T cell subpopulation that produces 1L-2.9 Normal natural killer cell function in MCTD also lends support to the normalcy of the TnR function in this disease,47 whereas the decreased response to IL-2 found in MCTD may reflect the abnormality of another T cell subpopulation since the main T cell subpopulation that responds to IL-2 is a non-T,1 T8+ cell subpopulation that’ could contain a natural killer cell population as well. In addition, the production of IL-1 is diminished, indicating a monocyte dysfunction, the nature of which is not clear but may be important. Anti-RNP antibody seems to contribute to the immunoregulatory disturbance by causing loss of Ty cells after penetrating them through their Fc receptor. Whether this antibody could also penetrate monocytes bearing receptors for the Fc portion of IgG has not yet been studied, but it is an interesting possibility that is worth further investigation. If so, with the exception of the
IMMUNOREGULATION
119
IN MCTD
abnormality in Tp messages found in the feedback inhibition assays, most of the abnormalities of immune regulation that were discovered in MCTD might be explained by the penetration of anti-RNP antibodies into Fey receptor-bearing cells. Therefore, perhaps, primary cellular abnormality in MCTD could reside in the function of Tp cells.
ACKNOWLEDGMENTS The following persons have contributed to the studies performed in our laboratory that have been reported here. Jorge Alcocer-Varela, Patricia Benveniste, Eugenia Fishbein, Roberto Gonzalez-Amaro, Armando Laff&t, Luis Llorente, Emma Melendro, Ronald Palacios, and Alejandro Ruiz-Arguelles. To them, I extend my gratitude and friendship.
REFERENCES 1. Sharp GC, Irvin WS, Tan EM, et al: Mixed connective tissue disease: An apparently distinct rheumatic disease syndrome associated with a specific antibody to an extractable nuclear antigen (ENA). Am J Med 52:148-159, 1972 2. Alar&n-Segovia D: Mixed connective tissue disease. A decade of growing pains. J Rheumatol8:535-540, 1981 3. Alar&n-Segovia D, Ruiz-Arguelles A: Decreased circulating thymus-derived cells with receptors for the Fc portion of immunoglobulin G in systemic lupus erythematosus. J Clin Invest 62:1390-1394, 1978 4. Alarcon-Segovia D, Ruiz-Arguelles A: Suppressor cell loss and dysfunction in mixed connective tissue disease. Arthritis Rheum 23:3 14-3 18, 1980 5. Gupta S, Malaviya AN, Rajogapalan P, et al: Subpopulations of human T lymphocytes IX. Imbalance of T cell subpopulations in patients with progressive systemic systemic sclerosis. Clin Exp Immunol 38:342-346, 1979 6. Alarc6n-Segovia D, Palacios R, Ibliiez de Kasep G: Human postthymic precursor cells in health and disease. VII. Immunoregulatory circuits of the peripheral blood mononuclear cells from patients with progressive systemic sclerosis. J Clin Lab Immunol5:143-148, 1981 7. Palacios R, Alar&-Segovia D, Ruiz-Arguelles A, et al: Human postthymic precursor cells in health and disease I. Characterization of the autologous rosette-forming T cells as postthymic precursors. Immunology 42: 127-l 35, 198 I 8. Palacios R, Llorente L, Ruiz-Arguelles A, et al: Human postthymic precursor cells in health and disease V. Stimulatory effect in serum thymic factor on the cell cycle of autologous rosette-forming and T-r cells. Immunology Lett 4:35-38, 1982 9. Fishbein E, Alcocer-Varela J, Alar&i-Segovia D: Cellular basesof the production of and response to interleukin-2. Role of autologous T cell subsets defined with monoclonal antibodies. Immunology (in press) 10. Palacios R, Llorente L, Alar&-Segovia D, et al: Autologous rosette-forming T cells as responding ceils in autologous mixed lymphocyte reaction. J. Clin Invest 65:152-155.1980 I 1. Palacios R, Alar&n-Segovia D, Llorente L, et al: Human postthymic precursor cells in health and disease II. Their loss and dysfunction in systemic lupus erythematosus and their partial correction with serum thymic factor. J Clin Lab Immunol5:71-80.1981 12. Alar&i-Segovia D, Palacios R, Benveniste P, et al: T cell subpopulations and immunoregulatory disturbances in primary Sjogren’s syndrome. (in press) 13. Palacios R, Ruiz-Arguelles A, Alar&n-Segovia D: Human postthymic precursor cells in health and disease IX. Immunoregulatory T cell circuits in peripheral blood of
patients with rheumatoid 43:473-479.1981
arthritis.
Clin
Exp
Immunol
14. Kung P, Goldstein G, Reinherz EL, et al: Monoclonal antibodies defining distinctive human T cell surface antigens. Science 206:347-351, 1979 15. Moretta LM, Webb SR, Grossi CE, et al: Functional analysis of two human T cell subpopulations: Help and suppression of B cell responses by T cells having receptors for IgM or IgG. J Exp Med 146:184-200, 1977 16. Warrington RJ, Olivier SL, Sander PJ, et al: A selective effect upon IgG synthesis by Ty lymphocytes and their products in man. Clin Immunol Immunopathol 26:415422.1983 17. Melendro El, Saldate C, River0 SJ, et al: T-cell subpopulations in the peripheral blood of patients with connective tissue diseases as determined by flow cytometry using monoclonal antibodies. Clin Immunol Immunopathol (in press) 18. Ruiz-Arguelles A, Alar&n-Segovia D, Llorente L, et al: Heterogeneity of the spontaneously expanded and mitogen-induced generation of suppressor cell function of T cells on B cells in systemic lupus erythematosis. Arthritis Rheum 23:1004-l 109, 1980 19. Alarcon-Segovia D, Palacios R: Human Postthymic precursor cells in health and disease. IV. Abnormalities in immunoregulatory T cell circuits in mixed connective tissue disease. Arthritis Rheum 24: 1486-1494, 1981 20. Alar&n-Segovia D, Ruiz-Arguelles A, Llorente L: Antibody penetration into living cells II. Antiribonucleoprotein IgG penetrates into Ty lymphocytes causing their deletion and the abrogation of their suppressor function. J Immunol 122:1855-1862,1979 21. Opelz G, Kuchi M, Takasugi M, et al: Autologous stimulation of human lymphocyte subpopulation. J Exp Med 142:1327-1333, 1975 22. Weksler ME, Kosak R: Lymphocyte transformation by autologous cells. V. Generation of immunologic memory and specificity during the autologous mixed lymphocyte reaction. J Exp Med 146: 1833-l 838, 1977 23. Sakane T, Steinberg AD, Green J: Failure of autologous mixed lymphocyte reactions between T and non-T cells with systemic lupus erythematosus. Proc Nat Acad Sci USA 75:3464-3468,1978 24. Kuntz MM, Innes JB, Weksler ME: The cellular basis of the impaired autologous mixed lymphocyte reaction in patients with systemic lupus erythematosus. J Clin Invest 63:151-153, 1979 25. Sakane T, Steinberg AD, Arnett FC, et al: Studies of immune functions of patients with systemic lupus erythema-
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tosus. III. Characterization of lymphocyte subpopulations responsible for defective autologous mixed lymphocyte reactions. Arthritis Rheum 22:770-776, 1979 26. Palacios R, Alar&n-Segovia D: Human postthymic precursor cells in health and disease VI. Effect of serum thymic factor on the response of cells from patients with systemic lupus erythematosus of mixed connective tissue disease in autologous mixed lymphocyte reaction. Clin lmmunol lmmunopathol 18:362-367, 1981 27. Miyasaka N, Sauvezie B, Pierce DA, et al: Decreased autologous mixed lymphocyte reaction in Sjiigren’s syndrome. J Clin Invest 66:928-933, 1980 28. Lal%n A, Alcocer-Varela J, Alar&i-Segovia D: Differences in the kinetics of the autologous mixed lymphocyte reaction between the various connective tissue diseases. Rheumatol Int, 1983 (in press) 29. Fitzharris P, Knight RA: Generation of suppressor cells in the autologous mixed lymphocyte reaction. Clin Exp Immunol46:185-195.1981 30. Huber C, Merkenschlager M, Gattringer C, et al: Human autologous mixed lymphocyte reactivity is primarily specific for xenoprotein determinants absorbed to antigenpresenting cells during rosette formation with sheep erythrocytes. J Exp Med 155:122221227, 1982 3 1, Laff6n A, Alcocer-Varela J, Alarc6nSegovia D: The autologous mixed lymphocyte reaction is not primarily due to xenoantigenic stimulation. Clin lmmunol lmmunopathol (in press) 32. Moody CE, Gupta S, Wekslen ME: Lymphocyte transformation induced by autologous cells. XV xenoantigens are not required for the proliferative response observed in the autologous mixed lymphocyte reaction. J Clin lmmunol 3:100-102,1983 33. Gillis S: Interleukin-2: Biology and biochemistry. J Clin lmmunol 3:1-13, 1983 34. Watson J, Mochizuki D: Interleukin-2. A class of T cell growth factors. lmmunol Rev 51:257-276, 1980 35. Alcocer-Varela J, Alar&-Segovia D: Decreased production of and response to interleukin-2 by cultured lymphocytes from patients with systemic lupus erythematosus. J Clin Invest 69:1388-1391, 1982 36. Alcocer-Varela J, Laff6n A, Alar&i-Segovia D: Differences in the production of and/or response to interleukin-2
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by T lymphocytes from patients with the various connective tissue diseases. (in press) 37. Alcocer-Varela J, Laff6n-A, Alar&n-Segovia D: Defective monocyte production of, and T lymphocyte response to, interleukin-1 in the peripheral blood of patients with systemic lupus erythematosus. (in press) 38. Katz P, Zaytoun AM, Lee JH, et al: Abnormal natular killer cell activity in systemic lupus erythematosus. An intrinsic defect in the lytic event. J Immunol 129:19661969,1982 39. Minato N, Takeda A, Kano S, et al: Studies of the functions of the natural killer-interferon system in patients with SjBgren’s syndrome. J Clin Invest 69:58 l-588, I982 40. Gonzalez-Amaro R, Alcocer-Varela J, Martinez Cordero E, et al: Natural cytotoxicity in mixed connective tissue disease and its enhancement by interleukin-2. (submitted for publication) 4 1. Alar&-Segovia D: Cellular immunity and its regulation in SLE. Clin Rheum Dis 8:63-75, 1982 42. Diaz-Jouanen E, Llorente L, Ramos-Niembro F, et al: Cold-reactive lymphocytotoxic antibodies in mixed connective tissue disease. J Rheumatol4:&10, 1977 43. Alar&n-Segovia D, Ruiz-Arguelles A, Fishbein E: Antibody to nuclear ribonucleoprotein penetrates live human mononuclear cells through Fc-receptors. Nature 27 I :67-69, 1978 44. Alar&n-Segovia D, Ruiz-Arguelles A, Fishbein E: Antibody penetration into living cells. I. Intranuclear immunoglobulin in peripheral blood mononuclear cells in mixed connective tissue disease and systemic lupus erythematosus. Clin Exp Immunol35:36&375, 1979 45. Tomonari KA, Wakisaka A, Sigawa M: Self-recognition by autologous mixed lymphocyte-reacting primed cells. J Immunol 125:159661600,1980 46. Fournier C, Charreire J: Autologous mixed lymphocyte reaction in man II. Autoreactive and alloreactive cells belong to two different T cell subsets. J Immunol 128:26982703, 1982 47. Palacios R, Alar&n-Segovia D: Human postthymic precursor cells in health and disease III. Role of autologous rosette-forming T cells in the generation of spontaneous killer cells in the autologous mixed lymphocyte reaction. Stand J Immunol 13:499-502, 198 I
Connective Tissue Disease: Immune Regulation
A Disorder
of
Donato Alar&n-Segovia
INCE mixed connective tissue disease (MCTD) was first recognized in 1972 by Sharp and his coworkers’ as a disease with features that were previously considered characteristic of various other connective tissue, or collagen-vascular, diseases, there has been much debate as to whether it is a distinct disease entity or a subset of one of the other collagen diseases.’ We approached this question by systematically studying the immunoregulatory T cell circuits in the various connective tissue diseases, our main thesis is that different disturbances leading to autoimmunity occur in each of disease. Should the disturbance found in MCTD be different from those occurring in the diseases it mimicks, the question of its entity could be solved by showing that it has distinct pathogenetic mechanisms. These studies have been done only of patients who were receiving no treatment, either because their disease had become quiescent or because they were studied before treatment was started. We believe this fact to be particularly important because medications such as corticosteroids, immunosuppressors, or even nonsteroidal antiinflammatory agents obviously would affect T cells. In addition, normal controls have been matched for sex and age of all patients who entered into these studies. In the process of acquiring this information, we have added new methods and systems as they become available. Therefore, the studies were done in different groups of patients who were not receiving concomitant treatment when a particular study was underway.
S
From the Department of Immunology and Rheumatology Institute National de la Nutricibn “‘Salvador Zubiran.” D. Alar&m-Segovia, MD, MS, FACP: Professor of Rheumatology and Immunology, Universidad National Auibnoma de Mexico. and Chairman, Department of Immunology and Rheumatology, lnstituto National de la Nutricion “Salvador Zubiran,” Mexico City, Mexico. Address reprint requests to Dr. D. Alar&n-Segovia, Instituto National de la Nutricibn ‘Salvador Zubiran. ” Vasco de Quiroga No. 15, Delegacibn Tlalpon. Mexico. D. F., 14000, Mexico. o I983 by Grune & Stratton. Inc. 0049-0172/83/1301~006$ I .00/O
114
The studies we performed can be divided as follows: Studies on T cell subpopuiations; studies of suppressor T cell functions; studies of functions of postthymic precursor cells; autologous mixed lymphocyte reactions, studies on interleukins- I and 2, natural killer cell function, and studies on the role of autoantibodies in immune regulation. The roles of these various cell subpopulations, functions and factors in T cell circuits are depicted in Fig. 1. Results that are available thus far in five of the consecutive tissue diseases, including MCTD, are summarized in Fig. 2.
STUDIES
ON T CELL SUBPOPULATIONS
As do patients with SLE,3 patients with MCTD have decreased numbers of T cells with surface receptors for the Fc portion of IgG (Ty cells),4 whereas their T cells with receptors for the Fc portion of IgM are normal, as opposed to what is found in scleroderma where these cells are decreased.5,6 We have previously shown7 that the autologous rosette-forming T cell (T,,, cell) behaves as a postthymic precursor cell capable of generating either help or suppression under the stimulus of the serum thymic facto? (Fig. 1). This cell is also the main cell that produces the T cell growth factor interleukin-2 (IL-2)9 and is an important responding cell in the autologous mixed lymphocyte reaction (AMLR).‘” T,* cell are decreased in SLE” and in Sjligren’s syndrome (SS)‘* but are increased in MCTD, the only connective tissue disease where this occurst3 The use of monoclonal antibodies has permitted the differentiation of several T cell subpopulations.14 T3 cells represent most, but not all, circulating T cells and probably exclude precisely the Ty cells; T4 cells are cells with a helper/inducer function-a function that is also probably exerted by Tp cells; and T8 cells are known to have an important role in suppressor and cytotoxic functions’ although this is also not an absolute since Ty cells are important, too. Indeed, there are many indications that Tr are suppressor ceIls,‘5~‘6 and a subpopulation of T4 cells may also have the suppressor function.
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Fig. 1. lmmunoregulatory circuits in humans. Four main areas are identified: the thymus (T) derived cells with a first group of postthymic precursor (T-1 cells. Tti cells encompass a peanut agglutinin (+I and a peanut agglutinin negative (-1 subpopulation, as well as T8+ and T4+ Tti cell subpopulations. Within this area there are also groups of helper (H) and suppressor (8) subpopulations that are generated from TM cells. H cells include T4 and Tp cells, whereas S cells include Ty and T8 cells. as well as a subpopulation of T4 cells with suppressor capacity. Tp cells send messages to Tti cells in a feedback inhibition system that generates suppression through Ty cells. In this area various factors also participate: FTS, serum thymic factor that exerts it effects on Tti cells and to a lesser extent on Ty cells; IL-2 that is mainly produced by the T4 + Ta cell and acts primarily on a non-T& T8 + cell, and IL-l, coming from other areas, that of monocytes (M&L which acts primarily on the T&T8 + cell. As a result of the balance between Hand S cells. a net regulation is obtained to act on 8 cells in another (stippled) area. 8 cells proliferate to produce surface Ig-bearing 8 cells and plasma cells that produce autoantibodies which, in turn. have immunoregulatory potential. 8 cells and Mr$ participate as stimulator cells in AMLR which, in turn. generates R-2, suppression. and natural cytotoxicity (not shown).
Nevertheless, T3, T4, and T8 cells have been found to have normal proportions in the blood of patients with MCTD when compared to their age/sex-matched controls.‘7 In this regard, MCTD is also different from all the other connective tissue diseases that present with various
MCTD m
among
Tar :t
T,
T4
T8
these T cell subpopulations”
STUDIES ON SUPPRESSOR CELL FUNCTIONS
Patients with SLE’* and patients with MCTD19 have decreased suppressor T cell func-
CCCD~~CI
j----/
Ta %I
imbalances (Fig. 2).
mmCmC
;UJ~FEE&-EGE, INklIE.
HELP
AMLR BLOW-=9 HEARLY
IL-2
IL-2 FTS
IL-I PROD
IL-I RESP
Fig. 2. lmmunoregulatory patterns in the connective tissue diseases. To each of five connective tissue diseases there corresponds an horizontal line of rectangles. The black rectangles represent abnormalities: the empty ones, normalcy: the hatched ones, a peculiar abnormality that is explained below, and those with a line are functions that have not yet been studied. The half black/half empty rectangle of IL-2 production in MCTD represents a normal production with AMLR stimulus and a decreased production with mitogen stimulation.
116
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tions on B cells, either when this function is elicited by simply culturing the cells before adding them in a coculture to autologous fresh cells that are stimulated with pokeweed mitogen (spontaneous suppression), or when the T cells are precultured with the T cell mitogen concanavalin-A (Concanavalin-A-induced suppression). There are two main differences between SLE and MCTD patients in regard to this loss of suppressor T cell function. On the one hand, this function is more markedly reduced in SLE than in MCTD patients; on the other hand, patients with MCTD have a more homogeneous reduction of both these functions” than do patients with SLE who may have one or the other that is more markedly reduced.‘* We have also shown that the anti-RNP antibody that is the serologic marker, when present in high titers of MCTD, may cause abrogation of the suppressor function of normal T cells.*’ This abrogation of T suppressor cell functions caused by anti-RNP antibody has been shown to be due to its penetration into Tr cells and is less marked on T cells from MCTD patients than in normal T cells, presumably because of the prior (in vivo) effect of the anti-RNP antibody on MCTD patients’ cells. STUDIES ON FUNCTIONS OF POSTTHYMIC
cells because of a defect in thymic factors, but these cells otherwise are functionally intact in feedback inhibition. Similar findings were discovered in another system, generation of suppression,’ where T, cells, not themselves suppressors, generate the suppressor function in culture. In this system, again, MCTD Tti cells were found to be functionally normal in aliogeneic combinations of cells and the defective generation of suppression was found to be in mononuclear cells that were depleted of T, cells.13 AUTOLOGOUS MIXED LYMPHOCYTE REACTION (AMLR)
T cells cocultured with autologous non-T cells that are rendered inactive by radiation or mitomycin treatment respond by proliferating after seven days of coculture.*’ This reaction has been found to have both memory and specificity** and probably reflects in vitro the complex interactions of the immunoregulatory T cell circuits. Indeed, T& cells have been determined by ourselves and others to behave as important responding cells in AMLR.” This system may, therefore, give much insight into the differences and similarities of the immunoregulatory T cell circuits in the connective tissue diseases. Reports on the response at seven
PRECURSOR CELLS
We have determined that the human Tti cells behave as postthymic precursor cells in a manner similar to that of the Ly l+, 2+, 3+ cell of the mouse.’ This includes the participation in a system of feedback inhibition where, driven by PWM, that T, cells receive influences of T/1 cells to exert suppression (Fig. 1). Both patients with SLE” and patients with MCTD13 were found to have decreased feedback inhibition function, which in the case of MCTD was surprising since they have increased rather than decreased Tti cells.‘3 Conversely, patients with primary Sjiigren’s syndrome (SS) were found to have a normal feedback inhibition function despite having low Tee cells.‘* In coculture studies with normal and MCTD cells (Fig. 3) we were able to detect that as opposed to SLE where the T, cells are themselves defective, in MCTD the abnormality resides in the messages provided by Tp cells rather than in the Ta cells. Patients with SS seem to have decreased Tti
TO -
Ao50-
E
3
lo-
a
Em 2 t
20-
8
IO oIO20-
Q’
YCTO
NC10
Tar
UC10
NORMAL
NORYAL
NORMAL
UC10
NORYAL
Fig. 3. Feedback inhibition with diverse combinations of normal or MCTP Tp or T- cells. Two experiments (A) and (B) are shown.
IMMUNOREGULATION
IN MCTD
117
days have shown it to be decreased in both SLE23-26 and SS2’ We decided to study the kinetics of this response, rather than only the proliferation on day 7, which provided interesting findings.28 MCTD patients were found to have a peculiar pattern of response (Fig. 4), in that they had a proliferative response on the third day of culture and low normal mean proliferative response on day 7. This was unlike any of the kinetic curves that were discovered in other connective tissue diseases, except perhaps scleroderma, where a peak proliferative response occurred on the fourth day. This early response in AMLR suggests a prior autosensitization that took place in vivo, with behavior in vitro akin to that of an anamnestic, or secondary response. Indeed, if the cells are left in culture for six more days, a second peak of response occurs. This is also related to what happens in the experimental secondary AMLR.29 Recent observations have suggested that the AMLR may actually represent a proliferative response to xenoantigens present in sheep red blood cells and/or in fetal calf serum3’ but neither we3’ nor others32 have found this to be a primary factor in AMLR.
“-I “*
I-
? I
/c,
I
2
3
4 DAYS
5
d
r-
r
Fig. 4. Characteristic pattern of kinetics of AMLR throughout seven days with cells from an MCTD patient.
INTERLEUKINS
Interleukin-2 (IL-2) is a soluble product of T lymphocytes that supports the immunoregulatory functions of other T lymphocytes.33*34 Deviations from normal in the production of and/or the response to IL-2 may both reflect and contribute to the aberration of immune regulation in the connective tissue diseases. We have initially reported that both the production of and the response to IL-2 are deficient in SLE.35 Further study of the production of IL-2 in response to two
different stimuli, namely, activation with phytohemagglutinin (PHA) and activation in AMLR and in the response to purified IL-2 yielded interesting differences among the various connective tissue diseases.36 Thus, as opposed to SLE patients, those with scleroderma had normal production of and response to IL-2. Patients with MCTD had decreased production of IL-2 upon PHA stimulation but had normal production with AMLR stimulus. Response of their T cells to IL-2 was moderately diminished as compared to their age/sex-matched controls. Conversely, patients with RA showed moderately decreased production of IL-2 with both stimuli, but normal response to the soluble factor. Patients with primary SS behaved similarly to those with SLE in that both the production of and the response to IL-2 were diminished, although both significantly less so than in SLE. Patients with dermatopolymyositis showed decreased production of IL-2 by their T cells in AMLR but showed normal production upon PHA stimulation as well as normal response to IL-2. Thus, when studying both the production of and the response to IL-2, we found different patterns that were peculiar to each connective tissue disease. Interleukin-1 is a monocyte product with diverse amplifying effects on immune cell reactions by acting on T cells. Monocytes from SLE patients were found to produce significantly less IL- 1 than those of the controls. Response to IL- 1 by T cells from SLE patients was also found to have decreased as compared to those of normal controls.3 In preliminary studies, we have found that the monocyte production of and T lymphocyte response to IL-l are both normal in scleroderma, whereas the production is decreased but the response is normal in MCTD (J. Alcocer-Varela and D. Alarcon-Segovia, unpublished observations). NATURAL KILLER CELL FUNCTION
A subpopulation of T lymphocytes has an innate capacity to kill tumor target cells without the intervention of either complement or antibodies. This function has been found to be diminished in both SLE and Sjogren’s syndrome,38*39 but was normal in MCTD.40 Natural killer function, however, increases with 11-2, but in MCTD it was found to increase considerably less than it
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118
would in normal subjects, probably reflecting the abnormal response to IL-2 that was found with 3H-thymidine incorporation.36 STUDIES ON THE ROLE OF AUTOANTIBODIES IN IMMUNE
REGULATION
Autoantibodies may contribute to immune regulation, or dysregulation, by various mechanisms that have recently been reviewed.4’ Antilymphocytic antibodies are found in about three quarters of the patients with MCTD.42 More important, however, is the possible role of the marker anti-RNP antibody that is present in MCTD at such high titers. This antibody as well as other autoantibodies have been found to be capable of penetrating into Tr cells through their Fc receptor43 and, in so doing, abrogate their suppressor cell function, ultimately causing their T-y cells from MCTD death.*’ Circulating patients have been determined to have intranuclear IgG44 and the suppressor cell function of circulating T cells in MCTD patients is mitigated a little further by incubation in anti-RNP antibody (Fig. 5). SUMMARY
The findings resulting from these studies indicate significant differences between MCTD and
SPONTANEOUS
60
-60
CON-A-
lND”CE0
ANTIRNP
-
-
-NORMAL
ANTI-
NORMAL
IP6
RNP
106
Fig. 5. Effect of anti-RNP IgG or control normal IgG on spontaneously expanded or concanavalin-A (Con-A) induced suppressor functions on mononuclear cells from either MCTD patients or of normal controls (stippled bars). The bars represent the mean values of eight experiments with different individuals.
the other connective tissue diseases, so that there is a distinct disease entity that mimicks other diseases. In MCTD, the marked diminution of Ty cells may explain the uniformly diminished function of suppressor T cells on B cells in the absence of a decrease of the other recognized suppressor T cell subpopulation, the T8 cells, and in the absence of decreased or functionally incompetent suppressor-generating T,, cells. Thus, T,, cells from MCTD patients were determined to behave normally in feedback inhibition and generation of suppression assays where a defect at the level of T/1 cells and perhaps other T cells was discovered. Indeed, Tne cells were found to be increased in MCTD, indicating both a normal priming by serum thymic factor* as well as possibly a feedback mechanism in an attempt to replenish the Ty cells that they generate.’ Tti cells are also important responding cells in AMLR.‘0.45,46 Besides the possibility that the early AMLR may be due to in vivo self-recognition, there are the possibilities that increased proportions of T,. cells may have contributed to early AMLR or that the loss of supressor Tr cells may have caused it. The normal production of IL-2 upon AMLR stimulus is also indicative of the normalcy of TO+cells in MCTD, because they are both responding cells in this system as well as in the main T cell subpopulation that produces 1L-2.9 Normal natural killer cell function in MCTD also lends support to the normalcy of the TnR function in this disease,47 whereas the decreased response to IL-2 found in MCTD may reflect the abnormality of another T cell subpopulation since the main T cell subpopulation that responds to IL-2 is a non-T,1 T8+ cell subpopulation that’ could contain a natural killer cell population as well. In addition, the production of IL-1 is diminished, indicating a monocyte dysfunction, the nature of which is not clear but may be important. Anti-RNP antibody seems to contribute to the immunoregulatory disturbance by causing loss of Ty cells after penetrating them through their Fc receptor. Whether this antibody could also penetrate monocytes bearing receptors for the Fc portion of IgG has not yet been studied, but it is an interesting possibility that is worth further investigation. If so, with the exception of the
IMMUNOREGULATION
119
IN MCTD
abnormality in Tp messages found in the feedback inhibition assays, most of the abnormalities of immune regulation that were discovered in MCTD might be explained by the penetration of anti-RNP antibodies into Fey receptor-bearing cells. Therefore, perhaps, primary cellular abnormality in MCTD could reside in the function of Tp cells.
ACKNOWLEDGMENTS The following persons have contributed to the studies performed in our laboratory that have been reported here. Jorge Alcocer-Varela, Patricia Benveniste, Eugenia Fishbein, Roberto Gonzalez-Amaro, Armando Laff&t, Luis Llorente, Emma Melendro, Ronald Palacios, and Alejandro Ruiz-Arguelles. To them, I extend my gratitude and friendship.
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by T lymphocytes from patients with the various connective tissue diseases. (in press) 37. Alcocer-Varela J, Laff6n-A, Alar&n-Segovia D: Defective monocyte production of, and T lymphocyte response to, interleukin-1 in the peripheral blood of patients with systemic lupus erythematosus. (in press) 38. Katz P, Zaytoun AM, Lee JH, et al: Abnormal natular killer cell activity in systemic lupus erythematosus. An intrinsic defect in the lytic event. J Immunol 129:19661969,1982 39. Minato N, Takeda A, Kano S, et al: Studies of the functions of the natural killer-interferon system in patients with SjBgren’s syndrome. J Clin Invest 69:58 l-588, I982 40. Gonzalez-Amaro R, Alcocer-Varela J, Martinez Cordero E, et al: Natural cytotoxicity in mixed connective tissue disease and its enhancement by interleukin-2. (submitted for publication) 4 1. Alar&-Segovia D: Cellular immunity and its regulation in SLE. Clin Rheum Dis 8:63-75, 1982 42. Diaz-Jouanen E, Llorente L, Ramos-Niembro F, et al: Cold-reactive lymphocytotoxic antibodies in mixed connective tissue disease. J Rheumatol4:&10, 1977 43. Alar&n-Segovia D, Ruiz-Arguelles A, Fishbein E: Antibody to nuclear ribonucleoprotein penetrates live human mononuclear cells through Fc-receptors. Nature 27 I :67-69, 1978 44. Alar&n-Segovia D, Ruiz-Arguelles A, Fishbein E: Antibody penetration into living cells. I. Intranuclear immunoglobulin in peripheral blood mononuclear cells in mixed connective tissue disease and systemic lupus erythematosus. Clin Exp Immunol35:36&375, 1979 45. Tomonari KA, Wakisaka A, Sigawa M: Self-recognition by autologous mixed lymphocyte-reacting primed cells. J Immunol 125:159661600,1980 46. Fournier C, Charreire J: Autologous mixed lymphocyte reaction in man II. Autoreactive and alloreactive cells belong to two different T cell subsets. J Immunol 128:26982703, 1982 47. Palacios R, Alar&n-Segovia D: Human postthymic precursor cells in health and disease III. Role of autologous rosette-forming T cells in the generation of spontaneous killer cells in the autologous mixed lymphocyte reaction. Stand J Immunol 13:499-502, 198 I