out.pdf

Immunologic Research 1998; 17/1 &2:41-47

Mechanisms of Systemic Autoimmunity in Murine Models of SLE

Division of Rheumatology, Department of Medicine, University of Pennsylvania School of Medicine, Philadelphia, PA.

Abstract Our laboratory has utilized spontaneous and experimentally induced models of systemic autoimmunity in mice in order to elucidate the cellular deficiencies in immunoregulation that are essential to this process. In the spontaneously autoimmune mouse strains, genetic defects in T and B cell tolerance are the primary abnormalities that drive the syndrome. The induced chronic graft-vs-host model depends on abnormal T-B interactions resulting from allogeneic recognition of major histocompatibility complex (MHC) class II. Future inves- tigations will target the biochemistry of the loss of tolerance and the specificity of autoreactive T cells that provide help for autoantibody production.

Key Words Systemic lupus erythematosus Mouse models Apoptosis Immunoregulation Tolerance Autoimmunity Lymphoproliferation

Introduction Our laboratory investigates the mechanisms

of generalized autoimmunity in murine models of systemic lupus erythematosus (SLE) (1). We employ spontaneous models based on single genes (lpr, gld) or on multilple genes (MRL strain), and an induced model based on a chronic graft-vs-host reaction (GVHR) (2). We are particularly interested in the cellular expression of the genes that control the disease, and the cellular and molecular mechanisms that maintain tolerance or that result from failure of tolerance. In principle, the development of abnormal levels of autoimmunity implies a breakdown in the immunoregulation that nor-

mally preserves the distinction between self and nonself. The essential objective of this research is to determine where in the immune response this breakdown occurs (see Fig. 1 ) and what the mechanisms are that underlie its occurrence. In principle, certain key aspects of the immune response are failing to be appropriately regulated, and this is what allows autoimmunity to develop. Given these essential defects, the other elements of the immune response contribute in their normal fashion.

Ipr and gld Mice Much of our work has targeted the MRL/

Mp-lpr/lpr (MRL/lpr) strain (3). These mice

Dr, Robert Eisenberg Division of Rheumatology 909 Stellar Chance Laboratories, 422 Curie Drive Philadelphia, PA 19104-6100 E-mail: raemd @ mail.medmpenn.edu

© 1998 Humana Press Inc. 0257-277X/98/ 17/1 &2:41-47/$9.75

41

Fig. 1. Cellular events in autoimmunity. A T-dependent immune response occurs when antigen is pro- cessed by antigen-presenting cells (APCs) and presented to CD4 T cells in conjunction with MHC class II. These T cells can then provide help to specific B cells that recognize the autoantigen and are activated to secrete autoantibodies. Cellular interactions among the T cells, B cells, and APCs are critical, and are mediated by a variety of cell-surface receptors and coreceptors, as well as by soluble factors (lymphokines). Autoantibodies themselves can interact with antigen and influence the whole process to produce feedback enhancement. At some level, the role of APC is assumed by the autoreactive B cell itself. Features relevant to considerations of mechanisms of autoantibody formation are listed is smaller type. Our work has addressed the question of where in this physiological process do the fundamental (genetic) abnormalities express themselves so that this normal immune process is bent toward potentially pathologic self-recognition. Our results so far inculpates T cells (chronic GVHR), B cells (BXSB), or both together (lpr).

develop massive lymphadenopathy owing to the accumulat ion of nonmalignant T and B lymphocytes, particularly unusual CD4-, CD8-, TCRc~,~+ T cells. These double nega- tive T cells (DNT) probably arise in the periphery from polyclonal CD8 + precursors that may be autoreactive to major histocompatibility complex (MHC) class I-restricted peptides (4,5). MRL/Ipr also develop high titers of autoantibodies, including specificities characteristic of SLE, such as anti-Sin, anti-dsDNA, anti-ribosomal P protein, and anti-Su (6-8). They die at around 5-6 mo of age, mainly from diffuse proliferative glomerulonephritis and vasculitis. The SLE-like syndrome is dictated by a number of genes in the

MRL background, which curiously is itself the result of interbreeding four nonautoimmune strains, LG/J, C57BL/6, AKR, and C3H (9,10). The lpr gene is a mutant allele offas, which codes for a cell-surface protein that in- teracts with another cell-surface protein, fas ligand, to mediate a signal that results in apoptotic death (11). Fas and fas ligand are each expressed in various lymphoid tissues, as well as elsewhere (12). In MRL/lpr mice, the fas mutation itself produces the striking lymphoproliferation. It also enhances and accelerates the SLE-like systemic autoimmunity dictated by the MRL background genes.

The lpr gene has been backcrossed onto other genetic backgrounds, where it generally

42 Eisenberg

causes massive lymphoproliferation and some degree of autoantibody formation, although only with the MRL strain are the immunopathological consequences of SLE seen (/3). The C57BL/6-lpr strain has been particularly useful, since it develops lymphadenopathy, and high titers of antichromatin antibodies and IgM rheumatoid factors, although no vasculits or glomerulonephritis. The availability of a variety of T and B cell allelic markers, trans- genes, and knockout (null) loci on the C57BL/6 background has made possible many of the mechanistic studies that our laboratory has undertaken.

A related single-gene mutation termed gld has been described and also backcrossed to several inbred mouse strain backgrounds (14); gld codes for a mutant form of fas ligand. In gld mice, self-reactive cells fail to be appropriately deleted, because no functional fas ligand is available to react with their fas receptor. The phenotypic effect is entirely equivalent to that of lpr, which indicates that the fas/ fas ligand system has no redundancy.

Extrinsic Environmental Effects

A key general issue in potential processes that might influence autoimmunity is the distinction between intrinsic and extrinsic effects. On the organism level, this translates to genetics vs environment. Does the genetic background of a particular mouse strain determine completely the development of systemic auto-immunity, or does it merely provide a substrate of susceptibility that needs to be triggered by an environmental immune stimu- lus? Certainly, numerous models exists in which a clearly defined immunization protocol is required to induce disease in an appropriate mouse strain. Such examples include EAE, collagen arthritis, HgC12-induced autoimmunity, and chronic GVHR. With the so-called spontaneous SLE models, however, no purposeful experimental manipulation is

necessary, and similar disease occurs in diverse animal colonies.

We have recently tested the role of environmental antigens in the MRL/Ipr disease by deriving this strain into germ-free (sterile) iso- lators. Under these conditions, all bacteria and viruses were absent, including the normal flora of the gut or skin. Compared to a matched cohort housed in a conventional colony, germ-free mice had similar levels of autoantibodies, dermatitis, and glomerulonephritis. In addition, the proliferation of DNT was unaf- fected, as was the use of T-cell receptor genes reflected in the V[3 repertoire. We then carried this approach one step further, by removing, as much as possible, all antigens from the environment. To do this, the mice were weaned onto a chemically defined diet that contained amino acids, but no protein antigens (15). A cohort of animals fed in this way was compared to a contemporaneous group maintained germ-free, but fed autoclaved mouse chow. The antigen-free state is known to have a profound effect on the levels of serum immunoglobulins, and this was found to be the case in a small number of BALB/c mice maintained in these conditions in our experiments. Somewhat surprisingly, the MRL/lpr mice in the antigen-free state developed high levels of autoantibodies, equivalent to those seen in the control group except for lower levels of anti-single-stranded (ss)DNA and IgG3 anti-IgG2a rheumatoid factor. The lymphadenopathy was reduced about 50%, but was still dominated by the DNT cells in the expected proportions. Glomerulonephritis was substan- tially reduced. Overall these experiments indicate that much of the MRL/lpr syndrome is dictated by the intrinsic (mal)functioning of the immune system as determined by the genetic makeup of the strain. Antigenic stimu- lus from the environment may also play a role in certain auto-antibody specificities and the evolution of kidney involvement.

Murine Models of SLE 43

Intrinsic Cellular Effects

Another way to consider the expression of the genetic elements of the systemic disease is to determine which cell types are directly affected and which ones are influenced from without. That is, in which cells must the genetic abnormalities be expressed intrinsically to permit the development of the syndrome? We have examined this by a series of chimera experiments in which autoimmune mice were lethally irradiated to destroy their immune systems, and they were rescued by transfer of bone marrow cells from two genetically diverse donors, one autoimmune (lpr) and one not. The T or B cells of the two donors were differentiated by allelic cell-surface markers or immunoglobulin allotypes. These experiments have demonstrated that the lpr gene must be expressed intrinsically in the DNT cells, in the T cells that provide help for autoantibody production, and in the B cells that produce autoantibodies (16-18). Interest- ingly, the interaction between the helper T cells and the B cells that produce autoantibodies is cognate and MHC-restricted (19). When normal (i.e., non-lpr) cells differentiate in the presence of lpr cells, they fail to form DNT cells, they cannot provide help for autoantibody production, and they cannot become autoantibody-forming cells. These findings suggest that the defective lpr gene expressed in these cells prevents them from being deleted in vivo during the development of tolerance. In addition, simultaneous T and B cell abnormalities are required for autoantibody production. This is not true in one other murine SLE model, BXSB, in which intrinsic B cell genetic defects can interact with normal T cells to produce autoantibodies (20).

We have performed comparable experiments testing the expression of the MRL background genes, in comparison with the C57BL/6 background genes (21). Strikingly, the genotype of the B cells appeared to dictate the fine

specificity of autoantibodies that were made. In mixed bone marrow chimeras made with MRL/Ipr and C57BL/6-1pr donors, the MRL/ lpr B cells made those autoantibodies characteristic of the MRL/lpr strain, whereas the C57BL/6-1pr B cells made autoantibodies characteristic of the C57BL/6-lpr strain. These results point to the key role that B cells play in controlling the development of autoimmunity, even though T cell abnormalities are also necessary. We are currently examining more carefully an additional role of the genetic background of the T cell in controlling the fine specificity of autoimmunity.

Fas/Fas Ligand Interactions

A parallel series of experiments utilized similar double bone marrow chimeras made with normal donors and donors that were homozygous for the gld mutation in the fas ligand gene. Thus, the genetic defect is extrinsic to those cells, although the overall effect is the same, since fas/fas ligand interactions are interrupted. In the gld/normal double bone marrow chimera protocols, the normal lymphoid cells provide functional fas ligand to the gld cells and correct their defect (22,23). This allowed us to test which normal cells could provide functional fas ligand in vivo in order to suppress the manifestations of the lpr/gld autoimmune syndrome. Our work to date has indicated that Thy-l+ cells express fas ligand in vivo to prevent both autoantibody production and lymphadenopathy (24). The fact that a small number of gld T cells still became double-negatives suggested that an autocrine fas/fas ligand interaction in a single cell could mediate "suicidal" deletion. Both CD4 and CD8 contribute to the suppression of autoantibody formation, whereas CD8 cells are the major suppressors of lymphadenopathy. Additional experiments must now address the specificity of interaction of these fas ligand- positive CD4 and CD8 cells with their targets.

44 Eisenberg

How does the fas ligand-positive cell delete autoreactive cells? Is it because of limitation of fas receptor expression during a restricted period or anatomical location? Is it because the deleted cell and the deletor specifically recognize each other or a third antigen-presenting cell? These questions are being investigated with other double chimeras using normal strains with restricted T cell heterogeneity.

Stem Cells and Autoimmunity

Our work to date indicates that in the lpr model of autoimmunity, genetic abnormalities in both T and B cells are required for development of the syndrome. Our parallel experiments with gld mice, as well as in vitro protocols by others, suggest that the mechanism of the effect of fas deficiency is through failure ofT and B cells to be deleted as a result of their autoreactive potential (25). This implies that the early precursors of the lymphoid populations that have not rearranged their antigen receptor genes would not yet be committed toward autoimmunity. The consis- tent observation that the autoimmune syndrome in MRL/Ipr mice takes several months to evolve (median survival is 5-6 too) further implies that the expression of the genetically determined disease involves time-limited, stochastic processes (26). We are currently testing this possibility by a hematopoietic stem cell transplantation protocol that may prove to be a useful therapeutic approach. Autoimmune MRL/lpr mice at 4-5 mo of age are lethally irradiated and reconstituted with bone marrow from age-matched syngeneic donors. The marrow is depleted of mature T and B cells by pretreatment with appropriate monoclonal antibodies (MAbs) and complement. Control MRL/lpr mice are reconstituted with similarly treated bone marrow from young (6-wk-old) syngeneic mice. If indeed the development of the autoimmune syndrome occurs entirely by the failure of deletion of mature autoreactive

lymphocytes secondary to their fas deficiency and other abnormalities resulting from the MRL background, then reconstitution with depleted bone marrow from either young or old mice should equally prolong the survival of the recipients. If instead some aspects of the autoimmune syndrome evolve because of genetic abnormalities expressed in the stem cell or other early precursor population, then the recipients of young marrow should have a slower return of autoimmunity and improved survival. The therapeutic potential for this approach relies on the proven technical capa- bility to harvest hematopoietic stem cells from the peripheral blood of both mice and humans, such that repletion of the immune system of individuals aggressively treated with immuno- suppression is possible with autologous stem cells (27,28). Since human SLE develops after decades of time and not months, the possibility of"resetting the clock" to day 0 would have major clinical benefit.

Chronic Autoimmune GVHR

Our laboratory is also working with a related model of experimentally induced systemic autoimmunity. If spleen cells from an inbred mouse are transferred to another mouse that differs only by MHC class II genes, a chronic GVHR ensues that results in the production of a spectrum of autoantibodies similar to that found in spontaneous SLE in mice and in humans accompanied by the development of immune-complex-type diffuse proliferative glomerulonephritis (29). Our work and that of others has shown that this syndrome depends on the recognition of foreign class II antigens on the B cells of the recipient by the CD4 T cells of the donor (30). This causes an allogeneic effect, which stimulates the recipient B cells to produce autoantibodies, The specific class II antigen recognized determines the spectrum of autoantibodies that is pro- duced (3I). As in the spontaneous disease, the


full expression of the autoimmune chronic GVHR requires months, even though an initial polyclonal allogeneic effect occurs early after transfer. We are currently investigating two aspects of this syndrome that have strong relevance for spontaneous SLE. First, we wish to determine the fine specificity of the donor T cells that stimulate the autoreactive B cells. Do they see peptides from the same autoantigen as the B cells recognize? Do they see other specific, but ubiquitous cellular peptides, or can they see any self-peptide that might be expressed in conjunction with MHC class II on all B cells? Second, what is the mechanism of loss of tolerance by the recipient B cells? Presumably, the presence of alloreactive T cells provides abnormal T cell help that subverts B cell tolerance. When in B cell ontogeny does this occur? Where does it occur anatomically? What are the biochemical markers of B cells that are "rescued" from tolerance in this way? These two general issues are being addressed with chronic GVHRs generated in transgenic mice with restricted repertoires of T or B cells.

Curiously, we have already found that the CD4 T cells of the recipient are necessary for the autoimmune syndrome. Therefore, the cellular mechanisms must be more complex than merely an allogeneic recognition of recipient B cells by donor T cells. In any case, the initial anomaly is dictated by abnormal T cell recognitionof B cells (allogeneic help), which then secondarily leads to loss of B cell tolerance and autoantibody formation.

Conclusion

Our general goal is to establish how B and T cell tolerance is lost in the development of systemic autoimmunity. Once this distinction between self and foreign has broken down, the immune system functions as it should in producing antibodies that turn out to be autoantibodies. If we under- s tood the crit ical failures of immu- noregulat ion that underl ie this process, we could potentially intervene specifically in treating or preventing individual autoimmune diseases.

References 1 Cohen PL, Eisenberg RA: Lprand

gld: single gene models of systemic autoimmunity and lympho- proliferative disease. Ann Rev Immunol 1991 ;9:243-269.

2 Eisenberg RA, Cohen PL: Class lI major histocompatibility antigens and the etiology of systemic lupus erythematosus. Clin Immunol Immunopathol 1983;29:1-6.

3 Andrews BS, Eisenberg RA, Theofilopoulos AN, Izui S, Wilson CB, McConahey PJ, Murphy ED, Roths JB, Dixon FJ: Spontaneous murine lupus-like syndromes clinical and immunopathological manifestations in several strains. J Exp Med 1978;148:1198-1215.

4 Herron LR, Eisenberg RA, Roper E, Kakkanaiah VN, Cohen PL, Kotzin BL: Selection of the T-cell

receptor repertoire in Lpr mice. J Immunol 1993;151:3450-3459.

5 Maldonado MA, Eisenberg RA, Roper E, Cohen PL, Kotzin BL: Greatly reduced lymphoproliferation in lpr mice lacking major histocompatibility complex class I. J Exp Meal 1995;181:641-648.

6 Eisenberg RA, Tan EM, Dixon FJ: Presence of anti-Sin reactivity in autoimmune mouse strains. J Exp Med 1978;147:582-587.

7 Elkon KB, Bonfa E, Llovet R, Eisenberg RA: Association between anti-Sin and anti-ribosomal P protein autoantibodies in human systemic lupus erythematosus and MRL/lpr mice. J Immunol 1989;143:1549-1554.

8 Treadwell EL, Cohen P, Williams D, O'Brien K, Volkman A, Eisenberg RA: MRL mice produce anti-Su autoantibody a specific marker for

systemic lupus erythematosus. J lmmunol 1993;150:695-699.

9 Eisenberg RA, Craven SY, Fisher CL, Morris SC, Rapoport R, Pisetsky DS, Cohen PL: The genetics of autoantibody production in MRL/ lpr lupus mice. Clin Exp Rheu- matol 1989;7:$35~-0.

10 Watson ML, Rao JK, Gilkeson GS, Ruiz P, Eicher EM, Pisetsky DS, Matsuzawa A, Rochelle JM, Seldin MF: Genetic analysis of MRL-lpr mice: relationship of the Fas apoptosis gene to disease manifestations and renal disease modifying loci. J Exp Med 1992; 176:1645-1656.

11 Cohen PL, Creech E, Nakul- Aquaronne D, McDaniel R, Ackler S, Rapoport RG, et al.: Antigen nonspecific effect of major histocompatibility complex haplotype on autoantibody levels in systemic lupus

46 Eisenberg

erythematosus-prone Ipr mice. J CIin Invest 1993;91:2761-2768.

12 Nagata S: Apoptosis by death factor. Cell 1997;88:355-365.

13 Izui S, Kelley VE, Masuda K, Yoshida H, Roths JB, Murphy ED: Induction of various autoantibodies by mutant gene lpr in several strains of mice. J Immunol 1984;133:227-233.

14 Roths JB, Murphy ED, Eicher EM: A new mutation gld that produces lymphoproliferation and autoimmunity in C3H/HeJ mice. J Exp Med 1984;159:1-20.

15 Bos NA, Kimura H, Meeuwsen CG, De Visser H, Hazenberg MP, WostmannBS,et al.: Semmimmuno- globulin levels and naturally occuring antibodies against carbo- hydrate antigens in germ-free BALB/c mice fed chemically deemed ultrafiltered diet. Eur J Immunol 1989;19:2335-2339.

16 Katagiri T, Cohen PL, Eisenberg RA: The lpr gene causes an intrinsic T-cell abnormality that is required for hyperproliferation. J Exp Med 1988;167:741-751.

17 Sobel ES, Cohen PL, Eisenberg RA: lpr T-cells are necessary for autoantibody production in lpr mice. J Immunol 1993; 150:4160-4167.

18 Sobel ES, Katagiri T, Katagiri K, Morris SC, Cohen PL, Eisenberg RA: An intrinsic B cell defect is required for the production of autoantibodies in the lpr model of murine systemic autoimm-nnity. J Exp Med 1991;173: 1441-1449.

19 Sobel ES, Kakkanaiah VN, Kakkanaiah M, Cheek RL, Cohen

PL, Eisenberg RA: T-B collab- oration for autoantibody production in lpr mice is cognate and MHC-restricted. J lmmunol 1994;152:6011-6016.

20 Fossati L Sobel ES, Iwamoto M, Cohen PL, Eisenberg RA, Izui S: The Yaa gene-mediated acceler- ation of murine lupus: Yaa- T- cells from nonautoimmune mice collaborate with Yaa+ ET-cells to produce lupus autoantibodies in vivo. Eur J Immunol 1995;25: 3412-3417.

21 Kakkanaiah VN, Sobel ES, MacDonald GC, Cheek RL, Cohen PL, Eisenberg RA: B cell genotype determines the fine specificity of autoantibody in lpr mice. J Immunol 1997;159:1027-1035.

22 Sobel ES, Kakkanaiah VN, Cohen PL, Eisenberg RA: Correction of gld autoimmunity by co-infusion of normal bone marrow suggests that gld is a mutation of the Fas ligand gene. Int Immunol 1993; 5:1275 1278.

23 Sobel ES, Kakkanaiah VN, Kakkanaiah M, Cohen PL, Eisenberg RA: Co infusion of normal bone marrow partially corrects the gld T-cell defect Evidence for an intrinsic and extrinsic role for Fas ligand. J Immunol 1995;154:459M-64.

24 MacDonald GC, Kakkanaiah VN, Sobel ES, Cohen PL, Eisenberg RA: In vivo depletion of Thy-l-positive cells originating from normal bone marrow abrogates the suppression of gld disease in normal-gld mixed bone marrow chimeras. J lmmuno11995; 154:444449.

25 Singer GG, Abbas AK: The fas antigen is involved in peripheral but not thymic deletion of T lymphocytes in T cell receptor transgenic mice Immunity 1994; 1:365-371.

26 Eisenberg RA, Craven SY, Warren RW, Cohen PL: Stochastic control of anti-Sin autoantibodies in MRL/ Mp-lpr/Ipr mice. J Clin Invest 1987 ;80:691-697.

27 Licht T Aksentijevich I, Gottesman MM, Pastan I: Efficient expression of functional human MDRI gene in murine bone marrow after retroviral transduction of purified hematopoietic stem cells. Blood 1995;86:111-121.

28 Snowden JA Biggs JC, Brooks PM: Autologous blood stem cell transplantation for autoimmune diseases. Lancet 1996;348: 1112,1113.

29 van der Veen FM, Rolink AG, Gleichmann E: Autoimmune disease strongly resembling systemic lupus erythematosus (SLE) in FI mice undergoinggraft-versus-host reaction (GVHR). Adv Exp Med Biol 1982;149:669~;77.

30 Morris SC, Cheek RL, Cohen PL, Eisenberg RA: Autoantibodies in chronic graft versus host result from cognate T-B interactions. J Exp Med 1990;171:503-517.

31 Bradley DS, Jennette JC, Cohen PL, Eisenberg RA: Chronic graft versus host disease-associated autoimmune manifestations are independently regulated by diff- erent MHC class lI loci. J Immunol 1994;152:1960-1969.


Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.

out.pdf

Documents

Transcript of out.pdf