Perforin Protects Against Autoimmunity in Lupus-Prone Mice

Stanford L. Peng, Javid Moslehi, Marie E. Robert and Joe Craft
J Immunol January 15, 1998, 160 (2) 652-660;

Abstract

The roles of cytolytic regulatory mechanisms in the immune system of lupus-prone mice were examined in perforin-deficient animals bearing functional or defective (lpr) Fas Ag (CD95). Perforin-deficient Fas+ animals developed accelerated autoimmunity, characterized by increased hypergammaglobulinemia, autoantibody production, and immune deposit-related end-organ disease compared with perforin-intact counterparts. In comparison, perforin-deficient lpr animals had accelerated mortality compared with perforin-intact lpr mice, associated with the abnormal accumulation of CD3+CD4−CD8− αβ T cells in conjunction with unaltered hypergammaglobulinemia, autoantibody production, and immune complex renal disease. These results indicate that cytolytic lymphoid regulation plays critical roles in the immune homeostasis of lupus-prone animals, and identify perforin-mediated cytotoxicity as a specific mechanism in the regulation of systemic autoimmunity.

MRL/Mp-lpr/lpr (MRL/lpr) mice spontaneously develop a severe autoimmune syndrome closely resembling systemic lupus erythematosus, characterized by hypergammaglobulinemia, autoantibody production, lymphadenopathy, and end-organ disease associated with immune complex formation (1). Their lymphadenopathy consists of a nonmalignant lymphoaccumulation of CD4−CD8−B220+ T cells (2), a consequence of defective activation-induced cell death (3, 4, 5, 6) secondary to functional Fas (CD95) deficiency (7, 8, 9, 10, 11). End-organ disease includes both hepatic (12) and salivary gland (13) lesions, but renal disease, characterized by glomerulonephritis, interstitial nephritis, and perivasculitis (14, 15, 16), has been examined most intensely, and has been linked to an immune complex pathogenesis (14, 17, 18).

Several studies have established that MRL lupus is driven by pathogenic CD4+ αβ T cells that provide help to autoreactive B cells (19, 20, 21). A more complex dysregulation of the immune system, however, in which both pathogenic and down-regulatory T cells compete for control of autoimmunity, is most likely associated with disease pathogenesis (22). Proposed regulatory mechanisms include cytokine-mediated modulation of immune responses, such as the skewing of Th1- vs Th2-dominated environments (23), but most studies have indicated pathogenic roles for several cytokines in murine lupus, including IFN-γ, IL-4, IL-6, IL-10, and IL-12 (24, 25, 26, 27, 28, 29, 30). On the other hand, T cells may directly regulate pathogenic lymphocytes by cytotoxicity, as suggested by the Fas-mediated regulation of some activated T cells and B cells (31, 32, 33). Indeed, more recent work in MRL lupus has suggested that γδ T cells regulate pathogenic αβ T cells (34), while γδ T cells and subsets of regulatory αβ T cells regulate B cells (22, 34, 35), via both Fas-dependent and Fas-independent mechanisms (34, 35, 36).

Other than Fas, potential regulatory mechanisms in lupus include TNF and perforin (pfp);3 together, these three mediate the majority of cytotoxic lymphocyte responses (37). A role for TNF in the down-regulation of lupus has been strongly suggested by the exacerbation of disease in TNF receptor-deficient lpr animals (12); however, the regulatory role of pfp in systemic autoimmunity is unknown, despite its critical role in lymphocyte-mediated cytotoxicity (38, 39, 40, 41). Instead, investigations in murine lupus have focused upon the potential pathogenic role of pfp: one in MRL/lpr mice described the constitutive, functional expression of pfp by the expanded CD4−CD8− T cells (42), and two studies in New Zealand mice demonstrated the steroid-sensitive expression of pfp in T cells from disease lesions (43, 44).

The circumstantial evidence implicating a regulatory role for pfp in murine lupus (34, 35, 36) suggested that pfp deficiency may exacerbate, rather than ameliorate, murine lupus. To explore this possibility, pfp-deficient lupus-prone animals were generated by crossing pfp−/− mice (41) with MRL/lpr breeders. Pfp-deficient Fas+ animals developed accelerated autoimmune disease compared with pfp-intact Fas+ counterparts, characterized by increased hypergammaglobulinemia, autoantibody production, and end-organ disease. In comparison, in lpr animals pfp deficiency caused early mortality associated wtih T cell lymphoaccumulation reminiscent of lpr-related lymphoproliferative disease. These results demonstrate that pfp plays essential roles in the regulation of both humoral autoimmunity and lymphoid regulation, and emphasize the critical role of cytotoxicity in the modulation of systemic autoimmunity.

Materials and Methods

Mice

Pfp−/− (C57BL/6 × 129/Sv) mice (41) (Taconic, Germantown, NY) were crossed against MRL/lpr breeders (The Jackson Laboratory, Bar Harbor, ME) to generate pfp+/− Fas+/lpr double-heterozygotic offspring, which were intercrossed to generate pfp-intact (pfp+) and pfp-deficient (pfp−) Fas-intact (Fas+) or lpr/lpr (lpr) F2 animals. Genotypes were screened via PCR of tail DNA, using primers PFP451F (5′-GCCTACCTGTGGCAATCACCCACTG), PFP670R (5′-TCAGCTGCAAAATTGGCTACCTTGG), and NEOMS1 (45) (5′-CCTTGCGCAGCTGTGCTCGACGTTG). The PFP451F-PFP670R combination yielded a 219-bp fragment corresponding to the wild-type allele, while the PFP451F-NEOMS1 combination yielded an approximately 720-bp fragment corresponding to the mutant allele (not shown). Fas genotype was also determined by PCR, as described elsewhere (3). All animals were maintained under specific pathogen-free conditions at Yale University School of Medicine (New Haven, CT).

Cytotoxicity assays

To assay cytotoxic activities in vitro, NK cell-enriched splenocytes were used as effectors against 51Cr-labeled YAC-1 targets (46). Briefly, twice-washed, erythrocyte-cleared splenocytes were passed through a prewarmed, prewetted nylon wool column, incubated at 37°C for 45 min. NK effectors were prepared from nonadherent cells, which were expanded for 48 h at 2 × 106 cells/ml in DMEM (Life Technologies, Gaithersburg, MD) containing 10% FCS and 500 U/ml of mouse rIL-2 (Sigma Chemical Co., St. Louis, MO). YAC-1 targets (TIB-160; American Type Culture Collection, Rockville, MD) were labeled for 2 h with 100 μCi of Na51CrO4 (Amersham Life Science Corp., Arlington Heights, IL), washed thrice, and resuspended in DMEM containing 10% FCS. A quantity amounting to 5 × 103 labeled target cells in 100 μl was mixed with an equal volume of effector cells at varying ratios in a round-bottom 96-well plate (Falcon 3077; Becton Dickinson, Lincoln Park, NJ). Cells were centrifuged at 100 × g to ensure contact, incubated for 4 h at 37°C, 5% CO2/air atmosphere, and then centrifuged again at 100 × g. One hundred microliters of supernatant were assayed for 51Cr release by standard gamma counter.

Ab analyses

Serum Ig and autoantibody studies were performed as previously described (35). Briefly, antinuclear Abs were assayed by indirect immunofluorescence (FANA) on sera diluted at 1/50 using HEp-2 substrate cells (Quidel, San Diego, CA). Fluorescence was visualized with a Zeiss Axioskop microscope at ×1000 magnification, with intensity rated 0 to 4+, as determined by the light meter’s estimated required exposure time for 10 to 12 cells per high power field on ASA 400 film (>120 s, 0; <120 s, 1+; <60 s, 2+; <30 s, 3+; <15 s, 4+). Specific autoantibody titers (IgG anti-dsDNA, IgG anti-small nuclear ribonucleoprotein (snRNP), and κ rheumatoid factor) were determined by ELISA using specific substrates on sera at 1/100 dilution. Serum Ig titers were determined by ELISA (Pierce, Rockland, IL, or PharMingen, San Diego, CA). Statistical significance was evaluated by paired Student’s t test on mean Ig titers or autoantibody OD values. FANA comparisons utilized t test on proportions.

Cell analyses

Spleens and six peripheral nonmesenteric lymph nodes were weighed, homogenized, and cleared of erythrocytes by osmotic lysis. Live cell count was determined by trypan blue exclusion. For phenotypic analyses, cells were analyzed by FACSort flow cytometry and CellQuest 1.1 software (Becton Dickinson, Bedford, MA): Abs included H129.19 FITC or PE (anti-CD4), GL3 PE (anti-TCR-γδ), RA3-6B2 biotin (anti-CD45R/B220), H57-597 FITC, or PE (anti-TCR-β; all from PharMingen), and/or 53-6.7 Quantum Red (anti-CD8α; Sigma Chemical Co.), followed by streptavidin-Texas Red (Life Technologies).

Histopathology

Tissue specimens were fixed in 10% buffered Formalin, and sections were stained with hematoxylin and eosin by standard procedures at Department of Pathology, Yale University School of Medicine (15, 16, 35, 36). Disease was evaluated on a 0 to 4 scale in renal glomeruli, tubules, and perivascular regions; salivary gland acini and perivascular regions; and hepatic periportal regions. The degree and histopathologic character of lymphoid infiltrates were evaluated in kidney, liver, and salivary gland sections. Immunofluorescent studies were performed onOCT-embedded frozen sections, as described (35, 36), using FITC- and/or PE-conjugated 145-2C11 (anti-CD3)CD3), H129.19 (anti-CD4), 53-6.7 (anti-CD8α), RA3-6B2 (anti-B220), H57-597 (anti-TCR Cβ), RR8-1 (anti-TCR Vα11b,d), B20.6 (anti-TCR Vβ2), RR4-7 (anti-Vβ6), MR5-2 (anti-TCR Vβ8.1/Vβ8.2), 1B3.3 (anti-TCR Vβ8.3), B21.5 (anti-TCR Vβ10b), 14-2 (anti-TCR Vβ14), and PE-conjugated KJ25 (anti-Vβ3; all from PharMingen). Immune deposit analyses utilized FITC-conjugated polyclonal goat anti-mouse IgG (Sigma Chemical Co.). All immunofluorescent photographs were taken on ASA 400 film at an exposure time of 8 s.

Results

Generation of pfp−/− lupus-prone mice

Pfp−/− (41) animals were crossed once against MRL/lpr breeders, generating pfp+/− Fas+/lpr offspring. These F1 animals were intercrossed to generate pfp+/+ (pfp+) or pfp−/− (pfp−), Fas+/+ (Fas+), or lpr/lpr (lpr) F2 animals, generating four genotypic groups of similar genetic backgrounds (approximately 50% MRL). Since previous studies have demonstrated the dominance of MRL autoimmunity-inducing genes in genetic crosses (34, 35, 47, 48), the background of these animals conveys a lupus-like autoimmune phenotype. Indeed, the disease of these mice can be considered representative of typical MRL murine lupus, as indicated by their production of hypergammaglobulinemia, specific autoantibodies, and end-organ disease (34, 35, 48, and see below). PCR-assayed genotypes were confirmed by flow cytometry for Fas staining, as well as by NK cytotoxicity assay: consistently, NK cell-enriched splenocytes from pfp− animals failed to lyse pfp-sensitive YAC-1 cells, whereas those from pfp+ animals successfully achieved as high as 40% specific target cell lysis, using E:T ratios varying from 40:1 to 1.25:1, regardless of Fas genotype (Fig. 1⇓ and data not shown).

FIGURE 1.
FIGURE 1.

Cytotoxicity activity of pfp-deficient mice. NK-enriched splenocytes from three pfp+ and three pfp− animals were used as effectors against 51Cr-labeled YAC-1 target cells. In contrast to pfp+ cells, pfp− cells consistently lacked cytotoxic activity, as assayed by 51Cr release. The data here are representative of three similar experiments using both Fas+ and lpr cells; error bars represent SDs.

Survival of pfp− lupus-prone mice

Notably, the median survival of pfp−lpr animals (n = 8) was only 8 wk, whereas pfp+lpr animals (n = 12) had a median survival of 24 wk (p < 0.001), typical of most hybrid MRL lupus strains (30, 34, 36, 47, 48). In contrast, all nine pfp+Fas+ and nine pfp−Fas+ animals survived beyond 32 wk of age.

Abnormal lymphoaccumulation in pfp−lpr mice

The accelerated mortality of pfp−lpr mice was most likely explained by uncontrolled lymphoaccumulation that afflicted their kidneys, livers, and salivary glands, as well as lymphoid organs (Table I⇓ and Figs. 2⇓–4). In all organs examined, pfp−lpr mice had substantial organ destruction by a monomorphic, atypical lymphoid infiltrate, characterized by enlarged, vesicular nuclei with indistinct nucleoli and irregular nuclear contours (Fig. 2⇓ and data not shown). Occasional small mature lymphocytes were scattered throughout such lesions. In salivary glands, only rare residual acini and salivary ducts remained (Fig. 2⇓H and data not shown), while in kidneys and livers, similar infiltrates were restricted predominantly in a perivascular location with a lesser degree of interstitial involvement (Fig. 2⇓, G and J, respectively, and data not shown). These infiltrates were highly reminiscent of lpr-induced lymphoaccumulative disease.

FIGURE 2.
FIGURE 2.

End-organ disease in perforin-deficient mice. A, Renal tissues in pfp+Fas+ animals were typically normal or occasionally had perhaps mild glomerular hypercellularity, as seen in this 12-wk-old specimen. Age-matched pfp−Fas+ animals and 8-mo-old pfp+Fas+ animals had kidneys with similarly negligible histologic findings. B, Both pfp+Fas+ and pfp−Fas+ animal groups also had grossly normal salivary gland histology, as seen in this 12-wk-old pfp+Fas+ tissue. Eight-month-old pfp−Fas+ animals, however, developed moderate glomerular disease with mild-moderate interstitial and perivascular infiltrates, as demonstrated by a specimen with glomerulonephritis and mild periglomerular reaction (C); and also developed moderate sialoadenitis consisting of destructive lesions of acini as well as perivascular infiltrates (D). Pfp+lpr animals consistently developed severe renal and salivary gland disease, which was evident in both organs by 12 wk of age: mesangial proliferation and hypercellularity, as well as mild interstitial infiltrates, were typically seen in kidneys (E), and perivasculitis and acinar infiltration were typically seen in salivary glands (F). Renal perivasculitis was also evident (not shown). Twelve-week-old pfp−lpr mice displayed (H) diffuse replacement of the salivary gland by an atypical lymphoid infiltrate that left only rare salivary acini and ducts. Smaller perivascular infiltrates with identical cytology were present in the kidney (G) and liver (J), with focal extension into the parenchyma. I, Most pfp+Fas+ animals had grossly normal hepatic histology, but occasionally had mild periportal infiltrates, as seen in this 12-wk-old specimen. Numbers of animals examined histologically were 3, 3, 3, 4, 3, 3, and 2 for the pfp+Fas+ 12 wk, pfp−Fas+ 12 wk, pfp+lpr 12 wk, pfp−lpr 12 wk, pfp+Fas+ 8 mo, pfp−Fas+ 8 mo, and pfp+lpr 8 mo groups, respectively. Scale bar, 25 μm.

View this table:
Table I.

End-organ disease in perforin-deficient lupus-prone mice

In contrast, kidneys, salivary glands, and livers from pfp+lpr mice revealed only limited and variable parenchymal lymphoid infiltrates, again in a perivascular pattern, but consisting of a polymorphous lymphoplasmacytic population, typical of MRL/lpr autoimmune disease (Fig. 2⇑, E and F, and data not shown) (12, 13, 14). Although mildly enlarged lymphocytes were present, the cytologic atypia seen in pfp−lpr animals was uniformly lacking. The atypical cells in pfp−lpr animals were αβ+CD3+CD4−CD8−B220− T cells, as determined directly by immunofluorescence on frozen tissue sections and indirectly by flow cytometry on cell suspensions from spleen and lymph nodes (p < 0.001; Figs. 3⇓ and 4 and data not shown; note the expansion of a TCR−-αβ+CD4−CD8−B220− T cell subset in pfp−lpr compared with pfp+lpr lymph nodes). In comparison, the infiltrates of pfp+lpr mice consisted predominantly of CD3−B220+ B cells, although CD3+B220− and CD3+B220+ T cells were occasionally seen. Thus, pfp contributes to the regulation of cellular homeostasis in lpr animals.

FIGURE 3.
FIGURE 3.

Immunofluorescence characterization of pfp-deficient lpr infiltrates. A, Negative background fluorescence of an infiltrate from a pfp+lpr animal stained with anti-Vβ14 Ab (scale bar, 10 μm). Pfp−lpr infiltrates were characterized as predominately TCR-αβ+CD3+CD4−CD8−B220−B, Here, a predominantly Vβ14+ area is shown, which stained for occasional Vβ8.1/Vβ8.2+ cells (scale bar, 25 μm), and C, many Vβ14 cells (scale bar, 10 μm). Immunohistologic analyses were performed from animals at 12 wk of age; these results are representative of 3, 3, 3, and 4 animals examined for the pfp+Fas+, pfp−Fas+, pfp+lpr, and pfp−lpr groups, respectively. Kidney infiltrates are shown here.

Accelerated autoimmunity in pfp−Fas+mice

In contrast, Fas-intact animals required pfp primarily to regulate humoral and end-organ autoimmune disease. Pfp−Fas+ animals had more hypergammaglobulinemia, higher levels of autoantibodies, and more severe end-organ disease than their pfp+Fas+ counterparts (Tables I-III, Figs. 5⇓–7, and data not shown). At 3 mo of age, pfp−Fas+ mice had higher titers of IgM, IgG1, IgG2a, and IgG2b than pfp+Fas+ counterparts (p < 0.001), and 8-mo-old animals maintained higher levels of IgM, IgG1, and IgG2b (p < 0.005; Table III⇓ and Fig. 5⇓). Similarly, pfp−Fas+ animals also developed higher levels of autoantibodies: at both 3 and 8 mo of age, they contained higher levels of rheumatoid factor and anti-snRNP Abs (p < 0.01), and at 8 mo of age, they contained higher levels of anti-dsDNA Abs (p < 0.01; Fig. 6⇓). In addition, all nine of nine 8-mo-old pfp−Fas+ animals tested positive for antinuclear Abs, in comparison with only five of nine pfp+Fas+ animals (p < 0.001). In contrast, no clear differences in serum Ig and autoantibody titers were observed between pfp−lpr and pfp+lpr animals, which developed higher levels of hypergammaglobulinemia, particularly of the IgG2a and IgG3 isotypes (p < 0.001), and autoantibodies, particularly antinuclear Abs and rheumatoid factor (p < 0.001), compared with Fas+ animals.

FIGURE 4.
FIGURE 4.

Cell populations in pfp-deficient mice. Spleens and six peripheral nonmesenteric lymph nodes were weighed, and their cells were counted. Lymph node cells were further analyzed by flow cytometry for B cell, γδ T cell, and αβ T cell populations; the latter was further analyzed by CD4, CD8, and B220 staining, demonstrating the expansion of CD4−CD8−B220− αβ T cells in pfp−lpr vs pfp+lpr mice. Comparable findings were found among spleens of the same animals (not shown). In the lower right panel, αβ T cells were B220−, unless specified as expressing this marker. Analyses were performed from animals at 12 wk of age; these results are representative of 3, 3, 3, and 4 animals for the pfp+Fas+, pfp−Fas+, pfp+lpr, and pfp−lpr groups, respectively. Note the use of logarithmic scale in the two upper and lower left panels.

FIGURE 5.
FIGURE 5.

Serum Igs in pfp-deficient mice. Sera were assayed for Ig isotype titers by ELISA. Note that the two remaining pfp+lpr animals that were available for analysis at 31 wk of age most likely reflect milder disease than typical for pfp+lpr disease, since their median survival was only 24 wk in this study. Numbers of animals examined for Ig synthesis were 12, 12, 12, 4, 9, 9, and 2 for the pfp+Fas+ 12 wk, pfp−Fas+ 12 wk, pfp+lpr 12 wk, pfp−lpr 12 wk, pfp+Fas+ 8 mo, pfp−Fas+ 8 mo, and pfp+lpr 8 mo groups, respectively, as described in Table III⇓. Note the use of logarithmic scale.

FIGURE 6.
FIGURE 6.

Autoantibody production by pfp-deficient mice. Sera were assayed for antinuclear Abs by the indirect antinuclear Ab test (FANA). Specific autoantibodies were tested by specific ELISA for IgG anti-dsDNA, IgG anti-snRNP, and κ rheumatoid factor titers. Normals include B10.BR and/or RAG-1−/− mice. Note that the two remaining pfp+lpr animals that were available for analysis at 31 wk of age most likely reflect milder disease than typical for pfp+lpr disease, since their median survival was only 24 wk in this study. Numbers of animals examined for autoantibodies were as described in Table III⇓ and this figure.

View this table:
Table II.

Renal Ig deposition in perforin-deficient lupus-prone micea

View this table:
Table III.

Mean serum Igs in perforin-deficient lupus-prone micea

End-organ disease in pfp−Fas+ animals primarily consisted of more intense light-microscopic disease of kidneys and salivary glands in comparison with pfp+Fas+ counterparts (Table I⇑ and Fig. 2⇑ and data not shown). Although all Fas+ animals at 3 mo of age had only mild or absent end-organ disease, pfp+Fas+ animals at 8 mo of age developed mild glomerular disease and occasionally mild perivascular infiltrates in salivary glands. Age-matched pfp−Fas+ animals, however, consistently contained more glomerular, interstitial, and perivascular renal lesions and/or infiltrates, as well as greater acinar damage and perivascular infiltrates in salivary glands. Hepatic infiltrates also appeared more common and perhaps more intense among pfp−Fas+ animals as compared with pfp+Fas+ counterparts, even though disease remained generally mild among all Fas+ animals. Notably, pfp+lpr animals developed renal, salivary gland, and hepatic lesions typical of MRL/lpr autoimmune disease, including glomerulonephritis, interstitial infiltrates, and perivasculitis in kidneys; acinar atrophy with periductal and perivascular infiltrates in salivary glands; and mild-moderate periportal infiltrates in livers.

The accelerated end-organ disease of pfp−Fas+ animals most likely resulted from increased immune complex-related formation secondary to accelerated humoral autoimmunity, since their renal lesions consistently contained greater immune deposits, as assayed by immunofluorescent studies (Table II⇑ and Fig. 7⇓). Pfp−Fas+ animals generally had diffuse capillary wall and mesangial immune deposits affecting all glomeruli, significantly more prominent than their pfp+Fas+ counterparts. The former also had more significant tubular basement membrane as well as occasional intranuclear staining, which were not apparent in pfp+Fas+ specimens. By comparison, both pfp+lpr and pfp−lpr animals developed severe diffuse mesangial and capillary wall deposits as well as glomerular collapse, consistent with their proliferative glomerulonephritis, and also developed tubular and intranuclear deposits as well.

FIGURE 7.
FIGURE 7.

Renal immune deposits in pfp-deficient lupus-prone mice. A, Pfp+Fas+ kidneys generally contained negligible immune deposits. B, Age-matched, pfp−Fas+ kidneys, however, contained mild glomerular and tubular immune deposits, with mild antinuclear Ab staining. Pfp+lpr kidneys (C) and pfp−lpr kidneys (D) contained substantial glomerular, tubular, and usually antinuclear immune deposits, typical of MRL/lpr immune complex glomerulonephritis (14, 17, 18, 34, 35). Analyses were performed from animals at 12 wk of age; these results are representative of 3, 3, 3, and 4 mice for the pfp+Fas+, pfp−Fas+, pfp+lpr, and pfp−lpr groups, respectively. Photographs were taken on ASA 400 film at an exposure time of 8 s. Scale bar, 25 μm.

Of note, overall cell populations were largely unaffected by pfp deficiency, particularly in Fas+ animals (Fig. 4⇑). Comparable spleen and lymph node weights and cell counts were found between pfp+Fas+ vs pfp−Fas+ animals, as well as between pfp+lpr vs pfp−lpr animals. Furthermore, pfp+ animals also contained numbers of αβ T cells, γδ T cells, and B cells (B220+αβ−γδ− cells) similar to pfp− animals, comparing pfp+Fas+ with pfp−Fas+ animals, as well as pfp+lpr with pfp−lpr animals.

Discussion

This study has demonstrated a protective role for pfp in murine lupus. Pfp−lpr mice developed significantly more lymphoaccumulation than their pfp+lpr counterparts, although hypergammaglobulinemia, autoantibody production, immune complex renal disease, and lymphadenopathy were grossly similar. In comparison, pfp−Fas+ animals developed significantly higher degrees of hypergammaglobulinemia, autoantibodies, and end-organ disease than pfp+Fas+ animals. Relatively small numbers of animals were examined in each genotypic group, perhaps making broad, definitive conclusions regarding the regulation of autoimmunity difficult. Nevertheless, these findings provide evidence that cytotoxic interactions down-regulate systemic autoimmune processes, in contrast to the pathogenic roles of cognate Th cells (19, 20, 21), and specifically identify pfp as a mechanism for such regulation. Of note, the results obtained in this study do not simply reflect a stochastic effect of the various genetic backgrounds on disease penetrance, since the four groups of mice used in this study each consisted of approximately 50% MRL genes, creating comparable autoimmune-prone, lymphoproliferation-prone genetic environments for all groups. Consequently, the effects of pfp− and/or Fas− deficiency seen in this study most likely reflect underlying processes characteristic to this background (34).

The findings in Fas-intact animals indicate a significant role for pfp in the regulation of systemic humoral autoimmunity and the consequent development of end-organ disease. Pfp may play a direct role in the regulation of autoreactive B cells by T cells or NK cells, such that pfp deficiency results in increased generalized autoreactive B cell hyperactivity, which previously has been suggested as pathogenic in murine lupus models (49, 50, 51). Pfp appears subordinate to that of Fas in the regulation of autoreactive B cells, however, since pfp deficiency did not result in humoral and end-organ disease similar to lpr disease; indeed, the grossly unchanged lymphocyte populations in pfp−Fas+ animals (Fig. 4⇑) argue against an overt regulatory defect in the T cell or B cell lineages. Rather, Fas-dependent regulation of B cells by T cells (31, 32, 33) or by NK cells (52, 53) most likely helps to prevent otherwise severe autoimmunity in pfp−Fas+ animals. As an alternative, pfp may provide a mechanism by which regulatory T cells, either αβ and/or γδ (22, 34, 35, 36), eliminate activated, pathogenic αβ T cells (22, 34).

The acceleration of end-organ disease in spite of relatively preserved humoral autoimmune parameters in pfp−lpr animals reinforces a role for pfp in cellular regulation, and suggests that pfp primarily regulates autoreactive T cells, rather than B cells, at least in the setting of Fas deficiency. Accordingly, autoreactive B cell activation appeared unchanged in pfp−lpr animals, as assayed by B cell counts, hypergammaglobulinemia, autoantibodies, and renal immune deposits, in comparison with the acceleration of autoimmunity by pfp deficiency in Fas+ animals. Indeed, Fas may be the primary mechanism by which B cells are regulated (31, 32, 33, 52, 53), and thus, pfp deficiency may not significantly affect lpr B cells. On the other hand, lpr T cells appear to rely heavily upon pfp for self-surveillance in the absence of Fas-Fas ligand interactions, resulting in T cell lymphoaccumulation in double-deficient states, even though other, tertiary pathways for cytotoxicity most likely remain (54). Alternatively, if pfp acts via other immune cell populations, such as NK cells, cellular effects may similarly predominate over humoral effects due to the roles of these cell types in primarily cellular immune effects, such as Ab-dependent or complement-mediated cytotoxicity.

Such considerations raise interest in the origin of the T cells that comprise the lymphoaccumulation syndromes of both pfp+lpr and pfp−lpr animals. Pfp deficiency may simply exacerbate lpr disease, such that the defective activation-induced cell death characteristic of lpr T cells is simply further propagated in double-deficient animals, resulting in the lymphoaccumulation of multiple autoreactive T cell clones previously stimulated by autoantigen(s) (3, 4, 5, 6). Pfp may also, however, play a role in the regulation of T cells undergoing different types of immune responses, such as during infection or tumor progression, such that combined pfp and Fas deficiency result in global dysregulation of ongoing immune responses. For example, the T cell infiltration in pfp−lpr animals may actually reflect an additional reactive lymphoproliferation to a distinct underlying malignancy, such as the hyperproliferation of atypical, clonal T cells in response to underlying EBV-induced B cell lymphoma in lymphomatoid granulomatosis (55, 56, 57), as suggested by the presence of a T cell-regulated B cell lymphoma in lpr animals (36). These considerations may be relevant to tumor progression in settings of immunodeficiency, such as AIDS, in which defective lymphocyte-mediated suppression may permit the development of malignant lymphoma (58, 59).

These demonstrations of the importance of cytotoxic mechanisms in systemic autoimmunity also provide new directions of study for therapeutic interventions. Traditional approaches have involved global down-regulation of the immune system, such as by anti-inflammatory drugs, corticosteroids, or antimetabolic agents (60, 61). The acceleration of autoimmunity in both TNF-deficient (12) and pfp-deficient lupus-prone animals (this study) strongly suggests that particular lymphocyte subsets provide significant regulatory functions that may be enhanced by specific therapeutic techniques. The identification and utilization of such cells will be of substantial interest to future investigations.

Acknowledgments

We thank Tom Taylor for assistance with flow cytometry.

Footnotes

  • ↵1 This work was supported in part by grants from National Institutes of Health (AR40072 and AR44076) and from the Arthritis and Lupus Foundations, their Connecticut chapters, and donations to Yale Rheumatology in the memories of Irene Feltman, Albert L. Harlow, and Chantal Marquis (to J.C.). S.L.P. was supported by Medical Scientist Training Program, Yale University School of Medicine.

  • ↵2 Address correspondence and reprint requests to Dr. Joe Craft, P.O. Box 208031, LCI 609, 333 Cedar Street, New Haven, CT 06520-8031. E-mail address: joseph.craft{at}yale.edu

  • 3 Abbreviations used in this paper: pfp, perforin; FANA, fluorescent antinuclear Ab; PE, phycoerythrin; snRNP, small nuclear ribonucleoprotein.

  • Received February 19, 1997.
  • Accepted September 30, 1997.

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The Journal of Immunology
Vol. 160, Issue 2
15 Jan 1998
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