Abnormal thymocyte development and production of autoreactive T cells in T cell receptor transgenic autoimmune mice.

Development of a C57BL/6-+/+ TCR transgenic mouse containing the rearranged TCR alpha- and beta-chain specific for the Db + HY male Ag results in production of a nearly monoclonal population of early thymocytes expressing the Db + HY reactive TCR. These thymocytes are autoreactive in H-2Db male mice and undergo clonal deletion and down-regulation of CD8. To study the effect of the lpr gene on development of autoreactive T cells, these transgenic mice were backcrossed with C57BL/6-lpr/lpr mice. T cell populations in the thymus and spleen were analyzed by three-color flow cytometry for expression of CD4, CD8, and TCR. The thymus of TCR transgenic H-2b/b lpr/lpr male mice had an increase in percent and absolute number of CD8dull thymocytes compared to TCR transgenic H-2b/b +/+ male mice. However, there was not a complete defect in clonal deletion, because clonal deletion and down-regulation of CD8 was apparent in both +/+ and lpr/lpr H-2Db HY+ male mice compared to H-2Db HY- female mice. The phenotype of splenic T cells was almost identical in TCR transgenic +/+ and lpr/lpr males with about 50% CD4-CD8- T cells and 50% CD8+ T cells. However, there was a dramatic increase in the SMLR proliferative response of splenic T cells from TCR transgenic lpr/lpr males compared to TCR transgenic +/+ males. To determine the specificity of this response, spleen cells from TCR transgenic lpr/lpr and +/+ mice were cultured with irradiated H-2b/b and H-2k/k male and female spleen cells. T cells from TCR transgenic C57BL/6-lpr/lpr male mice had an increased proliferative response to H-2b/b male spleen cells compared to T cells from TCR transgenic C57BL/6(-)+/+ male mice, but both lpr/lpr and +/+ mice had a minimal response to irradiated H-2b/b female or H-2k/k male or female stimulator cells. The splenic T cells from TCR transgenic lpr/lpr mice also had an increased specific cytotoxic activity against H-2b/b male target cells compared to TCR transgenic +/+ mice. These results demonstrate that there is a defect in negative selection of self-reactive T cells in the thymus of lpr/lpr mice and a defect in induction or maintenance of clonal anergy of self-reactive T cells in the periphery of lpr/lpr mice.     

Increased membrane immunoglobulin capping of B cells from C57Bl/6 lpr/lpr and C57Bl/6 nu/nu mice

When the capping of membrane immunoglobulin on spleen B cells from normal C57Bl/6 mice (B6) is taken as reference, a faster capping rate is found for cells of age-matched B6 mice which are congenic at the lymphoproliferation (lpr) or nude (nu) loci. Though both congenic strains can be characterized by an abnormal T-lineage cell content, the nature of the abnormality itself is very different since B6 nudes lack thymus-processed/influenced lymphocytes whereas B6 mice with the lpr phenotype suffer from an invasion of all lymphoid organs with cells of a particular T-cell subset. Moreover, the more "normal" capping rate of B cells from the double congenic B6 mice (nu/nu, lpr/lpr) is intriguing. Since other mice homozygous at the lpr locus (MRL-1) or at the nu locus (BALB/c nude) also cap faster than their congenic controls (MRL-n and BALB/c, respectively), the observed effects do not appear to depend on a peculiarity of the B6 genetic background. If the faster capping of B cells of nu congenic and of lpr congenic mice had a common origin, it might be that T cells would control in some way the mobility of B-cell membrane immunoglobulins: both congenic mice have in their spleen a very low proportion of mature T cells together with a very high proportion of prethymic/thymic immature T-cell types, either of which might affect B-cell behavioral responses to membrane immunoglobulin clustering.     

Autoreactive T cells in MRL/Mpr-lpr/lpr mice. Characterization of the lymphokines produced and analysis of antigen-presenting cells required

Lymph node cells from 4-wk-old MRL/Mp-lpr/lpr mice, but not from MRL/Mp-+/+ mice, when cultured in vitro for 5 to 7 days, will spontaneously proliferate and produce IL-2. We examined the expression of several cell surface Ag on lymph node cells from MRL/Mp-lpr/lpr mice before and after in vitro culture. There is an increase in the expression of Thy-1, L3T4, IL-2R, T cell activating protein, T cell receptor, and T3 complex on the surface of cultured cells. Cultured cells produced IL-3, IFN-gamma, and small but detectable amounts of IL-1 in addition to IL-2. Gamma irradiation of APC from young MRL/Mp-lpr/lpr mice or treatment of APC with a mAb (J11D) and C, completely abrogated their stimulatory capacity. These experiments suggest that B cells are the predominant APC responsible in the activation of autoreactive T cells in MRL/Mp-lpr/lpr mice. Lymph node cells from C57BL/6-lpr/lpr or C3H-lpr/lpr mice were unable to spontaneously proliferate or produce IL-2. Lymph node cells from (MRL/Mp-lpr/lpr x C57BL/6-lpr/lpr) F1 mice or (C3H-lpr/lpr x MRL/Mp-lpr/lpr) F1 mice did proliferate and produced IL-2 after in vitro culture. Using T cells from these F1 animals and APC from each parental haplotype, we found that APC from MRL/Mp-lpr/lpr mice induced more proliferation and greater amounts of IL-2, when compared to APC from F1 animals. APC from C57BL6-lpr/lpr mice or C3H-lpr/lpr were unable to induce spontaneous proliferation and IL-2 production. Therefore, B cells from MRL/Mp-lpr/lpr mice appear to possess unique features that enable them to activate autoreactive T cells more effectively than B cells from other mice bearing the lpr/lpr gene.     

High level expression of the plasma cell antigen PC.1 on the T-cell subset expanding in MRL/MpJ-lpr/lpr mice: detection with a xenogeneic monoclonal antibody and alloantisera

MRL/MpJ-lpr/lpr (MRL-lpr/lpr) mice spontaneously develop a systemic lupus erythematosus-like syndrome associated with the expansion of a T-cell subset exerting helper activity for autoantibody production. Several studies have demonstrated that these T cells have unusual phenotypic characteristics including the expression of the B220 B-cell marker. To further characterize the antigenic profiles of these T cells, we have generated monoclonal antibodies (MAb) by immunizing rats with MRL-lpr/lpr T cells. Using flow cytofluorometry analysis, one of these MAb (4G6), described here, was found to react strongly with T cells of MRL-lpr/lpr mice but weakly with T cells of congenic mice lacking the lpr mutation (MRL/MpJ-+/+ mice). This MAb also stained brightly T cells from C3H/Hej-lpr/lpr mice and dimly those from normal C3H/Hej mice. However, it failed to react with T cells from C57Bl/6-lpr/lpr mice or normal C57Bl/6 (B6) mice. Analysis of 4G6 reactivity (weak vs negative) of T cells in a series of inbred strains demonstrated a correlation with the Pca-1a genotype known to result in expression of the PC.1 antigen on plasma cells. Immunoprecipitation studies revealed that the 4G6 antigen has a mean apparent molecular weight of 115,000, under reducing conditions, similar to that of PC.1. Moreover, high expression of 4G6 was found on plasmacytoma lines and B blasts but not on T blasts. Identity of the 4G6 antigen with PC.1 was confirmed by the finding that conventional anti-PC.1 alloantisera could block the cell surface binding of the 4G6 MAb. Therefore, T cells from MRL-lpr/lpr (and C3H-lpr/lpr) mice aberrantly carry high levels of a plasma cell antigen, detected by the 4G6 MAb, which substantiates further that these T cells represent a unique subset with some surface properties of the B-cell lineage.     

Does depression of NK activity cause lymphadenopathy in lpr mice?

B6-lpr/lpr mice develop massive T cell lymphoproliferation, as associated with autoimmune disease. We found a reduced NK activity in the spleen of B6-lpr/lpr mice. Neonatal thymectomy markedly retarded the development of lymphoproliferation and the development of autoantibodies in the B6-lpr/lpr mice. These animals had a higher level of NK activity in the spleen. When the neonatally thymectomized B6-lpr/lpr mice were given anti-asialo GM1 serum (30 microliter) four times at 6-day intervals, initiated at the 8th-10th postnatal week, these mice developed lymphoproliferative disorders and splenomegaly, concomitantly with depression of NK activity. It is therefore tempting to speculate that NK cells are involved in the regulation of the occurrence of lymphoproliferative disorders.     

Genetic diversity of murine rheumatoid factors

Anti-Ig autoantibodies (rheumatoid factors, RF) have been implicated in the pathogenesis of human and murine rheumatoid arthritis as well as in the regulation of normal immune responses. Their genetic origin, clonal diversity, and inducing agents, and the relatedness between RF associated with disease and those occurring under physiologic conditions are not well understood. In this study, the genetic and clonotypic origin of 34 monoclonal IgM RF-secreting hybridomas from arthritic MRL-lpr/lpr and nonarthritic MRL-+/+ and C57BL/6-lpr/lpr mice was examined by RNA hybridization. For this purpose, we used probes for 10 VH and 13 Vk gene families as well as all JH and Jk gene segments. The majority of hybridomas expressed distinct Ig gene segment patterns and, hence, were clonally unrelated. Overall, a variety of different V and J gene segments were expressed in the hybridoma panel, suggesting that a large number of distinct genetic elements participates in expression of RF-like activity. RF from arthritic mice expressed Vk messages from the overlapping Vk22 and Vk28 gene families more frequently than did those from nonarthritic mice. RF from autoimmune MRL mice, both arthritic MRL-lpr/lpr and nonarthritic MRL-+/+, showed skewed JH4 segment usage, whereas those from C57BL/6-lpr/lpr preferentially expressed JH2.     

Studies of lymphoproliferation in MRL-lpr/lpr mice

MRL-lpr/lpr mice develop massive lymphoproliferation and an associated autoimmune process that includes anti-DNA formation, vasculitis, and glomerulonephritis. We have investigated the lymphoproliferation of MRL-lpr/lpr mice and have found that multiple factors are operative. Although neonatal thymectomy markedly retards the syndrome, chronic injection of poly rI.rC could substitute for the thymus. The resulting cells had the phenotype characteristic of the abnormal MRL-lpr/lpr T cells, Thy-1+, dull Ly-1+, Lyt-2-, 6B2+, Ig-. Splenectomy at 2 wk of age markedly retarded the development of this syndrome; however, splenectomy at birth did not. Some protection was afforded by splenectomy at 5 wk. Thus, there appears to be a critical period in the life of an MRL-lpr/lpr mouse when the spleen contributes importantly to the lymphoproliferation. A most remarkable observation was that an MRL-lpr/lpr spleen graft under the kidney capsule could induce lymphadenopathy characteristic of lpr/lpr mice in MRL +/+ recipients. Cells within the graft had to be able to proliferate for the adenopathy to occur because irradiation of the spleen with 800 R just before grafting abrogated the lymphadenopathy-inducing potential. No adenopathy was induced by +/+ spleen grafts placed into +/+ mice. Although MRL-lpr/lpr males develop disease slightly more slowly than female littermates, the differences are small. Manipulations that retard disease, such as splenectomy at 2 wk or neonatal thymectomy, magnified the sex differences. Male MRL-lpr/lpr mice that were thymectomized and splenectomized and given polyclonal immune activators failed to develop either anti-DNA or lymphadenopathy, whereas their female littermates expressed both abnormalities. We conclude from these studies that multiple factors serve to modulate the magnitude of the lymphoproliferation and autoimmune syndrome of MRL-lpr/lpr mice.     

Comparison of immunological effects of cholera toxin on autoimmune MRL/lpr and BXSB mice.

MRL/lpr and BXSB mice were treated weekly or biweekly with cholera toxin (CT) in intravenous dose of 2 micrograms/mouse. CT treatment notably alleviated proteinuria in MRL/lpr mice, but did not influence the course of lupus nephritis in BXSB male mice. Flow cytometric analysis showed that anomalous B220+ T cells in spleen and thymus were reduced in CT-treated MRL/lpr mice while no significant change in lymphocyte populations was induced in BXSB male mice by this treatment. The suppressive effect of CT treatment on Con A response and the augmentative action on LPS response were observed in MRL/lpr mice. The latter may reflect increased B cells in relative number in the peripheral lymphoid organs. Mitogenic responses in CT-treated BXSB male mice remained unchanged in comparison with those of untreated group. Increased production of IL-6 by spleen cells was demonstrated in MRL/lpr mice treated with CT while in BXSB mice the level of IL-6 was not changed by the treatment with CT. Production of IFN gamma was suppressed by CT treatment in both strains of mice. This may be attributed to the inhibitory effect of CT on IFN gamma-producing Th1 cells as reported previously (Munoz et al, J. Exp. Med. 172: 95-103, 1990). However, CT treatment did not inhibit anti-DNA antibody production in BXSB mice, whereas the autoantibodies were markedly decreased in MRL/lpr mice treated with CT.     

Opposing pharmacological actions of cepharanthin on lipopolysaccharide-induced histidine decarboxylase activity in mice spleens

A biscoclaurin alkaloid preparation, cepharanthin (Ceph), is reported to have opposing pharmacological effects, enhancement or depression, on several cells and tissues, although detailed mechanisms remain unclear. Previously, we reported that Ceph enhanced lipopolysaccharide (LPS)-induced histidine decarboxylase (HDC) activity in mice spleens by consecutive pre-administration. In this study, we examined the pharmacological effects on HDC activity of a single Ceph pre-administration to test the influence of the administration method. Consequently, HDC activities were decreased by a single administration 15 minutes before LPS challenge in ddY and ICR mice spleens. Moreover, to further examine this suppressing effect, we employed genetically mast cell-deficient WBB6F1 W/Wv (W/Wv) mice to avoid the influence of mast cells. In W/Wv mice, HDC activity was enhanced, but not in the congenic WBB6F1 +/+ mice. These findings suggest that mast cells influence the depressant effect on HDC activity by a Ceph single administration in mast cell sufficient mice.     

Increased protection from vaccinia virus infection in mice genetically prone to lymphoproliferative disorders

Mutations in the genes that encode Fas or Fas ligand (FasL) can result in poor restraints on lymphocyte activation and in increased susceptibility to autoimmune disorders. Because these mutations portend a continuously activated immune state, we hypothesized that they might in some cases confer resistance to infection. To examine this possibility, the immune response to, morbidity caused by, and clearance of vaccinia virus (VACV) Western Reserve was examined in 5- to 7-week-old Fas mutant (lpr) mice, before an overt lymphoproliferative disorder was observable. On day 6 after VACV infection, C57BL/6-lpr (B6-lpr) mice had decreased morbidity, decreased viral titers, and an increased percentage and number of CD4(+) and CD8(+) T cells. As early as day 2 after infection, B6-lpr mice had decreased liver and spleen viral titers and increased numbers of and increased gamma interferon (IFN-γ) production by several different effector cell populations. Depletion of individual effector cell subsets did not inhibit the resistance of B6-lpr mice. Uninfected B6-lpr mice also had increased numbers of NK cells, γδ(+) T cells, and CD44(+) CD4(+) and CD44(+) CD8(+) T cells compared to uninfected B6 mice. Antibody to IFN-γ resulted in increased virus load in both B6 and B6-lpr mice and eliminated the differences in viral titers between them. These results suggest that IFN-γ produced by multiple activated leukocyte populations in Fas-deficient hosts enhances resistance to some viral infections.     

Resistance to tolerance induction in B cells of autoimmune mice—Abnormal expansion of low affinity IgG-producing B-cell population

Several strains of mice are known to develop spontaneous autoimmune diseases like lupus erythematosus and they show various immunological abnormalities as well. Despite different genetic backgrounds, they manifest various immunological abnormalities in common, e.g., polyclonal B-cell activation (PBA) and resistance to tolerance induction. To elucidate mechanisms of the development of autoimmunity, tolerance inducibility was examined in autoimmune and normal mice using trinitrophenylated carboxymethyl cellulose (TNP-CMC) as tolerogen which is known to induce TNP-specific B-cell tolerance without the participation of T cells. NZB and MRL/Mp-lpr/lpr mice were used as autoimmune mice and C57BL/6, BALB/c, and MRL/Mp-+/+ mice as nonautoimmune mice. When TNP-CMC-injected mice were challenged with T-independent antigens, all of the mice tested were shown to be tolerant. In contrast, when TNP-CMC-injected mice were challenged with T-dependent antigen and secondary IgG responses were assessed, autoimmune mice showed rather hyperreactivity, while nonautoimmune mice showed hyporesponsiveness. Cyclophosphamide improved this defective tolerance inducibility. By the solid-phase radioimmunoassay it was revealed that average affinity of serum anti-TNP antibodies produced in TNP-CMC-injected mice was low. Such low affinity antibodies were produced in large amount in autoimmune mice. Hence, it was suggested that B-cell clones destined to produce low affinity IgG antibodies were responsible for the resistance to tolerance induction and such clones were expanding in autoimmune mice.     

Fas death receptor signaling represses monocyte numbers and macrophage activation in vivo

Over 1 billion monocytes are produced daily, with a small percentage differentiating into macrophages, suggesting that excess monocytes are deleted through a tightly regulated process. Although the in vivo mechanism governing monocyte/macrophage homeostasis is unknown, deletion of monocytes in culture is mediated by the Fas death pathway and is blocked by M-CSF. To determine the in vivo significance of Fas in monocyte development, mice lacking Fas (lpr/lpr) and mice deficient in Fas and M-CSF were examined. Compared with congenic control C57BL/6 (B6) mice, lpr/lpr mice displayed increased numbers of circulating monocytes. The lack of Fas in M-CSF-deficient mice resulted in an enhanced percentage, but not total numbers, of monocytes. Fas deficiency led to an increase in myeloid bone marrow progenitor potential only in M-CSF-intact mice. Although lpr/lpr and B6 mice had similar numbers of tissue macrophages, the loss of Fas in M-CSF-deficient mice was sufficient to increase the number of macrophages in a subset of tissues. Additionally, after stimulation with thioglycolate, lpr/lpr and B6 mice showed equivalent numbers of peritoneal macrophages. However, Fas-deficient peritoneal macrophages displayed a marked increase in spontaneous and LPS-induced proinflammatory molecule production. Moreover, Fas-deficient mice showed enhanced systemic inflammatory arthritis associated with up-regulation of IL-1beta and CCL2 secretion, elevated numbers of inflammatory monocytes, and increased numbers of tissue macrophages. Collectively, these data suggest that Fas may be required for maintaining circulating monocytes and for suppressing macrophage activation and recruitment that are stimulus dependent.     

Characterization of reciprocal Lmb1-4 interval MRL-Faslpr and C57BL/6-Faslpr congenic mice reveals significant effects from Lmb3

Susceptibility to severe lupus in MRL-Fas(lpr) mice requires not only the lpr mutation but also other predisposing genes. Using (MRL-Fas(lpr) x B6-Fas(lpr))F2 (where B6 represents C57BL/6) intercrosses that utilize the highly susceptible MRL and poorly susceptible B6 backgrounds, we previously mapped CFA-enhanced systemic lupus-like autoimmunity to four loci, named Lmb1-4, on chromosomes 4, 5, 7, and 10. In the current study, we generated and analyzed reciprocal interval congenic mice for susceptibility to CFA-enhanced autoimmunity at all four Lmb loci. Although all loci had at least a slight effect on lymphoproliferation, only Lmb3 demonstrated a major effect on lymphoproliferation and anti-chromatin Ab levels. Further characterization of Lmb3, primarily by comparing MRL-Fas(lpr) with MRL.B6-Lmb3 Fas(lpr) congenic mice, revealed that it also played a significant role in spontaneous lupus, modifying lymphoproliferation, IgG and autoantibody levels, kidney disease, and survival. The less susceptible B6 Lmb3 locus was associated with a marked reduction in numbers of CD4(+) and double-negative (CD4(-)CD8(-)) T cells, particularly in lymph nodes, as well as reduced T cell proliferation and enhanced T cell apoptosis, both in vivo and in vitro. IFN-gamma-producing CD4(+) T cells were also reduced in MRL.B6-Lmb3 Fas(lpr) mice. Further mapping using subinterval congenic mice placed Lmb3 in the telomeric portion of chromosome 7. Thus, Lmb3, primarily through its effects on CD4(+) and double-negative T cells, appears to be a highly penetrant lupus-modifying locus. Identification of the underlying genetic alteration responsible for this quantitative trait locus should provide new insights into lupus-modifying genes.     

Influence of the Ig H chain locus on autoantibody production in autoimmune mice.

Autoimmune disease is influenced by multiple genes. In this study, we investigated the role of one genetic locus, Ig H chain. IgG2a antichromatin, anti-ssDNA, and antihistone autoantibodies (autoAb) from (MRL/Mp-lpr/lpr x C57BL/6-lpr/lpr), (Ighj/b); (C57BL/6-lpr/lpr x C57BL/6-lpr/lpr-Igha), (Ighb/a); and (MRL/Mp-lpr/lpr x MRL/Mp-lpr/lpr-Ighb), (Ighj/b) mice were determined using allotype-specific ELISA. Strikingly, antichromatin and antihistone antibodies (Ab) were comprised of significantly more b allotype than either a or j allotype in all cohorts of F1 mice examined. In mice that produced anti-Sm Ab, the b allotype was used preferentially for these autoAb as well. However, no allotype skewing was observed in IgG2a Ab directed against TNP or DNA, or for total IgG2a. An Igh recombinant locus was utilized to examine the genetic control of b allotype skewing in lpr mice and in chronic graft vs host disease. In both models, the VH region did not appear to be responsible for the preferential use of b allotype. These results indicate a contribution to autoimmunity by the Igh locus and raise the possibility that Ig allotype may influence autoimmune disease by its effect on the production of certain autoAb.     

Lpr T cell hyporesponsiveness to mitogens linked to deficient receptor-stimulated phosphoinositide hydrolysis

The T lymphocytes that expand with age in the peripheral lymphoid organs of autoimmune disease-prone mice homozygous for the lpr mutation display deficient activation and proliferation in response to mitogenic lectins or antigen. In the present study, an attempt was made to correlate the deficient agonist-induced proliferation of these lpr T cells with early transmembrane signaling events mediated by receptor-coupled phosphoinositide hydrolysis. lpr T cells were capable of binding the agonistic lectin, phytohemagglutinin, in a normal manner. In addition, they expressed on their surface the antigen-specific T cell receptor-CD3 complex, which is required for T cell activation, albeit at a lower density than that found on congenic +/+ T cells. Furthermore, lpr T cells contained normal levels of the Ca2+- and phospholipid-dependent enzyme, protein kinase C, and the enzyme was translocated from the cytosol to the particulate fraction upon phorbol ester treatment. On the other hand, the lpr T cells displayed a markedly deficient agonist-induced phosphoinositide hydrolysis in comparison with their congenic +/+ counterparts, as indicated by the minimal accumulation of the phosphoinositide-derived second messengers, inositol phosphates and diacylglycerol. The defective step(s) in transmembrane signaling was bypassed by a combination of phorbol ester plus Ca2+ ionophore, which reconstituted proliferative responses of lpr T cells to normal levels, suggesting that: (a) the phosphoinositide signaling pathway plays an obligatory role in T cell activation; and (b) signaling events subsequent to phosphoinositide hydrolysis are, for the most part, intact in lpr T cells. The deficient step(s) in lpr T cell activation precedes, therefore, the generation of phosphoinositide-derived second messengers and could be due to defective function of the T cell receptor-CD3 complex, GTP-binding proteins, and/or phosphoinositide-specific phosphodiesterase. It remains to be determined whether the deficient signaling event(s) in lpr T cells is a direct pathologic consequence of the lpr gene, or rather, reflects the immature status of a normally minor thymic subset that is aberrantly exported and expanded in lpr mice.     

Neutrophil apoptosis in autoimmune Fas-defective MRL lpr/lpr mice

The apoptosis-defective lpr (fas) mutation in MRL mice causes the early onset of a lupus-like autoimmune disease with concomitant inflammation. In order to analyse the consequences of the impaired Fas-dependent apoptosis on inflammation, the susceptibility to apoptosis of polymorphonuclear leukocytes (PMN), obtained from MRL lpr/lpr mice, has been studied. Peritoneal PMN from lpr/lpr and control (+/+) mice were recruited with a mild inflammatory stimulus. The number of cells collected from the peritoneal cavity of young lpr/lpr mice was comparable to that obtained from age-matched control mice, indicating that PMN homeostasis is maintained regardless of the loss-of-function Fas mutation. Recruited neutrophils were exposed in culture to apoptosis-inducing stimuli. Treatment with agonist anti-Fas antibody increased apoptosis of +/+ PMN, but did not affect lpr/lpr PMN which do not express Fas on their surface. However, lpr/lpr PMN could undergo both spontaneous and stimulus-induced apoptosis in a fashion comparable to or higher than that of control +/+ mice. Analysis of mRNA expression revealed that lpr/lpr PMN have reduced expression of IL-18, whereas IL-1beta, IFNgamma, caspase 1 and caspase 3 are expressed at levels comparable to those of +/+ cells. However, caspase-3-like activity was higher in PMN from lpr/lpr mice than in +/+ cells, and correlated with enhanced apoptosis. It could be concluded that in young, uncompromised lpr/lpr mice, PMN homeostasis is still fully regulated through the involvement of Fas-independent, compensatory, apoptotic mechanisms. This could include an increased participation of caspase 3 in the apoptotic pathway, consequent to enhanced activation of the enzyme and to the decreased production of IL-18, which acts as a competitive caspase 3 substrate.     

Functional analysis of T cell subsets from mice bearing the lpr gene

The autosomal recessive lpr (lymphoproliferation) gene is responsible for a thymus-dependent massive lymphoproliferation associated with the development of lupus-like autoimmune disease. Phenotypic analysis of adult lpr/lpr lymph nodes has demonstrated accumulation of a dull Lyt-1+, Thy-1+ population that expresses neither Lyt-2 nor L3T4 antigens. With the use of a depletion method based on complement-mediated lysis with an anti-Lyt-2 monoclonal antibody (31 M) and a new anti-L3T4 monoclonal antibody (RL 172.4), we have purified the Lyt-2- L3T4- subset from lymph nodes or spleens of C57BL/6-lpr/lpr mice and determined whether they are immunologically functional in vitro. Production of neither interleukin 2 nor interferon-gamma was detected by the double-negative subset after stimulation with concanavalin A and/or phorbol myristate acetate. The frequencies of allospecific cytotoxic T lymphocyte (CTL) precursors and lectin-induced antigen-nonspecific CTL precursors were diminished to almost undetectable levels, whereas the Lyt-2+ population from lpr/lpr mice had CTL-precursor frequencies comparable with that of +/+ mice. These results show that the major cell subset of adult lpr/lpr lymph nodes or spleens is composed of lymphocytes with markedly limited potential for lymphokine production or antigenic stimulation.     

Tpl2 transduces CD40 and TNF signals that activate ERK and regulates IgE induction by CD40

Macrophages from Tpl2 knockout (Tpl2(-/-)) mice exhibit a defect in ERK activation by lipopolysaccharide (LPS). This impairs the nucleocytoplasmic transport of the tumor necrosis factor alpha (TNF-alpha) mRNA and prevents the induction of TNF-alpha by LPS. As a result, Tpl2(-/-) mice are resistant to LPS/D-galactosamine-induced shock. We demonstrate that Tpl2 is essential for ERK signals transduced by members of the TNF receptor superfamily, such as CD40 and the TNF receptor 1. Thus, ERK activation was impaired in Tpl2(-/-) B cells and macrophages stimulated with agonistic CD40 antibody or TNF-alpha, whereas the induction of other mitogen-activated protein kinases, such as JNK and p38, and the activation of NF-kappaB were unaffected. Tpl2 was recruited to a CD40/TRAF6 complex in response to CD40 stimulation. Moreover, TRAF6, which when overexpressed activates ERK, failed to do so in Tpl2(-/-) cells. The selective signaling defect resulting from the inactivation of Tpl2 allowed us to demonstrate that CD40-mediated ERK activation contributes to immunoglobulin production but is not essential for B-cell proliferation.