Hypoestoxide, a novel anti-inflammatory natural diterpene, inhibits the activity of IkappaB kinase.

Most inflammatory agents activate nuclear factor-kappaB (NF-kappaB), resulting in induction of genes coding for cytokines, chemokines, and enzymes involved in amplification and perpetuation of inflammation. Hypoestoxide (a bicyclo [9,3,1] pentadecane) is a diterpene from Hypoestes rosea, a tropical shrub in the family Acanthacea, several members of which are used in folk medicine in Nigeria. Here, we demonstrate that hypoestoxide (HE) abrogates the production of pro-inflammatory cytokines (IL-1beta, IL-6, and TNF-alpha) in lipopolysaccharide (LPS)-activated normal human peripheral blood mononuclear cells. Moreover, HE inhibits the production of nitric oxide (NO) by IL-1beta- or IL-17-stimulated normal human chondrocytes. In vivo, oral administration of HE to mice significantly ameliorated hind paw edema induced by antibodies to type II collagen plus LPS. Furthermore, topical administration of HE to mice also significantly inhibited phorbol ester-induced ear inflammation. The anti-inflammatory activity of HE may be due in part to its ability to inhibit NF-kappaB activation through direct inhibition of IkappaB kinase (IKK) activity. Thus, HE could be useful in treating various inflammatory diseases and may represent a prototype of a novel class of IKK inhibitors.

Differential control of autoantibodies and lymphoproliferation by Fas ligand expression on CD4+ and CD8+ T cells in vivo.

We have previously shown that the gld autoimmune syndrome is suppressed in lethally irradiated gld mice reconstituted with a mixture of normal and gld bone marrow (BM). Furthermore, in vivo depletion of normal Thy-1+ cells restores lymphoproliferation and autoantibody production in such chimeras, suggesting that T cells bearing Fas ligand are responsible for correcting the gld defect. In this study, mixed-BM chimeras lacking either normal CD4+ (B6CD4KO-B6gld) or normal CD8+ T cells (B6CD8KO-B6gld) were generated to determine the contribution of the normal T cell subsets to disease suppression. Lymphoproliferation was completely suppressed in B6CD4KO-B6gld chimeras but only modestly in B6CD8KO-B6gld chimeras. On the other hand, both types of mixed-BM chimeras had incomplete effects on the suppression of serum autoantibodies when compared with B6gld reconstituted with isologous BM. These results suggest that both T cell subsets provide Fas ligand to suppress immune cells responsible for autoantibody production; however, CD8+ T cells are mainly responsible for preventing lymphoproliferation.

Concentrations of circulating beta-chemokines do not correlate with viral load in human immunodeficiency virus-infected individuals.

The CC or beta-chemokines MIP-1alpha, MIP-1beta, and RANTES are the primary components of human immunodeficiency virus type 1 (HIV-1)-suppressive soluble factors in vitro. We studied the relationship between the concentrations of MIP-1alpha, MIP-1beta, and RANTES in plasma and HIV viral load in HIV-infected subjects. The HIV-positive patient group (n = 140) had significantly lower concentrations of all three beta-chemokines (MIP-1alpha, P < 0.0005; MIP-1beta, P < 0.005; RANTES, P < 0.0005) than the control group (n = 58 for MIP-1alpha, n = 27 for MIP-1beta, and n = 59 for RANTES). In addition, we divided the patient group into three subgroups (high, moderate, and low) based on the number of HIV-1 RNA copies in the plasma (as measured by quantitative HIV RNA PCR). Again, all three subgroups had significantly lower concentrations of the beta-chemokines than the HIV-negative control group. However, there was no significant difference in plasma beta-chemokine concentrations among the three subgroups within the patient group (P < 0.3). Although our results demonstrate that HIV-infected individuals had significantly lower concentrations of circulating beta-chemokines than healthy uninfected control subjects, we found no correlation between the concentrations of beta-chemokines in plasma and HIV-1 viral load in HIV-infected individuals.

Association of low concentrations of serum mannose-binding protein with recurrent infections in adults.

Low concentrations of mannose-binding protein (MBP; also known as mannose-binding lectin) are associated with common opsonic defect in immunodeficient children. We compared the concentrations of MBP in the sera of 47 adults with non-human immunodeficiency virus-related recurrent infections (group I) and 50 healthy adult controls. Mean serum MBP concentrations in the patient group did not differ significantly from those in the control group (P < 0.4). Nevertheless, the proportion of individuals with less than 5 ng of serum MBP per ml was significantly larger in the patient group (21%, P = 0.01) than in the control group (4%). Group II consisted of 73 pediatric and 56 adult patients with recurrent infections. Pediatric patients had significantly lower mean concentrations of serum MBP than their controls (P < 0.005), and there was no significant difference between the concentrations in sera of adult patients and adult controls (P < 0.4). Again, the proportion of individuals with less than 5 ng of serum MBP per ml was significantly larger in both pediatric (22%, P = 0.045) and adult (38%, P = 0.000016) patients than in their respective controls (4%). Our results demonstrate that, as in children, low concentrations of serum MBP can be associated with recurrent infections in adults.

Novel immunoregulatory B cell pathways revealed by lpr-+ mixed chimeras.

lpr, a murine mutation of the Fas apoptosis receptor, causes lymphadenopathy and autoantibody production, with lymphadenopathy primarily due to a population of CD4-CD8-B220+ T cells. Previous in vivo experiments, in which lpr and normal bone marrow cells were coinfused into lpr hosts, have demonstrated that only T cells of lpr origin accumulated abnormally and only B cells of lpr origin produced autoantibodies. Moreover, in these chimeras, B cells of normal origin were unable to respond to conventional, T cell-dependent exogenous Ag. To address the role of lpr B cells in regulation of lpr autoimmunity, we have prepared lpr-+ mixed chimeras and selectively eliminated lpr B cells using allele-specific, mAb treatment, thus allowing normal B cells to develop in an environment with lpr T cells. From these data, we arrived at four major conclusions: 1) Compared with control-treated chimeric mice, lpr B cell-depleted mice had greatly reduced total lymph node cell counts; 2) the T cells were derived equally from normal and lpr donors, and the percentage of lpr-derived CD4-CD8- T cells was greatly reduced; 3) despite the presence of the remaining lpr T cells, no autoantibodies were produced by the normal derived B cells; and 4) lpr T cells without lpr B cells were unable to prevent a normal B cell response to conventional Ag. These data demonstrate that B cells can play a critical and expansive regulatory role, not only for T cells, but for other B cells as well.

B cell genotype determines the fine specificity of autoantibody in lpr mice.

Anti-Sm Abs are specific markers of human systemic lupus erythematosus (SLE) and of murine models of this disease. In humans, anti-Sm Abs are mostly IgG1, and in MRL/lpr mice, IgG2a; both are T-dependent isotypes. Other lpr strains, such as B6/lpr, do not produce anti-Sm Ab spontaneously. The present study was aimed at identifying the cellular expression of background genes responsible for generation of the anti-Sm Ab response in MRL/lpr mice. We used double chimeric mice made by transferring MRL/lpr and B6/lpr bone marrows into irradiated allotype heterozygous F1 mice. Five mo after reconstitution, FACS analysis of lymph node (LN) and spleen cells revealed that both MRL/lpr and B6/lpr cells coexisted in roughly equal numbers. Ab produced by each donor could be distinguished by allotype-specific assays. IgG2a anti-Sm was made only by MRL-derived B cells despite the presence of T cells that might potentially provide help to the B6/lpr B cells. The frequency of anti-Sm Ab-producing individuals was similar to that of unmanipulated MRL/lpr mice (about 25%). IgG2a anti-chromatin and total IgG2a was mostly dominated by the MRL-derived B cells. B6-derived B cells produced more rheumatoid factor (RF) against their own IgG2b(b), while RF against IgG2a was dominated by MRL-derived B cells. This suggests that the control of the production of particular autoantibody specificities, such as anti-Sm, is determined by the expression of MRL or B6 background genes in B cells.

Suppression and reversal of gld disease by parabiosis with normal mice.

The disruption of the Fas receptor or Fas ligand by the lpr or gld mutations, respectively, results in severe autoimmune and lymphoproliferative disease due to the failure of Fas-mediated deletion of self-reactive lymphocytes. Recently, we have shown in mixed chimeras that gld-induced autoimmunity could be corrected by normal bone marrow, in particular by normal T cells. In contrast, lpr-mediated autoimmunity could not be influenced by normal bone marrow-derived cells. In the present report, we have studied the role of normal lymphocytes in suppressing or reversing gld-induced autoimmunity by parabiosis with normal mice. Our results show a suppression of lymphadenopathy, fewer CD4-CD8- T cells, and lower levels of autoantibody production in gld mice parabiosed with normal mice at 4-6 weeks of age. The gld mice parabiosed with normal mice at 4 months of age also exhibited a substantial reduction of both total and CD4-CD8- T cells in the periphery 2 months after surgery. However, they showed little reduction of autoantibodies compared to gld mice parabiosed with gld mice. In contrast, older lpr mice did not exhibit any reduction in lymphadenopathy or autoantibody production after parabiosis with normal mice. The prevention or reversal of lymphadenopathy in parabiosed gld mice suggests that ongoing Fas-mediated deletion in the periphery may play an important role in maintaining self-tolerance. The relative irreversibility of autoantibody synthesis in older parabiosed gld mice suggests that autoantibody-producing B cells or their committed precursors are long lived and do not express functional Fas receptor.

The abnormal lpr double-negative T cell fails to proliferate in vivo.

Mice homozygous for the autosomal recessive gene lpr develop marked lymphadenopathy and a systemic autoimmune disease resembling human systemic lupus erythematosus. The enlarged nodes are dominated by T cells with an unusual surface phenotype: dull Thy-1+, dull CD3+, CD4-, CD8-, B220+ (double-negative T cells or DNTs). Despite their massive accumulation in vivo, these cells fail to proliferate in response to conventional T-cell mitogens in vitro. The identification of the lpr mutation as a defect in the Fas apoptosis receptor gene suggests that DNT accumulation may result from abnormal persistence rather than overproliferation. To test in vivo whether DNTs persist abnormally or have a capacity to differentiate into single-positive T cells, we have performed cell transfer experiments between congenic strains of lpr and +/+ mice differentially marked by expression of the Ly-1 or Thy-1 alleles. Although transferred lpr lymph node cells were mostly DNTs at the time of injection, most recovered cells of donor origin were single positive, particularly CD8+, at all time points after transfer. Furthermore, transfer of purified DNTs resulted in recovery of relatively few cells of donor origin. Transfer of lpr T cells enriched for CD8 expression confirmed the preferential survival of this subset. Thus, DNTs are a surprisingly transient population and have little capacity for transformation to single positives. This would suggest that DNTs are constantly being renewed, perhaps from CD4+ and CD8+ precursors.

In vivo depletion of Thy-1-positive cells originating from normal bone marrow abrogates the suppression of gld disease in normal-gld mixed bone marrow chimeras.

Mice homozygous for gld develop an autoimmune syndrome characterized by hypergammaglobulinemia, massive accumulation of abnormal T cells and the production of autoantibodies. Previous studies in our laboratory have shown that reconstitution of lethally irradiated B6/gld recipients with a mixture of normal and gld bone marrow (BM) suppresses the gld-induced syndrome. In this report we extend this observation by demonstrating that the depletion of normal Thy-1+ cells, but not normal B cells, restores gld disease in mixed BM chimeras congenic for Thy-1 and IgH alleles. These results strongly suggest that normal T cells suppress the development of gld-related abnormalities. It is probable that the mechanism by which normal Thy-1+ cells mediate the suppression is Fas ligand dependent.

Co-infusion of normal bone marrow partially corrects the gld T-cell defect. Evidence for an intrinsic and extrinsic role for Fas ligand.

Ipr and gld mice develop systemic autoimmune diseases with nearly indistinguishable manifestations, including the accumulation of massive numbers of CD4-CD8- T lymphocytes. In vivo chimera experiments have shown that the Ipr mutation is functionally expressed in both T and B cells. When lethally irradiated Ipr mice were given a combination of normal and Ipr bone marrow, only Ipr-derived B cells produced autoantibodies and only Ipr-derived T cells hyperproliferated. In contrast, analogous experiments with gld mice showed that the co-infusion of normal bone marrow greatly reduced autoantibody production. These results indicated that the gld B cell defect was extrinsic to those cells producing autoantibodies, in agreement with the recent molecular data showing that the normal gene products of the Ipr and gld loci form an interacting receptor-ligand pair. In the present study, we have extended our functional studies with gld mice using T cell-marked congenic donors. Lymphadenopathy was reduced three- to fourfold in gld mice given a combination of congenic normal and gld bone marrow compared with mice given gld bone marrow alone, and the absolute number of CD4-CD8- T cells was reduced by a factor of 7. Surprisingly, the residual CD4-CD8- T cells present in the mixed chimeras were derived entirely from the gld donor marrow. This suggests that the gld mutation results in both an extrinsic and intrinsic defect in T cells.

T-B collaboration for autoantibody production in lpr mice is cognate and MHC-restricted.

A central question in autoimmunity is the mechanism of T cell help for autoantibody production. For responses to exogenous Ag, T-B collaboration is restricted by MHC class II molecules. To determine whether T cell help that leads to autoantibodies in murine SLE is also MHC-restricted, we have constructed bone marrow chimeras with Ig heavy chain (lgh) allotype- and I-A-congenic donor B6/lpr mice and I-A-congenic recipients. Developing T cells were thus positively selected in the host thymus to interact with B cells bearing I-A of one haplotype or the other. Additional control host mice were heterozygous for I-A expression, allowing T helper cell selection for both I-A haplotypes. Five months after reconstitution, serum total IgG2a, IgM, IgG2a antichromatin, and IgM rheumatoid factor were quantitated by allotype-specific ELISA. Data showed that whereas substantial numbers of B cells were present from both donor strains in all mice, autoantibody production was overwhelmingly from those donor B cells expressing the same I-A haplotype as the host. Sera from the I-A heterozygous control recipient group had roughly equal quantities of autoantibodies of both allotypes, as expected. The finding of MHC class II restriction implies that the T cell help that drives autoantibody production in lpr mice is delivered through cognate (cell-to-cell) interactions and not by soluble factors alone.

Selection of the T cell receptor repertoire in Lpr mice.

The development of double-negative (CD4-, CD8-) T cells and other T cells subsets in lymphoproliferation (lpr) mice continues to be poorly defined. Recent studies indicate that lpr is a mutation of a receptor mediating apoptosis. It has thus been hypothesized that T cell development in the thymus should be abnormally affected. In this study, we analyzed the TCR V beta repertoire of double-negative T cells as well as CD4+ and CD8+ single-positive subsets in various lpr and matched non-lpr strains. Particular comparisons were made to determine the influence of different class I and class II molecules on repertoire formation. The data demonstrate that positive and negative selection of the CD4+ and CD8+ subsets are normal in lpr mice when compared with non-lpr congenic mice. Surprisingly, the result also suggest that double-negative T cells are mostly selected on class I MHC molecules in a pattern similar to the CD8+ population, and that T cells positively selected on class II MHC antigens may be absent from the double-negative population. In all lpr strains, we also found an increased percentage of double-negative V beta 8.3+ cells out of proportion to levels in the CD4+ or CD8+ subsets. Longitudinal studies and studies in thymectomized animals showed that this increase reflects a peripheral process selectively affecting V beta 8.3+ double-negative T cells. Together, these repertoire data provide new insight into the effect of the lpr genetic defect on T cell development and the derivation of double-negative T cells. Despite the role of Fas in apoptosis and the abnormal expression of this gene in lpr mice, the present results support the hypothesis that thymic events are relatively normal in lpr mice, and that the double-negative T cells are mostly class I MHC selected and expanded by abnormal peripheral processes.

Correction of gld autoimmunity by co-infusion of normal bone marrow suggests that gld is a mutation of the Fas ligand gene.

lpr and gld mice develop phenotypically indistinguishable systemic autoimmune diseases and marked lymphadenopathy dominated by CD4-CD8- T cells. In vivo chimera experiments have demonstrated that both lpr T and lpr B cells are intrinsically defective. Analogous experiments were conducted using gld mice. Lethally irradiated gld mice were given mixtures of congenic gld and normal (+/+) bone marrow differentially marked by Ig heavy chain allotype. In sharp contrast to lpr-(+)/+ mixed chimeras, gld-(+)/+ chimeras had little autoantibody production at 5 months and minimal adenopathy at 6 months, indicating that the normal marrow-derived cells corrected the gld defect. Thus, aberrant autoantibody production is due to a defect extrinsic to the gld B cell and lymphoproliferation is due to a defect extrinsic to the gld T cell. These data support the hypothesis that gld mice lack an apoptosis-inducing ligand. The receptor for this ligand may be the Fas molecule, which is defective in lpr mice.

Murine lymphocytes exhibit heterogeneity in their proliferative responsiveness to calcium ionophore.

The calcium ionophore, A23187, when used alone was found to induce proliferation of murine T cells, at concentrations of 0.5-1 mM. This response required the presence of syngeneic splenic adherant cells (SAC) as a source of accessory cells. Interestingly, only CD4+ T cells but not CD8+ T cells or B cells responded to the calcium ionophore by proliferation. The inability of CD8+ T cells or B cells to respond was not related to decreased elevation in the intracellular ionized calcium [Ca2+]i concentration induced by the ionophore, because activated CD4+ T, CD8+ T and B cells all exhibited similar elevation in [Ca2+]i. The inability of CD8+ T cells to respond to calcium ionophore was probably due to insufficient production of autocrine growth factors, such as IL-2, inasmuch as the addition of exogenous IL-2 could completely restore the CD8+ T cell responsiveness. Also, exogenous rIL-1 could partially restore purified T cell response to calcium ionophore, whereas, rIL-6 failed to do so. IL-2, but not IL-4, acted as an autocrine growth factor for T cells responding to the calcium ionophore in the presence of SAC, since, antibodies against IL-2 or IL-2 receptor (IL-2R) but not against IL-4, could inhibit the T cell proliferation. Furthermore, exogenous rIL-2 but not rIL-4 supported the proliferation of T cells to calcium ionophore in the absence of accessory cells. Our results suggest that murine lymphocytes exhibit heterogeneity in their proliferative responsiveness to calcium ionophore and that this may not depend on the early activation signal such as the elevation in [Ca2+]i) induced by the ionophore but may depend on subsequent signals which regulate endogenous growth factor production.

CD4-CD8- thymocytes from MRL-lpr/lpr mice exhibit abnormal proportions of alpha beta- and gamma delta-TCR+ cells and demonstrate defective responsiveness when activated through the TCR.

MRL-lpr/lpr (lpr) mice develop profound lymphadenopathy resulting from the accumulation of CD4-CD8- (double-negative, DN) cells in the peripheral lymphoid organs. Earlier studies from our laboratory demonstrated an increased proportion of DN cells in the thymus of lpr mice with age. Inasmuch as the DN thymocytes constitute a heterogenous population of cells, in the present study, we investigated the TCR phenotype of DN thymocytes and their responsiveness to activation through the TCR. The DN thymocytes of young (1 month of age) lpr mice contained approximately 65% CD3+ cells of which approximately 60% were alpha beta-TCR+ and approximately 39% were gamma delta-TCR+ as detected by using pan anti-TCR mAbs. In old (4-6 months of age) or young MRL-(+/+) mice, similar proportions of CD3+, alpha beta- or gamma delta-TCR+ DN thymocytes were detected. Interestingly, however, in old (4-6 months of age) lpr mice, the CD3+ T cells increased to approximately 86% and the majority of these (approximately 81%) were alpha beta-TCR+ and only approximately 3% were gamma delta-TCR+. Also, in old lpr mice, there was a 10-fold increase in the absolute number of alpha beta-TCR+ DN cells in the thymus, whereas, the absolute number of gamma delta-TCR+ DN cells in the thymus did not alter significantly. Furthermore, a majority (approximately 84%) of the old lpr DN thymocytes expressed CD45R, similar to the peripheral DN T cells. In contrast, only a small number (approximately 1%) of DN thymocytes from young lpr or MRL-(+/+) mice expressed CD45R. The DN thymocytes from young lpr or MRL-(+/+) mice demonstrated strong and similar proliferative responsiveness to stimulation with PMA + calcium ionophore or PMA + IL-2, or to immobilized mAb directed against the TCRs (CD3, alpha beta and gamma delta). In contrast, the DN thymocytes and the DN peripheral T cells from old lpr mice demonstrated marked defect in responding to the above stimuli. The present study suggests that with the onset of lymphadenopathy, the DN cells in the thymus of old lpr mice are increasingly skewed toward the alpha beta-TCR repertoire, the majority of which express CD45R and respond poorly to mitogenic stimuli or when activated through the TCR. It is suggested that migration of such cells continuously to the periphery may result in severe lymphadenopathy seen in old MRL-lpr/lpr mice.

Association of pulmonary tuberculosis and HLA in south India.

In 204 patients with smear-positive pulmonary tuberculosis HLA-A10, B8 and DR2 were more frequently found than in 404 control subjects (p = 0.01); the greatest attributable risk (0.29) was associated with HLA-DR2. The radiographic extent of disease was also associated with HLA-DR2 (p = 0.0001). In 152 patients with smear-negative pulmonary tuberculosis, the frequencies of HLA-A10 and B8, but not DR2, were greater in the control subjects (p = 0.001 and 0.01 respectively). HLA-DR2 may be involved in the pathogenesis of advanced pulmonary tuberculosis. Study of endogamous, genetically disparate populations (caste) revealed other HLA associations (A3, B12 and DR4) unique to them, suggesting that genes linked with the HLA complex might also be significant in the pathogenesis of tuberculosis.

Immunosuppression by aldicarb of T cell responses to antigen-specific and polyclonal stimuli results from defective IL-1 production by the macrophages.

In the present study we investigated the immunomodulatory effects of aldicarb, a carbamate pesticide, on T cells activated by a number of different ways. When C3H mice were injected intraperitoneally with a single dose of Aldicarb, 0.005-50 micrograms/kg body wt, and their spleen cells were stimulated with T cell mitogens such as concanavalin A (ConA)3 or anti-CD3 monoclonal antibodies (mAb), a decreased responsiveness was detected when compared to the control mice. Aldicarb administered at concentrations less than 0.005 microgram/kg body wt failed to cause significant immunosuppression. Interestingly, when purified T cells from immunosuppressive doses of aldicarb-treated mice were stimulated with ConA in the presence of irradiated control macrophages, the defective T cell response was no longer demonstrable. Also, purified control T cells stimulated with ConA in the presence of irradiated macrophages from aldicarb-treated mice showed decreased responsiveness. Similar observations were made using anti-CD3 mAb to activate the T cells, inasmuch as whole spleen cells from aldicarb-treated mice showed decreased responsiveness to anti-CD3 stimulation, whereas purified T cells in the presence of irradiated control macrophages showed normal reactivity. The fact that aldicarb did not directly affect the T cell functions was further confirmed by stimulating purified T cells from aldicarb-treated mice with phorbol myristate acetate and calcium ionophore, a response which is independent of the accessory cells and which was found to be normal in aldicarb-treated mice. It was observed that the macrophages from aldicarb-treated mice demonstrated a decreased capacity to stimulate conalbumin-specific T helper cell clone, D10.G4, and when activated produced decreased amounts of IL-1 when compared to control macrophages. Also, the decreased stimulation of D10.G4 clone by aldicarb-treated macrophages was reconstituted when exogenous recombinant IL-1 was added to the cultures. These data together suggested that aldicarb affects the macrophage functions by interfering with IL-1 production and that it does not affect the T cell functions directly.

Autoreactive T cell clones isolated from normal and autoimmune-susceptible mice exhibit lymphokine secretory and functional properties of both Th1 and Th2 cells.

Recent studies have suggested the existence of two mutually exclusive subpopulations of T helper (Th) cells in the murine immune system, called Th1 which produces interleukin (IL)-2 and interferon (IFN)-gamma but not IL-4 and Th2 which secretes IL-4 and IL-5 but not IL-2. Also, functionally, Th1 cells generally activate the macrophages and mediate delayed-type hypersensitivity whereas Th2 cells provide help efficiently to B cells. In the present study, we investigated the lymphokine secretory properties of two well-characterized autoreactive (self-Ia reactive) T cell clones isolated from normal DBA/2 mice and autoimmune-susceptible MRL-lpr/lpr mice. It was observed that both the autoreactive T cell clones, following activation, produced IL-2, IL-4, and IFN-gamma. They induced hyper-Ia expression and cell proliferation in syngeneic B cells as well as activated the macrophages to exhibit tumoristatic properties. Both clones could also induce T-T network interaction in which syngeneic naive CD4+ T cells responded directly to stimulation with autoreactive T cell clones. The T-T interaction was demonstrable in 1-month-old MRL-lpr/lpr mice prior to the onset of the autoimmune disease but not in 6-month-old mice having lymphadenopathy and autoimmune disease. Unlike Th1 and Th2 cells which upon antigenic stimulation respond to exogenous IL-2 and IL-4, the autoreactive T cell clones responded only to IL-2 but not to IL-4. Our data suggest the existence of a unique subset of immunoregulatory CD4+ Th cells having the lymphokine secretory and functional properties of both the murine Th1 and Th2 subsets.

Evidence for major alterations in the thymocyte subpopulations in murine models of autoimmune diseases.

Thymocytes can be divided into four major subpopulations: CD4+CD8+ (double-positive), CD4-CD8- (double-negative), CD4+CD8- (CD4+) and CD4-CD8+ (CD8+) cells. Recent studies have shown that T-cell development in the thymus progresses as: CD4-CD8(-)----CD4+CD8(+)----CD4+ or CD8+ cells. In the present study we investigated these and other subpopulations of thymocytes in autoimmune MRL(-)+/+, MRL-lpr/lpr, C57BL/6-lpr/lpr, BXSB and NZB mice before (1-month old) and after (4-6-months old) the onset of lymphadenopathy and autoimmune disease. All the autoimmune strains at one month of age and other H-2, sex and age-matched controls (C3H, DBA/2, and C57BL/6) demonstrated normal proportions of thymocyte subsets with approximately 75% double-positive cells, 5-7% double-negative cells, 11-15% CD4+ cells and 3-5% CD8+ cells. By 4-6 months of age, MRL(-)+/+ mice demonstrated a moderate increase in double-negative cells (approximately 13%) and a decrease in double-positive cells (approximately 46%). Interestingly, in the presence of the lpr gene, as seen in MRL-lpr/lpr mice, the double-negative cells increased to approximately 47% and the double-positive cells decreased to approximately 16%. In contrast, 4-6-month-old C57BL/6-lpr/lpr mice failed to demonstrate any alterations in the thymocyte subsets thereby suggesting that background genes, in addition to the lpr gene, played a role in the thymocyte differentiation. BXSB male mice with severe lymphadenopathy behaved very similarly to MRL-lpr/lpr mice, inasmuch as their thymus contained approximately 48% double-negative cells and only approximately 8% double-positive cells. In contrast to MRL-lpr/lpr and BXSB strains, NZB mice at 6 or 10 months of age had normal composition of thymocyte subsets. In MRL and BXSB animals, although there was a significant increase in CD4+ cells (approximately 23-33%), due to a consequent increase in CD8+ cells (approximately 11%), the ratio of CD4+:CD8+ cells remained 2-3:1, similar to that seen in normal mice. Furthermore, using the J11d marker expressed by the majority of the double-negative and all double-positive thymocytes but not by mature functional T cells, we confirmed the above findings and demonstrated further that MRL-lpr/lpr mice at 4-6 months of age had an increased percentage of J11d- double-negative cells and a decrease in J11d+ double-negative cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Evidence for the existence of distinct heterogeneity among the peripheral CD4-CD8- T cells from MRL-lpr/lpr mice based on the expression of the J11d marker, activation requirements, and functional properties.

Autoimmune-susceptible, MRL-lpr/lpr (lpr) mice develop a profound lymphadenopathy resulting from the accumulation of CD4-CD8- (double-negative, DN) cells in peripheral lymphoid organs. The source and the mechanism of this abnormal accumulation of cells is still unknown. Recently, we reported that a significant number (approximately 35%) of the CD4-CD8- cells expressed J11d, a marker expressed by immature thymocytes but not by mature functional peripheral T cells. In the present study, we investigated the phenotype, growth requirements, and functional properties of purified J11d+ and J11d- subpopulations. Using the mAb, F23.1, which recognizes a TCR determinant encoded by the V beta 8 gene family, it was observed that approximately 30% of the J11d+ and J11d- DN cells expressed this determinant. Further studies on the thymus revealed that J11d+ DN cells from lpr thymus also contained F23.1+ cells (approximately 25%), whereas, similar cells from normal MRL(-)+/+mice were all F23.1-, consistent with earlier reports in other normal strains. Further phenotypic studies revealed that the peripheral J11d+ and J11d- cells from lpr mice were similar in expressing CD3, Ly-5 (B220), and Ly-24 (Pgp-1) determinants. When stimulated with phorbol myristic acetate (PMA) and recombinant IL-2 (rIL-2), only J11d- cells but not J11d+ cells responded by proliferation. However, in the presence of calcium ionophore (A23187) and PMA, both J11d+ and J11d- subpopulations proliferated by producing and responding to endogenous IL-2 but not IL-4. The lymph node T cells from 1-month-old MRL-lpr/lpr mice responded strongly when stimulated with PMA + rIL-4 or PMA + rIL-6. In contrast both J11d+ and J11d- subpopulations failed to respond when similarly stimulated. The J11d+ but not J11d- cells demonstrated spontaneous cytotoxic activity against the NK-sensitive YAC-1 tumor targets. The J11d- cells did not exhibit cytotoxic potential in spite of culture with PMA + rIL-2. Even after repeated culture in vitro with PMA + A23187 or PMA + rIL-2, both J11d+ and J11d- subpopulations failed to express the mature phenotype bearing CD4 and/or CD8 antigens. The present study demonstrates the expansion of unique J11d+, alpha beta-TCR+, DN T cells with cytotoxic potential in lpr mice and further suggests the existence of phenotypic and functional heterogeneity among the abnormal lpr DN cells.