Therapeutic effect of CpG motifs on the development of chronic graft-versus-host disease in mice

Transferring DBA/2 spleen cells into (C57BL/10xDBA/2) F1 (referred to as BDF1) mice induces a chronic graft-versus-host disease (GVHD), characterized by the production of Th(2) cytokines, hypergammaglobulinemia, and immune complex-mediated glomerulonephritis that resembles systemic lupus erythematosus. DNA motif consisting of an unmethylated CpG dinucleotide flanked by two 5' purines and two 3' pyrimidines (CpG ODN) induces Th(1) cytokine production in mice. This study examines the effect of administering CpG ODN to mice undergoing chronic GVHD, based on the premise that altering Th(1)/Th(2) activity might beneficially impact on disease progression.GVHD BDF1 mice injected with DBA/2 spleen cells were treated with weekly intraperitoneal injection of 50 microg CpG ODN. This treatment significantly suppressed the production of IgG anti-DNA autoantibody and reduced the development of glomerulonephritis. Serum IgG2a titers were higher in the CpG ODN than in non-CpG control group, whereas IgG1 titers were unchanged. As predicted, IFN-gamma levels were significantly higher in the CpG ODN-treated group, while IL-4 levels were lower, resulting in a shift in the Th(1)/Th(2) cytokine ratio. Results suggest that CpG ODN administration may be of therapeutic benefit in chronic GVHD.     

Effect of IL-12 encoding plasmid administration on tight-skin mouse

The tight-skin (Tsk/+) mutant mice, a putative murine model of scleroderma, are characterized by the excessive deposition of collagen and the presence of antinuclear antibodies. Type 2 cytokines, such as IL-4 and IL-6, are capable of regulating the synthesis of various matrix molecules, including type I collagen, by fibroblasts. IL-12 is well known to induce type 1 cytokine production and to reduce type 2 activity. Here, we examined the effect of IL-12 encoding plasmid (pCAGGSIL-12) on the disease progression of Tsk/+ mice. pCAGGSIL-12 plasmid or pCAGGS parental vector was injected intramuscularly 7 times at 3 week intervals into Tsk/+ mice. One week after the last injection, pCAGGSIL-12 administered Tsk/+ mice exhibited a marked decrease in the skin thickness compared with the mice treated with pCAGGS vector. The serum levels of antinuclear antibodies were diminished in pCAGGSIL-12 treated mice. IL-4 production by spleen cells from pCAGGSIL-12 plasmid treated mice was significantly lower than that from vector treated mice. These results indicate that pCAGGSIL-12 administration into Tsk/+ mice had beneficial effects in preventing the collagen accumulation in the skin and suppressing the autoimmunity via improvement of Th1/Th2 balance. The present study suggests that the IL-12 encoding plasmid administration might have a therapeutic effect on systemic sclerosis.     

IL-18 prevents the development of chronic graft-versus-host disease in mice

The development of chronic graft-versus-host disease (GVHD), which is induced by the transfer of DBA/2 spleen cells into (C57BL/6 x DBA/2)F1 (BDF1) mice, is closely related to diminished donor anti-host CTL activity and host B cell hyperactivation. Therefore, an approach which activates donor CD8+ T cells or suppresses donor CD4+ T cell-host B cell interaction may have clinical utility in the treatment of chronic GVHD. We have previously demonstrated that IL-18 induces the development of naive CD8+ T cells into type I effector cells in DBA/2 anti-BDF1 MLC. In this paper we examined the effect of IL-18 administration on the development of chronic GVHD in mice. The treatment was started before or after the onset of clinical evidence of the disease. Regardless of the treatment schedule, IL-18 significantly decreased immunological parameters indicative of chronic GVHD, such as elevated serum IgG antinuclear Abs, IgG1, and IgE levels, and host B cell numbers and their activation. Importantly, IL-18-treated mice did not show the same acute GVHD-like symptoms reported for IL-12 treatment, because there was no weight loss, death, or severe immunodeficiency as indicated by a decrease in IL-2 and IFN-gamma production by Con A-stimulated spleen cells. In contrast, IL-18 treatment partially but significantly restored the production of these cytokines. Data further suggested that these IL-18-mediated therapeutic effects may be due to the induction of donor CD8+ CTL, the decrease in donor CD4+ T cell numbers, and a down-regulation of host B cell MHC class II expression. Thus, our results suggest that IL-18 has beneficial effects in the prevention and treatment of chronic GVHD.     

Mechanisms of strain-dependent development of mast cells from mouse splenocytes

Mast cell development from spleen cells was not triggered by prostaglandin E1 (PGE1) or dibutyryl cAMP (db-cAMP) during a 12 day culture when the spleen cells were obtained from C57BL/6N and DBA/1 mice, but mast cells did develop when the spleen cells were obtained from C3H/HeN, BALB/c and ICR mice. A lack of endogenous IFN-gamma in the initial 2 days of the culture period was responsible for the failure. This was confirmed by adding neutralizing anti-IFN-gamma antibody and rIFN-gamma to the cultures and by determining IFN-gamma levels in the spleen cell cultures. Th1 cells in the spleens of C57Bl and DBA/1 mice were much more sensitive to PGE1 and db-cAMP than Th1 cells from other inbred mice strains, and consequently, IFN-gamma production was inhibited in spleen cell cultures of C57BL and DBA/1 mice on addition of PGE1 or db-cAMP. Furthermore, the different sensitivities of Th1 cells to PGE and db-cAMP were dependent on the different levels of IL-12 p40 monomers or homodimers in the spleen cell cultures. As the endogenous specific inhibitors of IL-12 p70 (heterodimers of p40 and p35), large amounts of IL-12 p40 monomers or homodimers in the spleen cell cultures of C57BL and DBA/1 mice enhanced the ability of PGE1 and db-cAMP to inhibit IFN-gamma production by antagonizing the activity of IL-12 heterodimers. These results indicate that the strain-dependent development of mast cells from mouse splenocytes is related to endogenous IFN-gamma levels, which are regulated by PGE, db-cAMP, IL-12 p70 and IL-12 p40.     

Antagonist of secondary lymphoid-tissue chemokine (CCR ligand 21) prevents the development of chronic graft-versus-host disease in mice

The use of receptor antagonists for chemokines is an alternative approach to blocking chemokine actions and has the potential to provide novel therapeutics. We determined the receptor antagonist properties of murine N-terminally truncated secondary lymphoid tissue chemokine (SLC)/6Ckine/CCR ligand 21 analogs and evaluated the preventive effects of SLC antagonists on chronic graft-vs-host disease (GVHD) in a murine model by blocking the homing of donor CCR7-expressing T cells into the recipient's lymphoid organs. SLC analogs truncated >4 aa residues from the N terminus showed a loss of chemotaxis and Ca2+ influx of CCR7-expressing cells and also inhibited SLC-stimulated chemotaxis and SLC-induced Ca2+ influx completely. To determine whether SLC antagonist inhibits the development of chronic GVHD, chronic GVHD was induced by injecting DBA/2 spleen cells into (C57BL/6 x DBA/2) F1 mice. Total numbers of spleen cells and host B cells, serum levels of IgE, and of total IgG and IgG1 of anti-DNA Abs in SLC antagonist-treated GVHD mice were significantly lower than those in control PBS-treated GVHD mice. This was due to a reduction in the levels of activated donor CD4+ T cells and a decrease in IL-4 production, resulting in a reduction in the numbers of activated host B cells. Therefore, our results suggest that SLC antagonist has beneficial effects for the prevention of chronic GVHD.     

CD4+ and CD8+ regulatory T cells generated ex vivo with IL-2 and TGF-beta suppress a stimulatory graft-versus-host disease with a lupus-like syndrome

Regulatory T cells generated ex vivo from conventional mouse T cells have been used to prevent and alter the course of a stimulatory graft-vs-host disease with a lupus-like syndrome. DBA/2 mouse T cells induce this syndrome when injected into (DBA/2 x C57BL/6) F(1) mice. Stimulating DBA/2 T cells with irradiated C57BL/6 in the presence of IL-2 and TGF-beta induced both CD4(+) and CD8(+) cells to develop potent suppressive activity and enhanced their survival. The IL-2 and TGF-beta-treated T cells lost their ability to induce graft-vs-host disease and, instead, prevented other parental T cells from inducing lymphoid hyperplasia, B cell activation, and an immune complex glomerulonephritis. Moreover, a single transfer of TGF-beta-conditioned T cells to animals that had already developed anti-dsDNA Abs decreased the titer, suppressed proteinuria, and doubled survival. This study raises the possibility that autologous regulatory T cells generated ex vivo have the potential to be used as an adoptive immunotherapy to induce allograft tolerance and to control autoimmunity.     

Dendritic cell-derived IL-12p40 homodimer contributes to susceptibility in cutaneous leishmaniasis in BALB/c mice

Protection against Leishmania major in resistant C57BL/6 mice is mediated by Th1 cells, whereas susceptibility in BALB/c mice is the result of Th2 development. IL-12 release by L. major-infected dendritic cells (DC) is critically involved in differentiation of Th1 cells. Previously, we reported that strain differences in the production of DC-derived factors, e.g., IL-1alphabeta, are in part responsible for disparate disease outcome. In the present study, we analyzed the release of IL-12 from DC in more detail. Stimulated DC from C57BL/6 and BALB/c mice released comparable amounts of IL-12p40 and p70. In the absence of IL-4, BALB/c DC produced significantly more IL-12p40 than C57BL/6 DC. Detailed analyses by Western blot and ELISA revealed that one-tenth of IL-12p40 detected in DC supernatants was released as the IL-12 antagonist IL-12p40 homodimer (IL-12p80). BALB/c DC released approximately 2-fold more IL-12p80 than C57BL/6 DC both in vitro and in vivo. Local injection of IL-12p80 during the first 3 days after infection resulted in increased lesion volumes for several weeks in both L. major-infected BALB/c or C57BL/6 mice, in higher lesional parasite burdens, and decreased Th1-cytokine production. Finally, IL-12p40-transgenic C57BL/6 mice characterized by overexpression of p40 showed increased levels of serum IL-12p80 and enhanced disease susceptibility. Thus, in addition to IL-1alphabeta, strain-dependent differences in the release of other DC-derived factors such as IL-12p80 may influence genetically determined disease outcome.     

Hepatocyte growth factor prevents lupus nephritis in a murine lupus model of chronic graft-versus-host disease

Chronic graft-versus-host disease (GVHD) induced in (C57BL/6 × DBA/2) F1 (BDF1) mice by the injection of DBA/2 mouse spleen cells represents histopathological changes associated with systemic lupus erythematosus (SLE), primary biliary cirrhosis (PBC) and Sjogren's syndrome (SS), as indicated by glomerulonephritis, lymphocyte infiltration into the periportal area of the liver and salivary glands. We determined the therapeutic effect of hepatocyte growth factor (HGF) gene transfection on lupus using this chronic GVHD model. Chronic GVHD mice were injected in the gluteal muscle with either HVJ liposomes containing 8 μg of the human HGF expression vector (HGF-HVJ liposomes) or mock vector (untreated control). Gene transfer was repeated at 2-week intervals during 12 weeks. HGF gene transfection effectively prevented the proteinuria and histopathological changes associated with glomerulonephritis. While liver and salivary gland sections from untreated GVHD mice showed prominent PBC- and SS-like changes, HGF gene transfection reduced these histopathological changes. HGF gene transfection greatly reduced the number of splenic B cells, host B cell major histocompatibility complex class II expression, and serum levels of IgG and anti-DNA antibodies. IL-4 mRNA expression in the spleen, liver, and kidneys was significantly decreased by HGF gene transfection. CD28 expression on DBA/2 CD4+ T cells was decreased by the addition of recombinant HGF in vitro. Furthermore, IL-4 production by DBA/2 CD4+ T cells stimulated by irradiated BDF1 dendritic cells was significantly inhibited by the addition of recombinant HGF in vitro. These results suggest that HGF gene transfection inhibited T helper 2 immune responses and reduced lupus nephritis, autoimmune sialoadenitis, and cholangitis in chronic GVHD mice. HGF may represent a novel strategy for the treatment of SLE, SS and PBC.     

Progression from acute to chronic disease in a murine parent-into-F1 model of graft-versus-host disease

The parent-into-immunocompetent-F(1) model of graft-vs-host disease (GVHD) induces immune dysregulation, resulting in acute or chronic GVHD. The disease outcome is thought to be determined by the number of parental anti-F(1) CTL precursor cells present in the inoculum. Injection of C57BL/6 (B6) splenocytes into (B6 x DBA/2)F(1) (B6D2F(1)) mice (acute model) leads to extensive parental cell engraftment and early death, whereas injection of DBA/2 cells (chronic model) results in little parental cell engraftment and a lupus-like disease. This study demonstrated that injection of BALB/c splenocytes into (BALB/c x B6)F(1) (CB6F(1)) mice resulted in little engraftment of parental lymphocytes and the development of lupus as expected. Injection of B6 splenocytes into CB6F(1) initiated an initial burst of parental cell engraftment similar to that of B6 into B6D2F(1). However, the acute disease resolved, and the CB6F(1) mice went on to develop chronic GVHD with detectable Abs to ssDNA, dsDNA, and extractable nuclear Ags. Limiting dilution CTL assays determined that B6 splenocytes have CTL precursor frequencies of 1/1000 against both CB6F(1) and B6D2F(1), whereas DBA/2 and BALB/c splenocytes have a CTL precursor frequency of 1/20,000 for their respective F(1)s. The Th cell precursor frequency for B6 anti-DBA/2 was 3-fold higher than that for B6 anti-BALB/c determined by limiting dilution proliferation assays. These results indicate the importance of adequate allospecific helper as well as effector T cells for the induction and maintenance of acute GVHD in this model, and presents an unexpected model in which initial acute GVHD is replaced by the chronic form of disease.     

Identification of a single non-H-2 gene regulating graft-versus-host disease response

Non-MHC loci have been shown to play an important role in the development and regulation of graft-vs-host disease (GVHD). In the murine model of GVHD under study, injection of C57BL/6 spleen cells into unirradiated (C57BL/6 x DBA/2)F1 hybrid recipient mice results in an acute form of GVHD characterized by CTL, suppressor cells, and runting. In contrast, injection of DBA/2 spleen cells into the same recipients results in a chronic form of GVHD that is characterized by a lack of CTL and hyperproduction of Ig and autoantibodies. After preliminary studies with the use of congenic mice showed that non-MHC loci were controlling GVHD responses in this model, genetic analysis of GVHD response of BXD recombinant inbred strains and (B10.D2 x DBA/2) X DBA/2 BC mice identified a single locus, Gvh, on chromosome 7 that controls whether acute or chronic GVHD results from injection of parental lymphocytes into unirradiated (C57BL/6 x DBA/2)F1 recipient mice.     

DBA/2J and DBA/2Ha lymphocytes differ in their ability to induce graft-vs-host disease

Graft-vs-host disease (GVHD) is a common occurrence after bone marrow transplantation despite the use of MHC-matched donors and recipients. This indicates that non-MHC loci play an important role in the regulation and development of GVHD. Non-MHC loci have been shown to regulate GVHD in a murine model where acute GVHD results from i.v. injection of C57BL/6J spleen cells into B6D2F1/J [C57BL/6J X DBA/2J)F1) recipients while chronic GVHD results from injection of DBA/2J spleen cells. In contrast to the hyperproduction of Ig and auto-antibodies that is characteristic of the chronic GVHD that occurs after injection of DBA/2J cells, injection of DBA/2Ha cells was found to induce CTL and suppressor cells characteristic of the acute GVHD that results from injection of C57BL/6 cells into B6D2F1/J recipients. Genetic analysis indicated that one autosomal locus is responsible for the different GVHD responses of DBA/2J and DBA/2Ha cells and that the DBA/2Ha allele is dominant. Further studies indicate that the different responses by DBA/2J and DBA/2Ha cells is not due to functional differences between the two sets of cells but by a radiosensitive B6D2F1 recipient immune response which discriminates between the DBA/2J and DBA/2Ha spleen cells.     

Thymic hormone modulation of age-induced changes in the induction of graft-versus-host disease by DBA/2J lymphocytes

Aging induces a number of changes in the immune system, including the involution of the thymus which results in the loss of thymic hormone production and alteration in T cell function. One age-dependent change in immune response is the increasing risk of developing acute or chronic form of graft-versus-host disease (GVHD) following bone marrow transplantation as the age of the recipient increases. A murine model of GVHD that has been extensively studied is one in which injection of C57BL/6 spleen cells into unirradiated B6D2F1 mice results in an acute form of GVHD characterized by cytolytic T lymphocytes (CTL), suppressor cells, runting, and occasionally death. In contrast, injection of DBA/2J spleen cells results in a chronic form of GVHD characterized by a lack of CTL and hyperproduction of immunoglobulin and autoantibodies. This study shows that the GVHD response of DBA/2J spleen cells is dependent on the age of the donor DBA/2J mice. If spleen cells from DBA/2J mice older than 3 months are injected into B6D2F1 recipients, CTL and lack of immunoglobulin production indicative of acute GVHD result. Administration of thymosin fraction 5, a collection of thymic hormones, to DBA/2J mice older than 3 months caused spleen cells from these treated mice to give a GVHD response characteristic of the chronic form of GVHD in B6D2F1 recipients. Thus, thymic hormones were able to modulate the changes in GVHD responses of DBA/2 lymphocytes that occur as the mice age. Preliminary fractionation of TF5 has indicated that there are at least two active thymic peptides present in TF5.     

Breaking of CD8+ T cell tolerance through in vivo ligation of CD40 results in inhibition of chronic graft-versus-host disease and complete donor cell engraftment

In the DBA/2 --> unirradiated (C57BL/6 x DBA/2)F(1) model of chronic graft-vs-host disease (cGVHD), donor CD4(+) T cells play a critical role in breaking host B cell tolerance, while donor CD8(+) T cells are rapidly removed and the remaining cells fall into anergy. Previously we have demonstrated that in vivo ligation of GITR (glucocorticoid-induced TNF receptor-related gene) can activate donor CD8(+) T cells, subsequently converting the disease pattern from cGVHD to an acute form. In this study, we investigated the effect of an agonistic mAb against CD40 on cGVHD. Treatment of anti-CD40 mAb inhibited the production of anti-DNA IgG1 autoantibody and the development of glomerulonephritis. The inhibition of cGVHD occurred because anti-CD40 mAb prevented donor CD8(+) T cell anergy such that subsequently activated donor CD8(+) T cells deleted host CD4(+) T cells and host B cells involved in autoantibody production. Additionally, functionally activated donor CD8(+) T cells induced full engraftment of donor hematopoietic cells and exhibited an increased graft-vs-leukemia effect. However, induction of acute GVHD by donor CD8(+) T cells seemed to be not so apparent. Further CTL analysis indicated that there were lower levels of donor CTL activity against host cells in mice that received anti-CD40 mAb, compared with mice that received anti-GITR mAb. Taken together, our results suggest that a different intensity of donor CTL activity is required for removal of host hematopoietic cells, including leukemia vs induction of acute GVHD.     

Identification of a locus on distal mouse chromosome 12 that controls resistance to tumor necrosis factor-induced lethal shock

Administration of recombinant murine tumor necrosis factor (TNF) to mice results in lethal shock, characterized by hypotension, hypothermia, and dramatic induction of cytokines released in the circulation, such as interleukin-6 (IL-6). The sensitivity of mice to the effects of murine TNF varies from strain to strain. DBA/2 mice were found to be considerably more resistant to TNF than C57BL/6 mice. The resistance proved to be dominant since (C57BL/6 x DBA/2)F1 mice were also resistant. Using BXD recombinant inbred mice and a dose of TNF lethal for C57BL/6 but not for DBA/2 mice, we found that the resistance to TNF links to loci coding for corticosteroid-binding globulin (Cbg), alpha1-protease inhibitor (Spi1), contrapsin (Spi2) and the contrapsin-regulating gene Spi2r that form a gene cluster on chromosome 12. Quantitative trait-loci analysis of TNF-induced induction of IL-6 and of hypothermia also points to the importance of this locus (P < 0.0002 and P = 0.017, respectively), more particularly the Cbg and Spi2 loci, in the resistance to TNF. We propose to name the locus "TNF protection locus." The data suggest that endogenous protease inhibitors and/or glucocorticoids play a significant role in the attenuation of TNF-induced lethal shock. This study also demonstrates that loci affecting important biological responses can be identified with very high resolution using recombinant inbred mice.     

TGF-beta and IL-10 regulation of IFN-gamma produced in Th2-type schistosome granulomas requires IL-12

Interleukin-10 (IL-10) and transforming growth factor-beta (TGF-beta) regulate CD4+ T cell interferon-gamma (IFN-gamma) secretion in schistosome granulomas. The role of IL-12 was determined using C57BL/6 and CBA mice. C57BL/6 IL-4-/- granuloma cells were stimulated to produce IFN-gamma when cultured with IL-10 or TGF-beta neutralizing monoclonal antibody. In comparison, C57BL/6 wild-type (WT) control granuloma cells produced less IFN-gamma. IL-12, IL-18, and soluble egg antigen stimulated IFN-gamma release from C57BL/6 IL-4-/- and WT mice. IFN-gamma production in C57 IL-4-/- and WT granulomas was IL-12 dependent, because IL-12 blockade partly abrogated IFN-gamma secretion after stimulation. All granuloma cells released IL-12 (p70 and p40), and IL-12 production remained constant after anti-TGF-beta, anti-IL-10, recombinant IL-18, or antigen stimulation. C57 WT and IL-4-/- mouse granuloma cells expressed IL-12 receptor (IL-12R) beta1-subunit mRNA but little beta2 mRNA. TGF-beta or IL-10 blockade did not influence beta1 or beta2 mRNA expression. CBA mouse dispersed granuloma cells released no measurable IFN-gamma, produced IL-12 p70 and little p40, and expressed IL-12R beta2 and little beta1 mRNA. In T helper 2 (Th2) granulomas of C57BL/6 WT and IL-4-/- mice, cells produce IL-12 (for IFN-gamma production) and IL-10 and TGF-beta modulate IFN-gamma secretion via mechanisms independent of IL-12 and IL-12R mRNA regulation. We found substantial differences in control of granuloma IFN-gamma production and IL-12 circuitry in C57BL/6 and CBA mice.     

A polymorphism in the coding region of Il12b promotes IL-12p70 and IL-23 heterodimer formation

IL-12 and IL-23 are heterodimeric cytokines involved in the induction of Th1 and Th17 immune responses. Previous work indicated that a region on chromosome 11 encoding the IL-12p40 subunit regulates strain differences in susceptibility to murine trinitrobenzene sulfonic acid-induced colitis. In addition, this region determines strain differences in LPS-induced IL-12 responses. In this study, we investigated how polymorphisms in the coding region of murine Il12b influence IL-12 and IL-23 heterodimer formation. Transfection studies using constructs containing IL-12p35 linked to IL-12p40 from the colitis-resistant C57BL/6 strain or to the polymorphic p40 variant from the colitis-susceptible SJL/J strain demonstrated that SJL/J-derived p40 constructs synthesized significantly more IL-12p70 than did constructs harboring the C57BL/6-p40 variant. This could not be attributed to differences in synthesis rate or secretion, implicating a greater affinity of SJL/J-derived IL-12p40 for its IL-12p35 subunit. This greater affinity is also associated with increased IL-23 synthesis. In addition, C57BL/6 mice transgenic for the SJL/J 40 variant synthesized significantly more IL-12p70 upon LPS challenge and were more prone to develop colonic inflammation than did C57BL/6 mice transgenic for the C57BL/6-p40 variant. The more efficient binding of the polymorphic Il12b variant to p35 and p19 is most likely due to conformational changes following differential glycosylation as a consequence of the polymorphism. The high synthesis rate of the mature cytokines resulting from this efficient binding can lead to rapid proinflammatory skewing of immune responses and distortion of the homeostatic balance underlying the greater susceptibility for colitis.     

Mechanisms of enhanced macrophage-mediated prostaglandin E2 production and its suppressive role in Th1 activation in Th2-dominant BALB/c mice

PGE(2) has been known to suppress Th1 responses. We studied the difference in strains of mice in PGE(2) production by macrophages and its relation to Th1 activation. Macrophages from BALB/c mice produced greater amounts of PGE(2) than those from any other strains of mice, including C57BL/6, after LPS stimulation. In accordance with the amount of PGE(2) produced, macrophage-derived IL-12 and T cell-derived IFN-gamma production were more strongly suppressed in BALB/c macrophages than in C57BL/6 macrophages. When macrophages were treated with indomethacin or EP4 antagonist, Th1 cytokines were more markedly increased in cells from BALB/c mice than in those from C57BL/6 mice. Although cyclooxygenase-2 was expressed similarly after LPS stimulation in these mouse strains, the release of arachidonic acid and the expression of type V secretory phospholipase A(2) mRNA were greater in BALB/c macrophages. However, exogenous addition of arachidonic acid did not reverse the lower production of PGE(2) by C57BL/6 macrophages. The expression of microsomal PGE synthase, a final enzyme of PGE(2) synthesis, was also greater in BALB/c macrophages. These results indicate that the greater production of PGE(2) by macrophages, which is regulated by secretory phospholipase A(2) and microsomal PGE synthase but not by cyclooxygenase-2, is related to the suppression of Th1 cytokine production in BALB/c mice.     

Clear suppression of Th1 responses but marginal amelioration of autoimmune manifestations by IL-12p40 transgene in MRL-FAS(lprcg)/FAS(lprcg) mice

To investigate the effects of overproduction of IL-12p40, a potent antagonist against IL-12, on lupus-like autoimmune disease in vivo, we generated p40 transgenic MRL-Fas(lprcg)/Fas(lprcg) mice. Serum p40 and IL-12 levels were 600- to 8000-fold and 3- to 20-fold higher in transgenic (p40-lpr(cg)) than nontransgenic (lpr(cg)) mice, respectively. Serum IFN-gamma levels increased after 3 months of age in lpr(cg) and this age-related increase was completely abrogated in p40-lpr(cg). Serum IL-4 levels were the same in both mice. Production of IgM and IgG anti-double-stranded DNA (dsDNA) antibodies was significantly lower in p40-lpr(cg). Anti-dsDNA antibodies decreased in Th1-dependent IgG2a but increased in the Th2-dependent IgG1 subclass significantly in p40-lpr(cg). Proteinuria, glomerulonephritis, and survival were only marginally ameliorated in p40-lpr(cg). The results suggest that excess p40 production in vivo may suppress Th1 responses in autoantibody and IFN-gamma production but lead to minimal improvement of clinical manifestations of autoimmune disease in this mouse model.     

Roquinimex-mediated protection effect on the development of chronic graft-versus-host disease in mice is associated with induction of Th1 cytokine production and inhibition of proinflammatory cytokine production

Roquinimex is an immunomodulator that can effectively inhibit the development of several autoimmune diseases in animal models, but the mechanism is still unknown. In this study, we investigated the effect of roquinimex on chronic graft-versus-host disease (GVHD) in mice, a well-established model for human systemic lupus erythematosus (SLE). Oral administration of roquinimex significantly suppressed the development of proteinuria and ameliorated nephritis symptoms in chronic GVHD mice. In addition, renal histopathology and immunohistochemistry studies revealed reduced glomerulonephritis and decreased IgG deposition in chronic GVHD mice treated with roquinimex. Chronic GVHD is characterized by a predominance of Th2 cytokines, and proinflammatory cytokines that also play an important role in the pathology of tissue damage. Therefore, we focused on the effect of roquinimex on cytokine production. Chronic GVHD mouse splenocytes exhibited severely reduced interferon (IFN)-gamma production in response to Concanavalin (Con A) stimulation and an overt Th2 skewness. Roquinimex treatment, however, induced IFN-gamma production and restored the Th1/Th2 cytokine balance, although only a minimal effect of roquinimex on interleukin (IL)-4 secretion was observed. The production of the proinflammatory cytokines TNF-alpha and IL-1 beta by peritoneal macrophages from lipopolysaccharide (LPS)-treated GVHD mice was significantly inhibited by roquinimex treatment. These data suggested that the beneficial effect of roquinimex on lupus might, at least in part, result from a restoration of Th1/Th2 cytokine balance and inhibition of inflammatory cytokine production.