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.     

Administration of an IL-12-encoding DNA plasmid prevents the development of chronic graft-versus-host disease (GVHD)

The transfer of DBA/2 spleen cells into (C57BL/10 x DBA/2)F1 mice induces chronic graft-vs-host disease (GVHD), which is characterized by the production of Th2 cytokines, hypergammaglobulinemia, and immune complex-mediated glomerulonephritis like systemic lupus erythematosus. IL-12 strongly induces the production of Th1 cytokines and reduces Th2 activity in vivo. In this study, the effect of gene therapy on the development of murine chronic GVHD was examined using an IL-12-encoding plasmid (pCAGGSIL-12), with the expectation that it might regulate Th1/Th2 activity and have a beneficial impact on the clinical manifestations of disease. pCAGGSIL-12 or its p40 antagonist plasmid (pCAGGSp40) were injected i.m. every 3 wk in GVHD-induced (C57BL/10 x DBA/2)F1 mice. A total of 100 microg of pCAGGSIL-12 improved the Th1/Th2 balance in vivo, suppressed the production of IgG, and significantly reduced the development of glomerulonephritis. GVHD was exacerbated by injection of the pCAGGSp40 antagonist. Our results demonstrate that GVHD can be treated successfully by the administration of an IL-12-encoding plasmid, and that such therapy does not induce acute GVHD.     

The two sides of cytokine signaling and glaucomatous optic neuropathy

The mechanistic study of glaucoma pathogenesis has shifted to seeking to understand the effects of immune responses on retinal ganglion cell damage and protection. Cytokines are the hormonal factors that mediate most of the biological effects in both the immune and nonimmune systems. CD4-expressing T helper cells are a major source of cytokine production and regulation. Type 1 helper T (Th1) cells are characterized by the production of proinflammatory cytokines such as interferon-gamma, interleukin (IL)-2, IL-12, IL-23, and tumor necrosis factor-alpha while type 2 helper T (Th2) cells are characterized by the production of IL-4, IL-5, IL-6, and IL-10. The balance of Th1/Th2 cytokine production influences many pathological processes and plays both causative and protective roles in neuron damages. Growing evidence indicates that imbalances of Th1/Th2 cytokine production are involved in neural damage or protection in many neurological diseases. In this review, we discuss the possible roles of Th1/Th2 cytokine production and imbalance of Th1/Th2 cytokines in retina, especially glaucomatous optic neuropathy.     

The two sides of cytokine signaling and glaucomatous optic neuropathy

The mechanistic study of glaucoma pathogenesis has shifted to seeking to understand the effects of immune responses on retinal ganglion cell damage and protection. Cytokines are the hormonal factors that mediate most of the biological effects in both the immune and nonimmune systems. CD4-expressing T helper cells are a major source of cytokine production and regulation. Type 1 helper T (Th1) cells are characterized by the production of proinflammatory cytokines such as interferon-gamma, interleukin (IL)-2, IL-12, IL-23, and tumor necrosis factor-alpha while type 2 helper T (Th2) cells are characterized by the production of IL-4, IL-5, IL-6, and IL-10. The balance of Th1/Th2 cytokine production influences many pathological processes and plays both causative and protective roles in neuronal damage. Growing evidence indicates that imbalances of Th1/Th2 cytokine production are involved in neural damage or protection in many neurological diseases. In this review, we discuss the possible roles of Th1/Th2 cytokine production and imbalance of Th1/Th2 cytokines in retina, especially glaucomatous optic neuropathy.     

A study of cytokine production in acute graft-vs-host disease

The role of cytokines in the development of acute graft-vs-host disease (GVHD) was investigated in B6AF1 mice that were injected with parental A/J lymphocytes. Splenocytes from GVH mice exhibited an increased capacity to produce interleukin (IL)-1, IL-6, and TNF-a when stimulated in culture with lipopolysaccharide (LPS). This enhanced capacity was diminished following in vivo treatment with immunosuppressive drugs. Concanavalin A-stimulated GVH spleen cells produced significantly lower levels of IL-2 but higher levels of interferon-gamma (IFN-gamma) than did syngeneic spleen cells. Immunosuppressive therapy in vivo increased the capacity of GVH spleen cells to produce IL-2. However, immunosuppressants differed in their effects on IFN-gamma production. Sch 24937 (6-bromo-5-chloro-2-[1-(methylsulfonyl)acetyl] 3-(2-pyridyl)indole) enhanced or had no effect while cyclosporin A consistently decreased the capacity of splenocytes to produce this lymphokine. These results indicate that the capacity of GVH splenocytes for cytokine production can be differentially affected by the actions of some pharmacological agents. The data also indicate that there may be differential regulation of the production of IL-2 and IFN-gamma by the Th1 subset in the GVH spleen.     

Inactive systemic lupus erythematosus id associated with a normal stimulated Th(1)/Th(2) cytokine secretory pattern

OBJECTIVES: To determine the Th(1)/Th(2)balance in systemic lupus erythematosus (SLE) patients with inactive disease. METHODS: A comprehensive analysis of peak secretion, overall cytokine production and secretory pattern of Th(1)and Th(2)cytokines from stimulated PBMC of 10 SLE patients with inactive disease and 10 age- and sex-matched controls. RESULTS: No significant differences were found in the peak and total secretion of all cytokines, as well as in the Th(1)and Th(2)secretory patterns and proliferative response between the two groups. CONCLUSION: Th(1)and Th(2) balance in inactive SLE is normal.     

Elevated proinflammatory cytokine production by a skewed T cell compartment requires monocytes and promotes inflammation in type 2 diabetes

An appropriate balance between proinflammatory (Th17 and Th1) and anti-inflammatory (regulatory T cells [Tregs] and Th2) subsets of T cells is critical to maintain homeostasis and avoid inflammatory disease. Type 2 diabetes (T2D) is a chronic inflammatory disease promoted by changes in immune cell function. Recent work indicates T cells are important mediators of inflammation in a mouse model of T2D. These studies identified an elevation in the Th17 and Th1 subsets with a decrease in the Treg subset, which culminates in inflammation and insulin resistance. Based on these data, we tested the hypothesis that T cells in T2D patients are skewed toward proinflammatory subsets. Our data show that blood from T2D patients has increased circulating Th17 cells and elevated activation of Th17 signature genes. Importantly, T cells required culture with monocytes to maintain Th17 signatures, and fresh ex vivo T cells from T2D patients appeared to be poised for IL-17 production. T cells from T2D patients also have increased production of IFN-γ, but produce healthy levels of IL-4. In contrast, T2D patients had decreased percentages of CD4(+) Tregs. These data indicate that T cells in T2D patients are naturally skewed toward proinflammatory subsets that likely promote chronic inflammation in T2D through elevated cytokine production. Potential therapies targeted toward resetting this balance need to be approached with caution due to the reciprocal relationship between Th17 cells and Tregs. Understanding the unique aspects of T2D T cells is essential to predict outcomes of such treatments.     

Role of Th1 and Th2 cytokines in BCG-induced IFN-gamma production: cytokine promotion and simulation of BCG effect

Induction of a T-helper-type 1 (Th1) immune response is indispensable for successful treatment of superficial bladder cancer with BCG. In this study possible involvement of various cytokines in BCG action as well as their potential roles in enhancing and mimicking BCG effect were explored. In immunocompetent cell cultures, IFN-gamma, a major Th1 cytokine, appears to be a late responsive cytokine to BCG stimulation. Its induction requires involvement of various endogenously produced Th1 and Th2 cytokines. Functional abolishment of any one of these cytokines (IL-2, IL-6, IL-12, IL-18, GMCSF, TNF-alpha, or IFN-alpha, except IL-10) by neutralizing antibodies leads to reduced IFN-gamma production (19-82% inhibition in mouse and 44-77% inhibition in human systems, respectively). In mice cytokines IL-2, IL-12, IL-18, and GMCSF are observed to synergize with BCG for IFN-gamma production, whereas in human cytokines IL-2, IL-12, TNF-alpha, and IFN-alpha exhibit similar synergistic effects. Rational combinations of these Th1-stimulating cytokines (IL-12 plus IL-18 in mice and IL-2 plus IL-12 in humans, respectively) dramatically up-regulate IFN-gamma production that is incomparably superior to BCG for induction of this cytokine. These results suggest that combined Th1-stimulating cytokines and combinations of BCG plus selected Th1-stimulating cytokines are rational candidates for further study in the treatment of bladder cancer patients.     

Effect of the fungal immunomodulatory protein FIP-fve on airway inflammation and cytokine production in mouse asthma model

The allergy is dependent on the balance between Th1 and Th2. The fungal immunodulatory protein (FIP-fve) was isolated from Flammulina velutipes. FIP-fve has been demonstrated to skew the response to Th1 cytokine production. We investigated whether oral administrations of FIP-fve inhibited allergen (OVA)-induced chronic airway inflammation in the mouse asthma model. After intranasal challenge with OVA, the airway inflammation and hyperresponsiveness were determined by bronchoalveolar lavage fluid (BALF) analysis and ELISA assay. Both pre-treated and post-treated with FIP-fve suppressed the airway hyperresponsiveness by methacholine challenge and significantly decreased the number of infiltrating inflammatory cells and Th2 cytokines in bronchoalveolar lavage fluid (BALF) and serum compared with the OVA sensitized mice. In addition, FIP-fve reduced OVA-specific IgE levels in serum. FIP-fve markedly alleviated the OVA-induced airway hyperresponsiveness (AHR) to inhaled methacholine. Based on lung histopathological studies using hematoxylin and Liu’s staining, FIP-fve inhibited inflammatory cell infiltration compared with the OVA-sensitized mice. Oral FIP-fve had an anti-inflammatory effect on OVA-induced airway inflammations and might posses the potential for alternative therapy for allergic airway diseases.     

Modulation of macrophage cytokine production by ES-62, a secreted product of the filarial nematode Acanthocheilonema viteae.

Parasite survival and host health may depend on the ability of the parasite to modulate the host immune response by the release of immunomodulatory molecules. Excretory-secretory (ES)-62, one such well-defined molecule, is a major secreted protein of the rodent filarial nematode Acanthocheilonema viteae, and has homologues in human filarial nematodes. Previously we have shown that ES-62 is exclusively associated with a Th2 Ab response in mice. Here we provide a rationale for this polarized immune response by showing that the parasite molecule suppresses the IFN-gamma/LPS-induced production, by macrophages, of bioactive IL-12 (p70), a key cytokine in the development of Th1 responses. This suppression of the induction of a component of the host immune response extends to the production of the proinflammatory cytokines IL-6 and TNF-alpha, but not NO. The molecular mechanism underlying these findings awaits elucidation but, intriguingly, the initial response of macrophages to ES-62 is to demonstrate a low and transient release of these cytokines before becoming refractory to further release induced by IFN-gamma/LPS. The relevance of our observations is underscored by the finding that macrophages recovered from mice exposed to "physiological" levels of ES-62 by the novel approach of continuous release from implanted osmotic pumps in vivo were similarly refractory to release of IL-12, TNF-alpha, IL-6, but not NO, ex vivo. Therefore, our results suggest that exposure to ES-62 renders macrophages subsequently unable to produce Th1/proinflammatory cytokines. This likely contributes to the generation of immune responses with an anti-inflammatory Th2 phenotype, a well-documented feature of filarial nematode infection.     

Depressed IL-1 production by chronic GVHD dermal fibroblasts

Chronic Graft-versus-Host disease (GVHD) is characterized by overt immunosuppression. In addition, the skin is a major anatomical site affected in chronic GVHD for reasons not yet known. Increased collagen deposition, a mononuclear cell infiltrate in the dermis as well as loss of fat and appendages, are observed in the skin. The inflammatory cytokine IL-1 was shown to affect fibroblast proliferation and secretory activities. In the present study, IL-1 generation by dermal fibroblasts, of chronic GVHD or control mice, was assessed. It was shown that two sequential signals are needed for IL-1 generation by dermal fibroblasts; priming by lymphokines/cytokines followed by a challenge with LPS. A variety of recombinant lymphokines and cytokines (G/M-CSF, IL-2, TNF, IL-1 beta and IFNs alpha, beta and gamma) were shown to be efficient in priming dermal fibroblasts for IL-1 generation. IL-1 activity in dermal fibroblasts, most probably of the IL-1 alpha species, was located in frozen-thawed cell lysates or associated to the cell membrane, though not secreted into the culture fluids. Dermal fibroblasts from chronic GVHD mice manifested a pronounced depression in IL-1 generation upon stimulation with exogenous lymphokines/cytokines and LPS. This was observed over a wide range of concentrations of lymphokines/cytokines and LPS. The depressed ability of chronic GVHD fibroblasts to generate IL-1 was pronounced even after few passages of the cells in vitro, and upon stimulation in culture outside the suppressive milieu of the animal.(ABSTRACT TRUNCATED AT 250 WORDS)     

In vivo CD86 blockade inhibits CD4+ T cell activation, whereas CD80 blockade potentiates CD8+ T cell activation and CTL effector function

To address whether a functional dichotomy exists between CD80 and CD86 in naive T cell activation in vivo, we administered anti-CD80 or CD86 blocking mAb alone or in combination to mice with parent-into-F(1) graft-vs-host disease (GVHD). In this model, the injection of naive parental T cells into unirradiated F(1) mice results in either a Th1 cytokine-driven, cell-mediated immune response (acute GVHD) or a Th2 cytokine-driven, Ab-mediated response (chronic GVHD) in the same F(1) recipient. Combined CD80/CD86 blockade beginning at the time of donor cell transfer mimicked previous results seen with CTLA4Ig and completely abrogated either acute or chronic GVHD by preventing the activation and maturation of donor CD4(+) T cells as measured by a block in acquisition of memory marker phenotype and cytokine production. Similar results were seen with selective CD86 blockade; however, the degree of CD4 inhibition was always less than that seen with combined CD80/CD86 blockade. A more striking effect was seen with selective CD80 blockade in that chronic GVHD was converted to acute GVHD. This effect was associated with the induction of Th1 cytokine production, donor CD8(+) T cell activation, and development of antihost CTL. The similarity of this effect to that reported for selective CTLA4 blockade suggests that CD80 is a critical ligand for CTLA4 in mediating the down-regulation of Th1 responses and CD8(+) T cell activation. In contrast, CD86 is critical for the activation of naive CD4(+) T cells in either a Th1 or a Th2 cytokine-mediated response.     

Lactobacillus strains differentially modulate cytokine production by hPBMC from pollen-allergic patients

The objective of this study was to assess the potential immunomodulatory effect of six Lactobacillus strains on human peripheral blood mononuclear cells (hPBMC) isolated from allergic patients. hPBMC from patients allergic to birch pollen or grass pollen were cultured in vitro in the presence or absence of selective bacterial strains. Cultures were left unstimulated or stimulated with αCD3/αCD28 or Bet v 1. After 1, 4 and 8 days, cells and culture supernatants were harvested and the effect on cellular proliferation and the supernatant levels of several cytokines was assessed. All strains had the ability to repress IL-13 production but did show a differential effect on IFN-γ induction. Both strains B223 and B1697 showed a lower IFN-γ, IL-12 and TNF-α induction as compared with the other tested strains. Strain B633 showed the best proliferation-suppressive properties in αCD3/αCD28-stimulated cells. Suppression of the T-helper type 2 (Th2) cytokine induction and induction of the Th1 cytokine production by specific strains might be beneficial for allergic patients having a disturbed Th1/Th2 immune balance. Furthermore, hPBMC of patients with seasonal allergy outside the pollen season can be used to determine the immunomodulatory activities of probiotic bacteria.     

Stimulation of host NKT cells by synthetic glycolipid regulates acute graft-versus-host disease by inducing Th2 polarization of donor T cells

NKT cells are a unique immunoregulatory T cell population that produces large amounts of cytokines. We have investigated whether stimulation of host NKT cells could modulate acute graft-vs-host disease (GVHD) in mice. Injection of the synthetic NKT cell ligand alpha-galactosylceramide (alpha-GalCer) to recipient mice on day 0 following allogeneic bone marrow transplantation promoted Th2 polarization of donor T cells and a dramatic reduction of serum TNF-alpha, a critical mediator of GVHD. A single injection of alpha-GalCer to recipient mice significantly reduced morbidity and mortality of GVHD. However, the same treatment was unable to confer protection against GVHD in NKT cell-deficient CD1d knockout (CD1d(-/-)) or IL-4(-/-) recipient mice or when STAT6(-/-) mice were used as donors, indicating the critical role of host NKT cells, host production of IL-4, and Th2 cytokine responses mediated by donor T cells on the protective effects of alpha-GalCer against GVHD. Thus, stimulation of host NKT cells through administration of NKT ligand can regulate acute GVHD by inducing Th2 polarization of donor T cells via STAT6-dependent mechanisms and might represent a novel strategy for prevention of acute GVHD.     

T cell TRAIL promotes murine lupus by sustaining effector CD4 Th cell numbers and by inhibiting CD8 CTL activity

T cells play an essential role in driving humoral autoimmunity in lupus. Molecules such as TRAIL exhibit strong T cell modulatory effects and are up-regulated in lupus, raising the possibility that they may influence disease severity. To address this possibility, we examined the role of TRAIL expression on pathogenic T cells in an induced model of murine lupus, the parent-into-F(1) (P-->F(1)) model of chronic graft-vs-host disease (GVHD), using wild-type or TRAIL-deficient donor T cells. Results were compared with mice undergoing suppressive acute GVHD. Although chronic GVHD mice exhibited less donor T cell TRAIL up-regulation and IFN-alpha-inducible gene expression than acute GVHD mice, donor CD4(+) T cell TRAIL expression in chronic GVHD was essential for sustaining effector CD4(+) Th cell numbers, for sustaining help to B cells, and for more severe lupus-like renal disease development. Conversely, TRAIL expression on donor CD8(+) T cells had a milder, but significant down-regulatory effect on CTL effector function, affecting the perforin/granzyme pathway and not the Fas ligand pathway. These results indicate that, in this model, T cell-expressed TRAIL exacerbates lupus by the following: 1) positively regulating CD4(+) Th cell numbers, thereby sustaining T cell help for B cells, and 2) to a lesser degree by negatively regulating perforin-mediated CD8(+) CTL killing that could potentially eliminate activated autoreactive B cells.     

Pulmonary overexpression of IL-10 augments lung fibrosis and Th2 responses induced by silica particles

Chronic inflammation and proinflammatory cytokines as well as T helper type 2 (Th2) cytokines have been involved in the pathogenesis of pulmonary injury and lung fibrosis. The actual role of IL-10 in lung fibrosis is still unclear because this cytokine has been identified as Th2 but possesses strong anti-inflammatory properties. To better dissect the potential role of IL-10 in silica-induced lung fibrosis, IL-10 was overexpressed in the lung of mice by adenoviral gene transfer during the inflammatory (administered at day -1) or the fibrotic (administered at day +30) stages of the disease. Pulmonary overexpression of IL-10 during both silica-induced lung inflammation and fibrosis exacerbated the fibrotic lesions as estimated by the measurement of hydroxyproline and other biochemical and histological markers. Increased expression of IL-10 significantly enhanced the number of lung lymphocytes and bronchoalveolar lavage fluid IgG1 but not IgG2a levels, indicating the induction of a Th2-like immune response. In addition, the production of the profibrotic Th2 cytokines IL-4 and IL-13 was also significantly increased upon IL-10 overexpression. No difference in transforming growth factor-beta or PGE(2) production was noted after adenoviral IL-10 treatment of silica-treated mice. Together, these data indicate that the increased expression of IL-10 significantly contributed to silica-induced lung fibrosis by exacerbating the Th2 response and the production of the profibrotic cytokines IL-4 and IL-13.     

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.     

Estrogen treatment down-regulates TNF-alpha production and reduces the severity of experimental autoimmune encephalomyelitis in cytokine knockout mice.

A shift toward Th2 cytokine production has been demonstrated during pregnancy and high dose estrogen therapy and is thought to be the primary mechanism by which estrogen suppresses the development of experimental autoimmune encephalomyelitis. However, low dose estrogen treatment is equally protective in the absence of a significant shift in cytokine production. In this study cytokine-deficient mice were treated with estrogen to determine whether a shift in Th2 cytokine production was required for the protective effects of hormone therapy. Estrogen effectively suppressed the development of experimental autoimmune encephalomyelitis in IL-4 and IL-10 knockout mice and in wild type littermate mice with a similar potency of protection. Significant disease suppression was also seen in IFN-gamma-deficient mice. The decrease in disease severity was accompanied by a concomitant reduction in the number of proinflammatory cytokine- and chemokine-producing cells in the CNS. Although there was no apparent increase in compensatory Th2 cytokine production in cytokine-deficient mice, there was a profound decrease in the frequency of TNF-alpha-producing cells in the CNS and the periphery. Therefore, we propose that one mechanism by which estrogen protects females from the development of cell-mediated autoimmunity is through a hormone-dependent regulation of TNF-alpha production.