Increased Production Of GM-CSF Research Articles

Butanol Purified Food Allergy Herbal Formula-2 Has an Immunomodulating Effect ex-vivo in Pediatric Crohn's Disease Subjects.

Background: TNF-α has a major role in the pathogenesis of Crohn's disease (CD). In contrast, GM-CSF may be beneficial for its anti-inflammatory role in a subset of patients with CD with antibodies against GM-CSF as seen in prior trials of GM-CSF which resulted in clinical improvement in CD. We developed butanol purified Food Allergy Herbal Formula-2 (B-FAHF-2) by refining FAHF-2. FAHF-2 suppressed TNF-α production by human peripheral blood mononuclear cells (PBMCs) and colonic mucosa, and abrogated colitis in a murine model. We sought to examine the effect of B-FAHF-2 and the herbs that comprise it on TNF-α and GM-CSF production as a potential herbal therapy for the treatment of CD.Methods: B-FAHF-2 was examined using high pressure liquid chromatography (HPLC) and compared to the original formulation, FAHF-2. PBMCs from pediatric patients with CD were cultured with lipopolysaccharide and B-FAHF-2, individual herbs or medium alone. Colonic biopsy specimens were cultured with or without B-FAHF-2. TNF-α and GM-CSF were measured by enzyme-linked immunosorbent assay (ELISA). B-FAHF-2 efficacy was tested in vivo in the CD45Rbhi transfer model.Results: B-FAHF-2 had a similar HPLC fingerprint as FAHF-2 but decreased TNF-α production by PBMCs and colonic mucosa from pediatric CD subjects at 20% of the FAHF-2 dose. B-FAHF-2 increased GM-CSF production by PBMCs and colonic mucosa from pediatric CD subjects including those with antibodies to GM-CSF. Of B-FAHF-2's herbal constituents, only Huang Bai suppressed TNF-α and increased GM-CSF production. In the murine model, B-FAHF-2 treatment alleviated colitis.Conclusions: B-FAHF-2 decreased TNF-α production by PBMCs and colonic mucosa from pediatric subjects at a lower dose than FAHF-2. B-FAHF-2 also increased GM-CSF production by PBMCs independent of antibodies. B-FAHF-2 may have a benefit in CD patients.

Brg1 restrains the pro-inflammatory properties of ILC3s and modulates intestinal immunity

Group 3 innate lymphoid cells (ILC3s), a subset of the innate lymphoid cells, are abundantly present in the intestine and are crucial regulators of intestinal inflammation. Brg1 (Brahma-related gene 1), a catalytic subunit of the mammalian SWI-SNF-like chromatin-remodeling BAF complex, regulates the development and function of various immune cells. Here, by genetic deletion of Brg1 in ILC3s (Smarca4ΔILC3), we prove that Brg1 supports the differentiation of NKp46+ILC3s by promoting the T-bet expression in NKp46−ILC3s, which facilitates the conversion of NKp46−ILC3s to NKp46+ILC3s. Strikingly, Smarca4ΔILC3 mice of the Rag1−/− background develop spontaneous colitis accompanied with increased GM-CSF production in ILC3s. By construction of a mixed bone marrow chimeric system, we demonstrate that Brg1 enhances T-bet and inhibits GM-CSF expression in ILC3s through a cell-intrinsic manner. Blockade of GM-CSF ameliorates colitis in Rag1−/−Smarca4ΔILC3 mice, suggesting that the suppression of GM-CSF production from ILC3s by Brg1 serves as a critical mechanism for Brg1 to restrain intestinal inflammation. We have further demonstrated that Brg1 binds to the Tbx21 and Csf2 gene locus in ILC3s, and favors the active and repressive histones modifications on gene locus of Tbx21 and Csf2 respectively. Our work reveals the essential role of Brg1 in intestinal immunity by regulating ILC3s.

LILRA2 Selectively Modulates LPS-Mediated Cytokine Production and Inhibits Phagocytosis by Monocytes

The activating immunoglobulin-like receptor, subfamily A, member 2 (LILRA2) is primarily expressed on the surface of cells of the innate immunity including monocytes, macrophages, neutrophils, basophils and eosinophils but not on lymphocytes and NK cells. LILRA2 cross-linking on monocytes induces pro-inflammatory cytokines while inhibiting dendritic cell differentiation and antigen presentation. A similar activating receptor, LILRA4, has been shown to modulate functions of TLR7/9 in dendritic cells. These suggest a selective immune regulatory role for LILRAs during innate immune responses. However, whether LILRA2 has functions distinct from other receptors of the innate immunity including Toll-like receptor (TLR) 4 and FcγRI remains unknown. Moreover, the effects of LILRA2 on TLR4 and FcγRI-mediated monocyte functions are not elucidated. Here, we show activation of monocytes via LILRA2 cross-linking selectively increased GM-CSF production but failed to induce IL-12 and MCP-1 production that were strongly up-regulated by LPS, suggesting functions distinct from TLR4. Interestingly, LILRA2 cross-linking on monocytes induced similar amounts of IL-6, IL-8, G-CSF and MIP-1α but lower levels of TNFα, IL-1β, IL-10 and IFNγ compared to those stimulated with LPS. Furthermore, cross-linking of LILRA2 on monocytes significantly decreased phagocytosis of IgG-coated micro-beads and serum opsonized Escherichia coli but had limited effect on phagocytosis of non-opsonized bacteria. Simultaneous co-stimulation of monocytes through LILRA2 and LPS or sequential activation of monocytes through LILRA2 followed by LPS led lower levels of TNFα, IL-1β and IL-12 production compared to LPS alone, but had additive effect on levels of IL-10 and IFNγ but not on IL-6. Interestingly, LILRA2 cross-linking on monocytes caused significant inhibition of TLR4 mRNA and protein, suggesting LILRA2-mediated suppression of LPS responses might be partly via regulation of this receptor. Taken together, we provide evidence that LILRA2-mediated activation of monocytes is significantly different to LPS and that LILRA2 selectively modulates LPS-mediated monocyte activation and FcγRI-dependent phagocytosis.

HIV-1–Infected Peripheral Blood Mononuclear Cells Enhance Neutrophil Survival and HLA-DR Expression Via Increased Production of GM-CSF: Implications for HIV-1 Infection

HIV-1 bound to intact neutrophils efficiently infects activated peripheral blood mononuclear cells (PBMC). Here, we evaluated the effect of the local milieu created by activated PBMC before and after HIV-1 infection on neutrophil survival and HLA-DR expression, with emphasis placed on a role for GM-CSF. PBMC of healthy adult individuals were activated by phytohemagglutinin (PHA) or anti-CD3/anti-CD28 and were subsequently cultured without (HIV-1⁻) or with HIV-1 (HIV-1+). The effects of the culture supernatants or recombinant GM-CSF on survival and HLA-DR expression by neutrophils of healthy adult individuals and of HIV-1-infected individuals were evaluated using flow cytometry. Conditioned medium from PHA-activated PBMC (HIV-1⁻ and HIV-1+) increased neutrophil survival and induced HLA-DR expression by neutrophils of healthy individuals in a GM-CSF dependent fashion. HIV-1 infection variably, but consistently, increased GM-CSF production by PHA-activated PBMC but not GM-CSF production by anti-CD3/anti-CD28-activated PBMC. The latter was correlated with a loss of CD3+GMCSF+ cells after infection. Neutrophils of elite controllers exhibited a diminished HLADR response to GM-CSF in culture, whereas neutrophils of HIV-1+ subjects having a low viral load on anti-retroviral therapy or subjects with a high viral load exhibited a range of HLA-DR responses. GM-CSF production within the mucosa or draining lymph nodes may promote HIV-1 infection by facilitating sustained contact between viable neutrophils with bound HIV-1 and CD4 lymphocytes. The minimal effect of GM-CSF on HLA-DR expression by neutrophils of elite controllers provides indirect support for this conclusion.

Production and inhibition of eosinophil colony stimulating factor by lymphocytes from patients with bronchial asthma

To clarify the mechanism of the eosinophilia in patients with bronchial asthma (BA), we examined the production and inhibition of eosinophil colony stimulating factor (Eo-CSF) of mononuclear cells (MNC) and lymphocytes from BA. After bone marrow cells from normal volunteers (NA) were incubated with culture media of MNC or lymphocytes cultured in the presence of interleukin-2 (IL-2, 1 U/ml) or Dermatophagoides farinae (10 micrograms/ml, Df), the number of eosinophils was increased and GM-CSF production was increased after 5 days of culture. Interleukin 5 (IL-5) production was increased in some cases of BA, but did not differ between BA and NA delete. When MNC were incubated in the presence of IL-2 and interleukin 4 (IL-4), neither eosinophilia nor GM-CSF production occurred. These results suggest first that MNC and T cells in BA have increased responsiveness to IL-2 and Df, second that increased GM-CSF production partly causes eosinophilia, and third that IL-4 inhibited IL-2-induced eosinophil proliferation by inhibition of GM-CSF production.

Y box-binding factor promotes eosinophil survival by stabilizing granulocyte-macrophage colony-stimulating factor mRNA.

Short-lived peripheral blood eosinophils are recruited to the lungs of asthmatics after allergen challenge, where they become long-lived effector cells central to disease pathophysiology. GM-CSF is an important cytokine which promotes eosinophil differentiation, function, and survival after transit into the lung. In human eosinophils, GM-CSF production is controlled by regulated mRNA stability mediated by the 3' untranslated region, AU-rich elements (ARE). We identified human Y box-binding factor 1 (YB-1) as a GM-CSF mRNA ARE-specific binding protein that is capable of enhancing GM-CSF-dependent survival of eosinophils. Using a transfection system that mimics GM-CSF metabolism in eosinophils, we have shown that transduced YB-1 stabilized GM-CSF mRNA in an ARE-dependent mechanism, causing increased GM-CSF production and enhanced in vitro survival. RNA EMSAs indicate that YB-1 interacts with the GM-CSF mRNA through its 3' untranslated region ARE. In addition, endogenous GM-CSF mRNA coimmunoprecipitates with endogenous YB-1 protein in activated eosinophils but not resting cells. Thus, we propose a model whereby activation of eosinophils leads to YB-1 binding to and stabilization of GM-CSF mRNA, ultimately resulting in GM-CSF release and prolonged eosinophil survival.

Effect of 2,3,7,8-tetrachlorodibenzo- p-dioxin on anti-CD3-induced changes in T-cell subsets and cytokine production

The influence of 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD) exposure on the cytokine-dependent toxicity syndrome induced by the injection of 145-2C11 (anti-CD3), a hamster monoclonal antibody to the CD3 epsilon portion of the murine T-cell receptor, was studied. This syndrome has been attributed to the transient release of several cytokines including TNF-α, IFN-γ, IL-2, IL-3, IL-6, and GM-CSF. Exposure of C57B1/6 mice to TCDD (15 μg/kg) 2 days prior to anti-CD3 injection exacerbated anti-CD3-induced toxicity as evidenced by significantly enhanced and prolonged body weight loss and lymphoid tissue atrophy. Unexpectedly, TCDD exposure did not alter plasma levels of TNF or IL-2 at any time after anti-CD3 injection. However, plasma IFN-γ was significantly reduced at 24 h and plasma IL-6 levels were elevated 48 h after anti-CD3 injection in TCDD-treated mice. In addition, TCDD exposure resulted in elevated levels of plasma GM-CSF at 24 and 48 h. Since the body weight of TCDD-treated mice diverged from vehicle-treated mice at 48 h, it suggests that the increased IL-6 and GM-CSF may have contributed to the prolonged loss of body weight. The ability of spleen cells from vehicle- and TCDD-treated mice to produce cytokines was evaluated in vitro at various times after anti-CD3 injection. TCDD treatment resulted in reduced IL-2 and GM-CSF production at 90 min but increased GM-CSF production at 48 h post-anti-CD3 injection. In contrast, TCDD exposure did not influence cytokine production by spleen cells from mice injected with a control IgG and activated in vitro with anti-CD3. Flow cytometric analysis showed that the percentage of CD4 + cells in the draining lymph nodes from TCDD-treated mice was reduced 48–144 h post-anti-CD3 injection. In contrast, the percentage of CD8 + cells was not affected by TCDD exposure. A high fraction of lymph node cells (LNC) from TCDD-treated animals showed decreased forward angle light scatter and increased 90° light scatter following anti-CD3 injection, which is a pattern characteristic of cells undergoing apoptosis. In contrast, few LNC from vehicle-treated animals showed this light scatter profile. These data suggest that TCDD may be targeting T-helper cells during activation resulting in activation-driven cell death (apoptosis) rather than differentiation.