Florfenicol has been shown to possess anti-inflammatory activity. However, its possible use for asthma has not yet been studied. First we investigated the anti-inflammatory properties of florfenicol using mice asthma model. BALB/c mice were immunized and challenged by ovalbumin. Treatment with florfenicol caused a marked reduction in inflammatory cells and three Th2 type cytokines in the bronchoalveolar lavage fluids of mice. The levels of ovalbumin-specific IgE and airway hyperresponsiveness were significantly altered after treatment with florfenicol. Histological studies using H&E and AB-PAS staining demonstrate that florfenicol substantially inhibited ovalbumin-induced inflammatory cells infiltration in lung tissue and goblet cell hyperplasia in the airway. These results were similar to those obtained with dexamethasone treatment. We then investigated which signal transduction mechanisms could be implicated in florfenicol activity. Our results suggested that the protective effect of florfenicol was mediated by the inhibition of the p38 MAPK-mediated phosphorylation of GATA 3.

Tpl2 (cot/MAP3K8) is an upstream kinase of MEK in the ERK pathway. It plays an important role in Tumor Necrosis Factor-alpha (TNF-alpha) production and signaling. We have discovered that 8-halo-4-(3-chloro-4-fluoro-phenylamino)-6-[(1H-[1,2,3]triazol-4-ylmethyl)-amino]-quinoline-3-carbonitriles (4) are potent inhibitors of this enzyme. In order to improve the inhibition of TNF-alpha production in LPS-stimulated human blood, a series of analogs with a variety of substitutions around the triazole moiety were studied. We found that a cyclic amine group appended to the triazole ring could considerably enhance potency, aqueous solubility, and cell membrane permeability. Optimization of these cyclic amine groups led to the identification of 8-chloro-4-(3-chloro-4-fluorophenylamino)-6-((1-(1-ethylpiperidin-4-yl)-1H-1,2,3-triazol-4-yl)methylamino)quinoline-3-carbonitrile (34). In a LPS-stimulated rat inflammation model, compound 34 showed good efficacy in inhibiting TNF-alpha production.

Ceftiofur is a new broad-spectrum, third-generation cephalosporin antibiotic for veterinary use. Immunopharmacological studies can provide new information on the immunomodulatory activities of some drugs, including their effect on cytokine productions. For this reason, we investigated the effect of ceftiofur on cytokine productions in vitro. We found that ceftiofur can downregulate tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6), but did not affect interleukin-10 (IL-10) production. We further investigated signal transduction mechanisms to determine how ceftiofur affects. RAW 264.7 cells were pretreated with 1, 5, or 10mg/L of ceftiofur 1h prior to treatment with 1mg/L of LPS. Thirty minutes later, cells were harvested and mitogen activated protein kinases (MAPKs) activation was measured by Western blot. Alternatively, cells were fixed and nuclear factor-κB (NF-κB) activation was measured using immunocytochemical analysis. Signal transduction studies showed that ceftiofur significantly inhibited extracellular signal-regulated kinase (ERK), p38, and c-jun NH2-terminal kinase (JNK) phosphorylation protein expression. Ceftiofur also inhibited p65-NF-κB translocation into the nucleus. Therefore, ceftiofur may inhibit LPS-induced production of inflammatory cytokines by blocking NF-κB and MAPKs signaling in RAW264.7 cells.

To investigate the effects of rifampicin in a mouse model of endotoxin shock, mice were pretreated with rifampicin for at different times before and after challenging with a lethal dose of lipopolysaccharide (LPS). We found that rifampicin had a significant preventive effect and reduced the mortality of mice at early stages. To further understand the mechanism, plasma cytokine levels were examined. Mice treated with LPS alone showed markedly increased plasma levels of TNF, IL-1β, IL-6, and IL-10, while mice pretreated with rifampicin showed significantly lower plasma levels of these cytokines compared to the LPS alone. Our results suggest that rifampicin has a beneficial effect on septic shock caused by LPS through modulation of cytokines. This suggests that rifampicin could be a candidate for treatment of septicemia caused by gram-negative bacterial infection.

To investigate whether ivermectin, a semi-synthetic derivative of a family of macrocyclic lactones could inhibit lipopolysaccharide (LPS)-induced inflammation in vivo and in vitro.

Cytosolic phospholipase A2α (cPLA2α) is the rate-limiting enzyme for release of arachidonic acid, which is converted primarily to prostaglandins via the cyclooxygenase (COX) 1/2 pathways, and leukotrienes via the 5-lipoxygenase (LO) pathway. We utilized inhibitors of cPLA2α, COX-1/2 and 5-LO to determine the potential roles of these enzymes in development of experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS). Blocking cPLA2α prevented EAE development and greatly reduced antigen-induced production of Th1-type cytokines and IL-17. Blocking COX-1/2 delayed onset and reduced severity of EAE, and reduced production of Th1-type cytokines, but not IL-17. Blocking 5-LO delayed onset and reduced cumulative severity of EAE, but did not reduce production of Th1-type cytokines or IL-17. Finally, blockade of cPLA2α from the onset of clinical EAE reduced duration of EAE relapses. Therefore, cPLA2α represents a potential therapeutic target for treatment of MS.

Some antibacterials have been reported to regulate the host immune and inflammatory responses both in vitro and in vivo. Florfenicol is an antibiotics used in treatment of infection. We investigated the effects of florfenicol on cytokine production by lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages in vitro, and the results showed that florfenicol reduced tumor necrosis factor (TNF) and interleukin-6 (IL-6) production but had little effect on interleukin-1β (IL-1β) and interleukin IL-10 (IL-10) secretion. This inspired us to further study the effects of florfenicol in vivo. Florfenicol significantly attenuated TNF and IL-6 production in serum from mice challenged with LPS, and in consistent with the results in vitro. In murine model of endotoxemia, mice were prophylactically or therapeutically treated with florfenicol prior to or after LPS challenge. The results showed that florfenicol significantly increased mouse survival. Further studies revealed that florfenicol prevented the LPS-induced nuclear factor-κB (NF-κB) translocation from cytoplasm into nuclear in RAW 264.7 macrophages. These observations indicate that florfenicol modulates early cytokine responses by blocking NF-κB pathway, and thus, increases mouse survival. This effect of the drug may be of potential usefulness in treatment of bacterial shock.

Interactions between PD-1 and its two differentially expressed ligands, PD-L1 and PD-L2, attenuate T cell activation and effector function. To determine the role of these molecules in autoimmune disease of the CNS, PD-1-/-, PD-L1-/- and PD-L2-/- mice were generated and immunized to induce experimental autoimmune encephalomyelitis (EAE). PD-1-/- and PD-L1-/- mice developed more severe EAE than wild type and PD-L2-/- mice. Consistent with this, PD-1-/- and PD-L1-/- cells produced elevated levels of the pro-inflammatory cytokines IFN-gamma, TNF, IL-6 and IL-17. These results demonstrate that interactions between PD-1/PD-L1, but not PD-1/PDL-2, are crucial in attenuating T cell responses in EAE.

Background: T(H)2-mediated allergic asthma is characterized by eosinophilia, mucus overproduction, and airway hyperresponsiveness (AHR). Although it is clear that T(H)2 cells and their cytokines; play an important role in AHR, the roles of T(H)1 cells and neutrophils in AHR are controversial.Objective: We sought to determine the roles of T(H)1 cells and neutrophils in AHR.Methods: Ovalbumin-specific CD4(+) T cells were purified from DO11.10 mice, differentiated into TO cells, and injected into naive BALB/c, lL-4RalphaKO, or IL-8RKO mice. After ovalbumin antigen challenge, cytokine mRNA levels in lung samples, as well as inflammatory cell types and numbers in bronchoalveolar lavage fluid (BALF), were determined. AHR was assessed by measuring resistance in tracheostomized mice and enhanced pause in freely moving mice.Results: T(H)1 cells induced AHR as robust as T(H)2 cells. They also induced lung inflammation dominated by neutrophils. Neither AHR nor inflammation were reduced when T(H)1 cells were transferred into IL-4RalphaKO mice. When IL-8RKO mice were used as recipients of T(H)1 cells, neutrophilia was greatly reduced, but the AHR was as strong as that seen in wild-type mice. On the other hand, dexamethasone treatment had no effect on neutrophilia but has significantly reduced AHR. Reduction in AHR was accompanied by a reduction in the numbers of lymphocytes and macrophages in BALF.Conclusions: T(H)1 cells can induce strong AHR independent of IL-4 and IL-13. The AHR is associated with the presence of lymphocytes and macrophages, but not neutrophils, in BALF. Our results point to a pathway whereby T(H)1 cells mediate AHR independent of neutrophilic inflammation.

Experimental autoimmune encephalomyelitis (EAE), a Th1-mediated inflammatory disease of the central nervous system (CNS), is a model of human multiple sclerosis. Cytosolic phospholipase A2α (cPLA2α), which initiates production of prostaglandins, leukotrienes, and platelet-activating factor, is present in EAE lesions. Using myelin oligodendrocyte glycoprotein (MOG) immunization, as well as an adoptive transfer model, we showed that cPLA2α−/− mice are resistant to EAE. Histologic examination of the CNS from MOG-immunized mice revealed extensive inflammatory lesions in the cPLA2α+/− mice, whereas the lesions in cPLA2α−/− mice were reduced greatly or completely absent. MOG-specific T cells generated from WT mice induced less severe EAE in cPLA2α−/− mice compared with cPLA2α+/− mice, which indicates that cPLA2α plays a role in the effector phase of EAE. Additionally, MOG-specific T cells from cPLA2α−/− mice, transferred into WT mice, induced EAE with delayed onset and lower severity compared with EAE that was induced by control cells; this indicates that cPLA2α also plays a role in the induction phase of EAE. MOG-specific T cells from cPLA2α−/− mice were deficient in production of Th1-type cytokines. Consistent with this deficiency, in vivo administration of IL-12 rendered cPLA2α−/− mice susceptible to EAE. Our data indicate that cPLA2α plays an important role in EAE development and facilitates differentiation of T cells toward the Th1 phenotype.

Valpha14 natural killer T (NKT) cells produce large amounts of both IL-4 and IFN-gamma upon stimulation with a ligand, alpha-galactosylceramide (alpha-GalCer), and play a crucial role in various immune responses, including allergic diseases. Interestingly, Valpha14 NKT cells are not essential for the induction of IgE responses but rather induce suppression of specific IgE production upon activation. The suppression in the IgE production is not detected either in Valpha14 NKT cell-deficient mice or in IFN-gamma-deficient mice. Thus, activated Valpha14 NKT cells are likely to exert a potent suppressive activity on Th2 cell differentiation and subsequent IgE production by producing a large amount of IFN-gamma. In marked contrast, little regulatory effect of IL-4 produced by Valpha14 NKT cells on Th2 cell differentiation is suggested.

Murine Vα14 natural killer T (NKT) cells are thought to play a crucial role in various immune responses, including infectious, allergic, and autoimmune diseases. Because Vα14 NKT cells produce large amounts of both interleukin (IL)-4 and interferon (IFN)-γ upon in vivo stimulation with a specific ligand, α-galactosylceramide (α-GalCer), or after treatment with anti-CD3 antibody, a regulatory role on helper T (Th) cell differentiation has been proposed for these cells. However, the identity of the cytokine produced by Vα14 NKT cells that play a dominant role on the Th cell differentiation still remains controversial. Here, we demonstrate by using Vα14 NKT-deficient mice that Vα14 NKT cells are dispensable for the induction of antigen-specific immunoglobulin (Ig)E responses induced by ovalbumin immunization or Nippostrongylus brasiliensis infection. However, upon in vivo activation with α-GalCer, Vα14 NKT cells are found to suppress antigen-specific IgE production. The suppression appeared to be IgE specific, and was not detected in either Vα14 NKT– or IFN-γ–deficient mice. Consistent with these results, we also found that ligand-activated Vα14 NKT cells inhibited Th2 cell differentiation in an in vitro induction culture system. Thus, it is likely that activated Vα14 NKT cells exert a potent inhibitory effect on Th2 cell differentiation and subsequent IgE production by producing a large amount of IFN-γ. In marked contrast, our studies have revealed that IL-4 produced by Vα14 NKT cells has only a minor effect on Th2 cell differentiation.

A novel subset of peripheral T cells, peripheral NK T cells, is found to be a major population comprising 5% of splenic T and 40% of bone marrow T cells. The majority of peripheral NK T cells are characterized by the expression of an invariant TCR-alpha encoded by V alpha 14/J alpha 281 with a one nucleotide N region. Moreover, a specific reduction of V alpha 14+ NK T cells has been demonstrated to be tightly associated with various autoimmune diseases, indicating their decisive role in autoimmune disease development. In this study, we investigated the phenotypes of peripheral V alpha 14+ NK T cells and their TCR-beta repertoire. Peripheral V alpha 14+ NK T cells, comprise two populations, i.e., small and large sized cells, at an equal frequency, belonged to the CD4- CD8- fraction, and are heat stable antigen(bright), macrophage-1bright, B220bright, CD45RBdim, and Mel-14dim, but CD5-, distinct from thymic NK T cells. TCR-beta analysis clearly showed that peripheral V alpha 14+ NK T cells utilized two to three dominant invariant TCR-beta, such as V beta 8.2 D beta J beta 2.5/V beta 7 D beta J beta 2.1 in the spleen and liver, V beta 8.2 D beta J beta 2.5/V beta 8.3 D beta J beta 2.2/V beta 7 D beta J beta 2.6 in the bone marrow, and V beta 7 D beta J beta 2.1/V beta 3 D beta J beta 1.2 in intestinal intraepithelial lymphocytes. Judging from the unusual surface phenotypes, such as heat stable antigen, macrophage-1, B220, CD45RBdim, and Mel-14dim, which are known to be T cell activation markers, peripheral V alpha 14+ NK T cells may always be activated under physiologic conditions, resulting in the oligoclonal expansion of V alpha 14+ NK T cells with different invariant TCR-beta in different peripheral organs. The unique features of V alpha 14+ NK T cells are discussed.

Natural killer T (NKT) lymphocytes express an invariant T cell antigen receptor (TCR) encoded by the V α 14 and J α 281 gene segments. A glycosylceramide-containing α-anomeric sugar with a longer fatty acyl chain (C 26 ) and sphingosine base (C 18 ) was identified as a ligand for this TCR. Glycosylceramide-mediated proliferative responses of V α 14 NKT cells were abrogated by treatment with chloroquine–concanamycin A or by monoclonal antibodies against CD1d/V β 8, CD40/CD40L, or B7/CTLA-4/CD28, but not by interference with the function of a transporter-associated protein. Thus, this lymphocyte shares distinct recognition systems with either T or NK cells.

A lymphocyte subpopulation, the V α 14 natural killer T (NKT) cells, expresses both NK1.1 and a single invariant T cell receptor encoded by the V α 14 and J α 281 gene segments. Mice with a deletion of the J α 281 gene segment were found to exclusively lack this subpopulation. The V α 14 NKT cell–deficient mice could no longer mediate the interleukin-12 (IL-12)–induced rejection of tumors. Although the antitumor effect of IL-12 was thought to be mediated through natural killer cells and T cells, V α 14 NKT cells were found to be an essential target of IL-12, and they mediated their cytotoxicity by an NK-like effector mechanism after activation with IL-12.

A novel lymphoid lineage, NK T cells, was recently found. The NK T cells are the major population in the periphery comprising 5% of splenic T cells and 40% of bone marrow T cells. They express a unique TCR composed of invariant V alpha 14J alpha 281 and V beta 8.2 together with NK receptor (NKRPI). Surprisingly, the invariant V alpha 14+ TCR is exclusively expressed on NK T cells but not on conventional T cells. As the selective decrease in V alpha 14+ NK T cell population in the periphery is tightly correlated with autoimmune disease development, V alpha 14+ NK T cells control development of autoimmune diseases. We also found that V alpha 14 TCR gene rearrangement and transcripts were detected at an early embryogenesis (d9.5) before the thymus formation. Therefore NK T cells are in the distinct category from conventional T cells. The target of NK T cells is found to be CD1 (class 1b, monomorphic class I MHC-like molecule) present on bone marrow-derived cells and is killed by Fas-FasL interaction or perforin-mediated mechanisms. These results indicate that NK T cells consist of an immunoregulatory system different from defense system in terms of homogeneous repertoire, extrathymic development in early stage of gestation, and their regulatory functional role.

NK1.1+ T [natural killer (NK) T] cells express an invariant T cell antigen receptor alpha chain (TCR alpha) encoded by V alpha 14 and J alpha 281 segments in association with a limited number of V betas, predominantly V beta 8.2. Expression of the invariant V alpha 14/J alpha 281, but not V alpha 1, TCR in transgenic mice lacking endogenous TCR alpha expression blocks the development of conventional T alpha beta cells and leads to the preferential development of V alpha 14 NK T cells, suggesting a prerequisite role of invariant V alpha 14 TCR in NK T cell development. In V beta 8.2 but not B beta 3 transgenic mice, two NK T cells with different CD3 epsilon expressions, CD3 epsilon(dim) and CD3 epsilon(high), can be identified. CD3 epsilon(high) NK T cells express surface V alpha 14/V beta 8 TCR, indicating a mature cell type, whereas CD3 epsilon(dim) NK T cells express V beta 8 without V alpha 14 TCR and no significant CD3 epsilon expression (CD3 epsilon(dim)) on the cell surface. However, the latter are positive for recombination activating gene (RAG-1 and RAG-2) mRNA, which are only expressed in the precursor or immature T cell lineage, and also possess CD3 epsilon mRNA in their cytoplasm, suggesting that CD3 epsilon(dim) NK T cells are the precursor of V alpha 14 NK T cells.

A novel peripheral T cell subset characterized by the expression of a NK marker and invariant TCR encoded by V alpha 14 J alpha 281 gene segments with a 1-base N-region was investigated in relation to autoimmune disease development. First, we observed that invariant V alpha 14+ NK T cells are specifically reduced with aging in C57BL/6 lpr/lpr or MRL lpr/lpr mice, whereas no change was observed in age-matched control C57BL/6 or MRL +/+ mice as determined by FACS analysis and RNase protection assay. This reduction precedes the disease development and could also be detected in other autoimmune disease-prone mice, such as C3H gld/gld and (NZB x NZW)F1 mice. These results suggest that the specific decrease in invariant V alpha 14+ NK T cells correlates strongly with the development of autoimmunity. Second, injection of MRL lpr/lpr mice with anti-V alpha 14 mAb resulted in the early onset and exacerbation of lymphosplenomegaly due to the accumulation of abnormal CD3+ B220+ CD4-CD8- T cells as well as an increase in the titers of anti-dsDNA autoantibodies. These results indicate that V alpha 14+ NK T cells regulate autoimmune responses and play a crucial role in controlling the development of autoimmune diseases.