Cytosolic phospholipase A2α blockade abrogates disease during the tissue-damage effector phase of experimental autoimmune encephalomyelitis by its action on APCs.

Cytosolic phospholipase A(2)α (cPLA(2)α) is the rate-limiting enzyme for release of arachidonic acid, which is converted primarily to PGs via the cyclooxygenase 1 and 2 pathways and to leukotrienes via the 5-lipoxygenase pathway. We used adoptive transfer and relapsing-remitting forms of experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis, in two different strains of mice (SJL or C57BL/6) to demonstrate that blockade of cPLA(2)α with a highly specific small-molecule inhibitor during the tissue-damage effector phase abrogates the clinical manifestation of disease. Using the adoptive transfer model in SJL mice, we demonstrated that the blockade of cPLA(2)α during the effector phase of disease was more efficacious in ameliorating the disease pathogenesis than the blockade of each of the downstream enzymes, cyclooxygenase-1/2 and 5-lipooxygenase. Similarly, blockade of cPLA(2)α was highly efficacious in ameliorating disease pathogenesis during the effector phase of EAE in the adoptive transfer model of EAE in C57BL/6 mice. Investigation of the mechanism of action indicates that cPLA(2)α inhibitors act on APCs to diminish their ability to induce Ag-specific effector T cell proliferation and proinflammatory cytokine production. Furthermore, cPLA(2)α inhibitors may prevent activation of CNS-resident microglia and may increase oligodendrocyte survival. Finally, in a relapsing-remitting model of EAE in SJL mice, therapeutic administration of a cPLA(2)α inhibitor, starting from the peak of disease or during remission, completely protected the mice from subsequent relapses.

Evolutionary and biochemical differences between human and monkey acidic mammalian chitinases.

Acidic mammalian chitinase (AMCase), an enzyme implicated in the pathology of asthma, is capable of chitin cleavage at a low pH optimum. The corresponding gene (CHIA) can be found in genome databases of a variety of mammals, but the enzyme properties of only the human and mouse proteins were extensively studied. We wanted to compare enzymes of closely related species, such as humans and macaques. In our attempt to study macaque AMCase, we searched for CHIA-like genes in human and macaque genomes. We found that both genomes contain several additional CHIA-like sequences. In humans, CHIA-L1 (hCHIA-L1) is an apparent pseudogene and has the highest homology to CHIA. To determine which of the two genes is functional in monkeys, we assessed their tissue expression levels. In our experiments, CHIA-L1 expression was not detected in human stomach tissue, while CHIA was expressed at high levels. However, in the cynomolgus macaque stomach tissue, the expression pattern of these two genes was reversed: CHIA-L1 was expressed at high levels and CHIA was undetectable. We hypothesized that in macaques CHIA-L1 (mCHIA-L1), and not CHIA, is a gene encoding an acidic chitinase, and cloned it, using the sequence of human CHIA-L1 as a guide for the primer design. We named the new enzyme MACase (Macaca Acidic Chitinase) to emphasize its differences from AMCase. MACase shares a similar tissue expression pattern and pH optimum with human AMCase, but is 50 times more active in our enzymatic activity assay. DNA sequence of the mCHIA-L1 has higher percentage identity to the human pseudogene hCHIA-L1 (91.7%) than to hCHIA (84%). Our results suggest alternate evolutionary paths for human and monkey acidic chitinases.

MMP dependence of fibroblast contraction and collagen production induced by human mast cell activation in a three-dimensional collagen lattice.

Mast cell-fibroblast interactions may contribute to fibrosis in asthma and other disease states. Fibroblast contraction is known to be stimulated by coculture with the human mast cell line, HMC-1, or by mast cell-derived agents. Matrix metalloproteinases (MMPs) can also mediate contraction, but the MMP-dependence of mast cell-induced fibroblast contractility is not established, and the consequences of mast cell activation within the coculture system have not been fully explored. We demonstrate that activation of primary human mast cells (pHMC) with IgE receptor cross-linking, or activation of HMC-1 with C5a, enhanced contractility of human lung fibroblasts in a three-dimensional collagen lattice system. This enhanced contractility was inhibited by the pan-MMP antagonist, batimastat, and was transferrable in the conditioned medium of activated mast cells. Exogenously added MMPs promoted gel contraction by mediating the proteolytic activation of latent transforming growth factor-beta (TGF-beta). Consistent with this, fibroblast contraction induced by mast cell activation was enhanced by addition of excess latent TGF-beta to the cultures. Batimastat inhibited this response, suggesting that MMPs capable of activating latent TGF-beta were released following mast cell activation in coculture with fibroblasts. Collagen production was also stimulated by activated mast cells in an MMP-dependent manner. MMP-2 and MMP-3 content of the gels increased in the presence of activated mast cells, and inhibition of these enzymes blocked the contractile response. These findings demonstrate the MMP dependence of mast cell-induced fibroblast contraction and collagen production.

Binding characterization of the interleukin-13 signaling complex and development of a ternary time-resolved fluorescence resonance energy transfer assay.

Interleukin-13 (IL-13) is a critical mediator of pulmonary pathology associated with asthma. Drugs that block the biological function of IL-13 may be an effective treatment for asthma. IL-13 signals by forming a ternary complex with IL-13Ralpha1 and IL-4R. Genetic variants of IL-13 and of its receptor components have been linked to asthma. One in particular, IL-13R110Q, is associated with increased IgE levels and asthma. We characterized the interactions of the binary complexes composed of IL-13 or IL-13R110Q with IL-13Ralpha1 and the ternary complexes composed of IL-13 or IL-13R110Q and IL-13Ralpha1 with IL-4R using surface plasmon resonance and time-resolved fluorescence resonance energy transfer (TR-FRET). By both biophysical methods, we found no differences between IL-13 and IL-13R110Q binding in either the binary or the ternary complex. IL-4R bound to the IL-13/IL-13Ralpha1 complex with slow on and off rates, resulting in a relatively weak affinity of about 100nM. We developed a TR-FRET assay targeting the interaction between the IL-4R and the binary complex. Two antibodies with known binding epitopes to IL-13 that block binding to either IL-13Ralpha1 or IL-4R inhibited the TR-FRET signal formed by the ternary complex. This assay will be useful to identify and characterize inhibitory molecules of IL-13 function.

Modulation of the IL-12/IFN-gamma axis by IFN-alpha therapy for hepatitis C.

Although IFN-alpha forms the foundation of therapy for chronic hepatitis C, only a minority of patients has a sustained response to IFN-alpha alone. The antiviral activities of IFN-alpha formed the rationale for its use in viral hepatitis. However, IFN-alpha and the other Type I IFNs are also pleiotropic immune regulators. Type I IFNs can promote IFN-gamma production by activating STAT4 but can also inhibit production of IL-12, a potent activator of STAT4 and IFN-gamma production. The efficacy of IFN-alpha in the treatment of hepatitis C may therefore depend in part on the balance of IFN-gamma-inducing and IL-12-suppressing effects. We characterized the effects of pegylated IFN-alpha therapy for hepatitis C on the capacity of patients' PBMC to produce IL-12 and IFN-gamma ex vivo. Cells from patients with a sustained virological response to therapy had significantly greater levels of IFN-alpha-driven IFN-gamma production prior to treatment than those from nonresponding patients. No differences in pretreatment IL-12 productive capacity were seen between patient groups. However, therapy with IFN-alpha led to suppression of inducible IL-12 production throughout the course of therapy in both groups of patients.

A directly labeled TR-FRET assay for monitoring phosphoinositide-3-kinase activity.

Phosphoinositide 3-kinases (PI3Ks) comprise a family of kinases that transfer the terminal phosphate of adenosine triphosphate to phosphoinositides at the 3-hydroxyl of the inositol ring to form phosphoinositide (3,4,5) triphosphate (PIP3). The PI3Ks have been shown to play key roles in cell growth, motility, morphology, and survival and thus are of interest as targets in anti-inflammatory and anti-oncogenic drug development. To facilitate identification of novel and selective inhibitors of PI3Ks, we have developed a TR-FRET assay that uses directly labeled reagents. The assay makes use of the high affinity binding of phosphoinositides to a Pleckstrin homology (PH) domain in the general receptor for phosphoinositides 1 (Grp1) protein. It monitors PIP3 produced from the enzymatic reaction by measuring its competition with Bodipy-FL-labeled PIP3 for binding to Terbium chelate-labeled Grp1. By using directly labeled reagents, this assay configuration offers higher sensitivity and faster binding/dissociation kinetics than existing non-radioactive assays, which are critical for competitive assay formats. The assay is homogenous, robust (Z' = 0.88), and simple and, thus, compatible with high throughput screening (HTS) processes.

Combined antiangiogenic and immune therapy of prostate cancer.

Experimental studies of antiangiogenic or immune therapy of cancer have generated a great deal of optimism. However, the results of clinical testing of these therapies are below expectations. We hypothesized that the antitumor efficacy can be increased when immune destruction of tumor cell is combined with destruction of tumor vasculature by antiangiogenic drugs. In the present study the therapeutic efficacy of combined antiangiogenic and immune therapy has been tested against the highly aggressive, MHC class I negative murine RM1 prostate tumor. SU6668 was used as the antiangiogenic drug and recombinant murine B7.2-IgG fusion protein was used to stimulate T cell-mediated immune destruction of tumor cells. SU6668 is an inhibitor of the tyrosine kinase activity of three angiogenic receptors VEGFR2 (Flk-1/KDR), PDGFRbeta and FGFR1 that play a crucial role in tumor-induced vascularization. Our studies show that B7.2-IgG treatment of mice with established RM1 prostate tumors resulted in a significant inhibition of tumor growth. Both CD4+ and CD8+ T cells were responsible for this effect. SU6668 therapy substantially inhibited tumor vascularization and tumor growth. When tumor-bearing mice were treated with SU6668 in combination with B7.2-IgG, the antitumor effects were substantially higher than in mice treated separately with SU6668 or B7.2-IgG. Prolonged treatment of mice with SU6668 did not inhibit the immunoreactivity of T lymphocytes. On the contrary, T cells from mice treated with a combination of SU6668 and B7.2-IgG showed higher proliferative responses and cytokine production following anti-CD3 stimulation than T cells of mice treated separately with these modalities. These results indicate that antiangiogenic and immune therapies using SU6668 and B7.2-IgG are compatible and manifest complementary antitumor effects. Combined antiangiogenic and immune therapy might represent a new strategy for cancer treatment.

B7.2-Ig fusion proteins enhance IL-4-dependent differentiation of tumor-sensitized CD8+ cytotoxic T lymphocyte precursors.

The B7/CD28 co-stimulatory pathway plays a critical role in T cell activation and differentiation. Our previous study demonstrated that administration of B7.2-Ig fusion proteins to tumor-bearing mice elicits IL-4-dependent, CD8+ T cell-mediated tumor regression. Here, we investigated whether B7.2-Ig stimulation of tumor-sensitized CD8+ CTL precursors during in vitro antigen re-sensitization actually results in their differentiation into mature CTLs and if so, whether such a process depends on IL-4 signals. Splenocytes from tumor-sensitized (tumor-bearing or tumor-immunized) mice exhibited low levels of anti-tumor CTL responses upon culturing alone, but induced strikingly enhanced CTL responses when stimulated in vitro with B7.2-Ig fusion proteins. Because CTLs were not generated from normal splenocytes even by B7.2-Ig stimulation, the expression of the B7.2-Ig effect required the in vivo tumor sensitization of CD8+ CTL precursors. Administration of anti-CD4 or anti-CD40 ligand (CD40L) to mice before tumor sensitization resulted in almost complete inhibition of CTL responses generated in the subsequent culture containing B7.2-Ig. In contrast, anti-IL-4 did not influence in vivo tumor sensitization required for CTL induction. However, B7.2-Ig stimulation of tumor-sensitized splenocytes enhanced IL-4 production and neutralization of this IL-4 with anti-IL-4 potently down-regulated CTL responses. These results indicate that B7.2-Ig enhances IL-4-dependent differentiation of anti-tumor CD8+ CTL precursors that can be sensitized in vivo depending on collaboration with CD4+ T cells involving CD40L function.

The capacity of the natural ligands for CD28 to drive IL-4 expression in naïve and antigen-primed CD4+ and CD8+ T cells.

The B7/CD28 costimulatory pathway plays a critical role in T cell activation including Th1/Th2 differentiation. However, little is known about whether CD28 costimulation favors polarization of either Th1 and Th2 or both. Here, we show a critical role of the natural ligands for CD28 molecules (B7.2-Ig or B7.1-Ig fusion proteins), particularly in the induction of type 2 T cell polarization. Upon TCR-triggering with suboptimal doses of anti-CD3, costimulation of naïve CD4+ T cells with anti-CD28 mAb or B7-Ig fusion proteins led to comparable levels of IFN-gamma production. Naïve T cells could produce IL-4 when CD28 costimulation was done with B7-Ig, but not with anti-CD28. IL-4-selective upregulation was also observed when T cells from anti-OVA TCR transgenic mice were stimulated with OVA in the presence of B7-Ig. Correlating with IL-4 expression, GATA-3 expression was induced much more potently by costimulation with B7-Ig than with anti-CD28 mAb, while T-bet induction by these two costimulatory reagents was comparable. This B7 effect was also applied for naïve and antigen-primed CD8+ T cells: IL-4-expressing CD8+ T cells were generated when naïve and alloantigen-primed T cells were stimulated with anti-CD3 and recall antigens, respectively, in the presence of B7-Ig costimulation. Importantly, such CD8+ T cell differentiation required the coexistence of CD4+ T cells during the initial TCR stimulation. These observations indicate that both type 2 CD4 and CD8 T cell polarizations are efficiently induced via costimulation of CD28 with its natural ligands, although the differentiation of CD8+ T cells is dependent on CD4+ cells.

Induction of tumor regression by administration of B7-Ig fusion proteins: mediation by type 2 CD8+ T cells and dependence on IL-4 production.

CD28 signals contribute to either type 1 or type 2 T cell differentiation. Here, we show that administration of B7.2-Ig fusion proteins to tumor-bearing mice induces tumor regression by promoting the differentiation of antitumor type 2 CD8(+) effector T cells along with IL-4 production. B7.2-Ig-mediated regression was not induced in IL-4(-/-) and STAT6(-/-) mice. However, it was elicited in IFN-gamma(-/-) and STAT4(-/-) mice. By contrast, IL-12-induced tumor regression occurred in IL-4(-/-) and STAT6(-/-) mice, but not in IFN-gamma(-/-) and STAT4(-/-) mice. Moreover, B7.2-Ig treatment was effective in a tumor model not responsive to IL-12. B7.2-Ig administration elicited elevated levels of IL-4 production. Tumor regression was predominantly mediated by CD8(+) T cells, although the induction of these effector cells required CD4(+) T cells. Tumor regression induced by CD8(+) T cells alone was inhibited by neutralizing the IL-4 produced during B7.2-Ig treatment. Thus, these results indicate that stimulation in vivo of CD28 with B7.2-Ig in tumor-bearing mice results in enhanced induction of antitumor type 2 CD8(+) T cells (Tc2) leading to Tc2-mediated tumor regression.

Intranasal IL-12 produces discreet pulmonary and systemic effects on allergic inflammation and airway reactivity.

IL-12 modulates T cell responses between helper T cells Th2 and Th1; however, the therapeutic potential of IL-12 for allergic diseases either directly or as an adjuvant in allergen therapy has been controversial. The role of intranasal IL-12 as an adjuvant in modulating the grass pollen allergen (GAL) therapy-induced systemic immune response and lung-specific inflammation and airway reactivity was examined in this study using a mouse model of established allergic asthma. The effects of intranasal or nebulized IL-12 with or without intranasal anti-IFN-gamma antibody were examined in groups of control and allergen-sensitized or -challenged mice. T cell cytokine patterns, antibody response profiles, pulmonary inflammation and airway reactivity were examined. Intranasal IL-12 was found to be more effective in the Th2-Th1 shifting of immune response and anti-inflammatory activity in the lung compared to nebulized IL-12 at the given doses. Intranasal IL-12 significantly decreased production of IFN-gamma, eotaxin and LTC4/D4/E4 in the lung and decreased eosinophil infiltration, resulting in attenuated airway hyper-responsiveness in GAL-sensitized (GS) mice. In contrast, intranasal IL-12 significantly increased IFN-gamma production in the thoracic lymph node cultures and decreased the IL-5/IFN-gamma ratio, suggesting a Th2-Th1 shift. Also, intranasal IL-12 increased GAL-specific IgG2a antibody response, while the IgE response remained unaffected. The systemic effects of IL-12 were IFN-gamma dependent. IL-12 induces differential expression of its own receptor beta1 and beta2 subunits in the lung tissues to augment IL-12 responsiveness. Together, these results demonstrate that intranasal IL-12 is effective in shifting the systemic immune response in the direction of Th1 in IFN-gamma-dependent manner, while decreasing pulmonary inflammation and airway reactivity independent of IFN-gamma. Thus, intranasal delivery of IL-12 may provide an approach for the treatment of asthma and may be useful as an adjuvant in local nasal immunotherapy (IT) and in asthma.

Capacity of murine IL-12 to inhibit the development of primary melanoma tumors and to prevent lung metastases in the melanoma-challenged mice.

Interleukin-12 (IL-12) has the capacity to activate cytotoxic lymphocytes, stimulate natural killer cells, induce the production of INF-gamma, and be synergistic with IL-2. We have evaluated this cytokine in an experimental model for metastatic melanoma that approximates the major clinical stages of metastatic dissemination. To develop primary melanoma tumors, mice were injected subcutaneously with 5 x 10(5) cells in a volume of 25 microliters into the middle of the tail (11). In a month, mice were started to be treated for 4 weeks with recombinant murine IL-12 (R mIL-12) at the following doses: 0, 0.5, 2.5, 5.0, 15.0, and 50 micrograms/kg. Diameters of the primary melanoma tumors were measured at weekly intervals. At the end of 13 weeks (9 weeks from the start of treatment with R mIL-12), all surviving mice were sacrificed. Pathological examination of lung metastases (macroscopy) was done with all dead or sacrificed mice. Treatment of mice bearing melanoma at a dose of 300 ng/mouse (15 micrograms/kg) inhibited development of primary tumors in 40% of mice. The primary tumor diameters were significantly lower in the group treated with 300 ng/mouse (15 micrograms/kg) in comparison to controls. At the end of the observation period, groups treated with 0.5, 2.5, 15.0, and 50 micrograms/kg had mean primary tumor diameters smaller than the control group. Evaluation of IL-12 therapy on primary tumor growth, mean diameters of primary tumors, survival rate, and development of lung metastases showed that the best results were observed using 300 ng/mouse (15 micrograms/kg) R mIL-12.

Combined therapy of local and metastatic 4T1 breast tumor in mice using SU6668, an inhibitor of angiogenic receptor tyrosine kinases, and the immunostimulator B7.2-IgG fusion protein.

The therapeutic efficacy of combined antiangiogenic and immune therapy was tested against weakly immunogenic and highly metastatic 4T1 breast tumor using SU6668, an angiogenesis inhibitor and recombinant murine (rm) B7.2-IgG fusion protein, an immunostimulator. SU6668 inhibits the tyrosine kinase activity of three angiogenic receptors VEGFR2 (Flk-1/KDR), PDGFRbeta, and FGFR1, which play a crucial role in tumor-induced vascularization. rmB7.2-IgG is a fusion protein of the extracellular domain of B7.2, and the hinge and constant domains of murine IgG2a. Intermolecule disulfide linkages are maintained so that it forms a dimer. Our studies showed that three weekly immunizations of BALB/c mice bearing 0.5-0.8 cm 4T1 breast tumors with rmB7.2-IgG and irradiated 4T1 tumor cells (B7.2-IgG/TC) resulted in a significant inhibition of tumor growth and formation of pulmonary metastases. T-cell depletion experiments revealed that both CD4(+) and CD8(+) T lymphocytes are required for stimulation of the antitumor and antimetastatic immune response by B7.2-IgG/TC. Treatment of mice with SU6668 substantially inhibited tumor vascularization but did not inhibit tumor infiltration by T lymphocytes or the T-cell response to rmB7.2-IgG therapy. On the contrary, tumors in mice immunized with B7.2-IgG/TC and treated with SU6668 had higher numbers of tumor infiltrating T cells than tumors of other groups. SU6668 treatments initiated either on day 3 or day 10 after inoculation of 4T1 tumor cells resulted in a significant inhibition of tumor growth. Similarly, three weekly immunizations with B7.2-IgG/TC starting either on day 7 or 12 inhibited growth of 4T1 tumors. However, the most potent antitumor effects were observed in mice treated with a combination of SU6668 and B7.2-IgG/TC. Analysis of pulmonary metastases revealed that combined therapy also had a more potent antimetastatic effect than each modality alone. These results indicate that antiangiogenic and immune therapies using SU6668 and B7.2-IgG/TC are compatible, and manifest complementary antitumor and antimetastatic effects. Combined antiangiogenic and immune therapy might represent a new strategy for cancer treatment.