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Leukotriene A4 (LTA4) hydrolase catalyzes a rate-limiting final biosynthetic step of leukotriene B4 (LTB4), a potent lipid chemotactic agent and proinflammatory mediator. LTB4 has been implicated in the pathogenesis of various acute and chronic inflammatory diseases, and thus LTA4 hydrolase is regarded as an attractive therapeutic target for anti-inflammation. To facilitate identification and optimization of LTA4 hydrolase inhibitors, a specific and efficient assay to quantify LTB4 is essential. This article describes the development of a novel 384-well homogeneous time-resolved fluorescence assay for LTB4 (LTB4 HTRF assay) and its application to establish an HTRF-based LTA4 hydrolase assay for lead optimization. This LTB4 HTRF assay is based on competitive inhibition and was established by optimizing the reagent concentration, buffer composition, incubation time, and assay miniaturization. The optimized assay is sensitive, selective, and robust, with a Z' factor of 0.89 and a subnanomolar detection limit for LTB4. By coupling this LTB4 HTRF assay to the LTA4 hydrolase reaction, an HTRF-based LTA4 hydrolase assay was established and validated. Using a test set of 16 LTA4 hydrolase inhibitors, a good correlation was found between the IC50 values obtained using LTB4 HTRF with those determined using the LTB enzyme-linked immunoassay (R = 0.84). The HTRF-based LTA4 hydrolase assay was shown to be an efficient and suitable assay for determining compound potency and library screening to guide the development of potent inhibitors of LTA4 hydrolase.

Various subpopulations of T lymphocytes-i.e. Type 1, Type 2, Tr1 T cells-play a major role in the homeostasis of the immune system and in the pathogenesis of many inflammatory and auto-immune diseases. At present, in the absence of specific surface markers, these T cells can only be reliably distinguished on the basis of their cytokine production profile. The Elispot assay detects cytokine-producing cells, but in most cases can detect only one secreted cytokine, which represents a major limitation of this technique. We have developed a Fluorospot assay to detect single cells that simultaneously produce multiple cytokines. The Fluorospot assay permits the detection of regulatory T cells with an immunosuppressive activity, identified by their coexpression of IL-10 and IFNgamma. Polarized type 1 and type 2 specific tetanus toxoid T cells are also directly detected using a dual color Fluorospot. This technique will therefore be useful for detailed analysis of T lymphocytes in various disease states in which an imbalance of T cell subpopulations is suspected, but will also provide a better characterization of polarized specific immune responses.

Soluble GM-CSF receptor alpha subunit (sGMRalpha) is a soluble isoform of the GMRalpha that is believed to arise exclusively through alternative splicing of the GMRalpha gene product. The sGMRalpha mRNA is expressed in a variety of tissues, but it is not clear which cells are capable of secreting the protein. We show here that normal human monocytes, but not lymphocytes, constitutively secrete sGMRalpha. Stimulation of monocytes with GM-CSF, LPS, PMA, or A23187 rapidly up-regulates the secretion of sGMRalpha in a dose-dependent manner, demonstrating that secretion is also regulated. To determine whether sGMRalpha arose exclusively through alternative splicing of the GMRalpha gene product, or whether it could also be generated through ectodomain shedding of GMRalpha, we engineered a murine pro-B cell line (Ba/F3) to express exclusively the cDNA for cell surface GMRalpha (Ba/F3.GMRalpha). The Ba/F3.GMRalpha cell line, but not the parental Ba/F3 cell line, constitutively shed a sGMRalpha-like protein that bound specifically to GM-CSF, was equivalent in size to recombinant alternatively spliced sGMRalpha (60 kDa), and was recognized specifically by a mAb raised against the ectodomain of GMRalpha. Furthermore, a broad-spectrum metalloprotease inhibitor (BB94) reduced constitutive and PMA-, A23187-, and LPS-induced secretion of sGMRalpha by monocytes, suggesting that shedding of GMRalpha by monocytes may be mediated in part through the activity of metalloproteases. Taken together, these observations demonstrate that sGMRalpha is constitutively secreted by monocytes, that GM-CSF and inflammatory mediators up-regulate sGMRalpha secretion, and that sGMRalpha arises not only through alternative splicing but also through ectodomain shedding of cell surface GMRalpha.

Objective: To evaluate the safety and clinical efficacy of administering an anti-interleukin-10 (anti-IL-10) monoclonal antibody (mAb) to systemic lupus erythematosus (SLE) patients with active and steroid-dependent disease. In addition, we sought to assess the effects of in vivo IL-10 neutralization on biologic markers of SLE., Methods: Treatment consisted of 20 mg/day intravenous administration of an anti-IL-10 murine mAb (B-N10) for 21 consecutive days, with a followup period of 6 months. Six patients were studied., Results: Treatment was safe and well tolerated. All patients developed antibodies against B-N10. Cutaneous lesions and joint symptoms improved in all patients beginning during B-N10 administration and continuing to month 6. The SLE Disease Activity Index decreased from a mean +/- SEM of 8.83+/-0.91 on day 1 to 3.67+/-0.67 on day 21 (P = 0.001), 1.50+/-0.84 at month 2, and 1.33+/-0.80 at month 6 (P<0.001). At the end of followup, the disease was clinically inactive in 5 of the 6 patients. Prednisone administration was decreased from a mean +/- SEM of 27.9+/-5.7 mg/day on day 1 to 9.6+/-2.0 mg/day at month 6 (P<0.005). Activity of immune and endothelial cells rapidly decreased, as assessed by the early evolution of several biologic markers., Conclusion: This is the first report of IL-10 antagonist administration to humans. The study shows the involvement of IL-10 in the pathogenesis of SLE, and indicates that the use of IL-10 antagonists may be beneficial in the management of refractory SLE.

Interleukin (IL) 17 is a proinflammatory cytokine secreted mainly by activated human memory CD4 T cells that induces IL-6, IL-8, and nitric oxide. Because IL-6 and IL-8 have been implicated in the pathogenesis of cervical cancer, we investigated the action of IL-17 on human cervical tumor cell lines in vitro and in vivo. We showed that in vitro, IL-17 increases IL-6 and IL-8 secretion by cervical carcinoma cell lines at both protein and mRNA levels. No direct effect of IL-17 on in vitro proliferation of cervical tumor cell lines could be demonstrated. However, two cervical cell lines transfected with a cDNA encoding IL-17 exhibited a significant increase in tumor size as compared to the parent tumor when transplanted in nude mice. This enhanced tumor growth elicited by IL-17 was associated with increased expression of IL-6 and macrophage recruitment at the tumor site. A potential role of IL-17 in modulation of the human cervical tumor phenotype was also supported by its expression on the cervical tumor in patients with CD4 infiltration. IL-17 therefore behaves like a T-cell-specific cytokine with paradoxical tumor-promoting activity. This may partially explain previous reports concerning the deleterious effect of CD4 T cells in cancer.

We have previously shown that total T cells derived from lymph nodes (LN) involved by Hodgkin's disease (HD) secrete higher levels of colony-stimulating activity than total T cells present within benign hyperplastic (BH) LN and B-non-Hodgkin's lymphoma (B-NHL) LN, suggesting that T cells with particular properties accumulate in HD LN. To further characterize this T-cell population, we have quantified production of both granulocyte-macrophage colony-stimulating factor (GM-CSF) and macrophage colony-stimulating factor (M-CSF) production in a total of 98 T-cell clones (TCC) derived from CD25+ activated T cells present in HD LN; TCC derived from CD25+ T cells obtained from B-NHL LN(101 TCC), BH LN(95 TCC), and peripheral blood (PBL; 38 TCC) of healthy donors were used as controls. HD LN were characterized by the presence of an elevated number (44%) of TCC producing particularly high titers of both GM-CSF and M-CSF, whereas only a minority of such TCC was found in control groups (10% in B-NHL, 16% in BH, 8% in PBL). These observations support the hypothesis of a selection of T-cell families with particular properties occurring in contact with Reed-Sternberg (RS) cells. According to the biological properties of GM-CSF and M-CSF, it seems reasonable to suggest the involvement of this particular subset of T cells in the granulomatous process, the peripheral blood polynucleosis, and in the paracrine growth of RS cells.

A major problem encountered for quantification of IL2 production by stimulated T cells is its simultaneous consumption by these activated cells. In the present study, 40 T-cell clones (TCC) derived from normal peripheral blood, hyperplastic lymph nodes (LN) or lymph nodes involved by malignant lymphomas, were studied for their ability to produce IL2. When supernatants were generated in the presence of 20% fetal calf serum (FCS), no IL2 could be detected for 22 of the 40 TCC, whereas very low levels were found for the 18 other TCC (mean value 31 pg/ml; range from 10 pg/ml to 114 pg/ml); in contrast, when conditioned media were produced with reduced amounts of FCS (final concentration, 1%) as well as in the presence of an anti-CD25 monoclonal antibody (final concentration, 50 micrograms/ml), all TCC were found to release IL2, and very high quantities of this lymphokine were measured (mean value: 11,387 pg/ml; range, from 250 pg/ml to 37,000 pg/ml). Consequently, inhibition of IL2 consumption by PHA-stimulated TCC seems to be an absolute requirement for estimating the true capacity of T cells to produce this lymphokine.

We searched for the presence of IL2 receptor (CD25) on T cells as an activation marker in lymph nodes involved by B-cell non-Hodgkin's lymphomas (B-NHL). In 26 malignant lymph nodes studied, the number of CD25+ T cells among total T cells was usually low when assessed by immunofluorescence analysis (mean +/- SD: 6.7% +/- 11.2%), but greatly increased when an immunomagnetic rosette method was used (mean +/- SD: 17.5% +/- 16.6%). In six cases, CD25-/CD25/CD25+ cells were isolated by immunomagnetic separation, with a purity greater than 97% for both populations. Expansion of CD25-/CD25+ T cells was obtained with IL2 and PHA, then conditioned media (CM) were prepared. No IL2 activity was found in CM from both CD25-/CD25+ T cells when tested on CTLL2 cells. BCGF and BCDF mu/gamma activities were assayed on normal B cells stimulated with soluble or insolubilized anti-mu antibodies(BCGF) or with Cowan I (BCDF). Results of production of all these activities were comparable for both populations, and thus do not favour the possibility that CD25+ T cells closely associated with malignant B-NHL cells in lymph nodes may influence their proliferation (BCGF) or expression/secretion of heavy chain isotype (BCDF mu/gamma).

In vivo activated T cells (CD25+) present in lymph nodes involved by B-non-Hodgkin's lymphomas (B-NHL) were investigated here for their ability to proliferate in vitro. CD25-/CD25+ T cells were isolated using a rosette method with magnetic beads, then the frequency of proliferating T lymphocyte-precursors (PTL-P) in both populations was assessed by limiting dilution experiments, in the presence of IL2, PHA and allogeneic spleen cells as feeders. In a total of 16 cases studied, growing microcultures were observed in all cases for CD25- T cells (mean value of PTL-P frequency: 1/32; range 1/10 - 1/2899) but in 6 cases only for CD25+ T cells (mean value of PTL-P frequency: 1/441; range 1/119 - 1/3736); the absence of any proliferative cultures in the 10 other cases indicated that the number of PTL-P was inferior to 1/12480. These results suggest that the proliferative potential of CD25+ T cells infiltrating lymph nodes involved by B-NHL is paradoxically decreased.