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CRTH2 is one of the prostaglandin D₂ receptors and plays a proinflammatory role in allergic diseases. Gene expression markers in whole blood induced by CRTH2 activation have not previously been reported. Using microarray analyses of 54 675 genes, we revealed modest gene expression changes in human whole blood stimulated in vitro by a selective CRTH2 agonist, DK-PGD₂. Five genes were found to exhibit 1.5- to 2.6-fold changes in expression. The expression of Charcot-Leyden crystal protein/galectin-10 (CLC/Gal-10) in particular was consistently enhanced in human whole blood stimulated by DK-PGD₂, as confirmed by quantitative real-time polymerase chain reaction analyses. DK-PGD(2)-induced increases in blood CLC/Gal-10 mRNA levels were largely attenuated by the CRTH2 antagonist CAY10471.Thus, the DK-PGD₂-induced CLC/Gal-10 mRNA level can serve as a potential marker for monitoring pharmacodynamic effects of blood exposure to CRTH2 modulating agents.

Asthma, chronic obstructive pulmonary disease (COPD) and acute lung injury/acute respiratory distress syndrome (ALI/ARDS) are characterized by neutrophilic inflammation and elevated levels of leukotriene B4 (LTB4). However, the exact role of LTB4 pathways in mediating pulmonary neutrophilia and the potential therapeutic application of LTB4 receptor antagonists in these diseases remains controversial. Here we show that a novel dual BLT1 and BLT2 receptor antagonist, RO5101576, potently inhibited LTB4-evoked calcium mobilization in HL-60 cells and chemotaxis of human neutrophils. RO5101576 significantly attenuated LTB4-evoked pulmonary eosinophilia in guinea pigs. In non-human primates, RO5101576 inhibited allergen and ozone-evoked pulmonary neutrophilia, with comparable efficacy to budesonide (allergic responses). RO5101576 had no effects on LPS-evoked neutrophilia in guinea pigs and cigarette smoke-evoked neutrophilia in mice and rats. In toxicology studies RO5101576 was well-tolerated. Theses studies show differential effects of LTB4 receptor antagonism on neutrophil responses in vivo and suggest RO5101576 may represent a potential new treatment for pulmonary neutrophilia in asthma., (Copyright 2010 Elsevier Inc. All rights reserved.)

The inhibition of LTB(4) binding to and activation of G-protein-coupled receptors BLT1 and BLT2 is the premise of a treatment for several inflammatory diseases. In a lead optimization effort starting with the leukotriene B(4) (LTB(4)) receptor antagonist (2), members of a series of 3,5-diarylphenyl ethers were found to be highly potent inhibitors of LTB(4) binding to BLT1 and BLT2 receptors, with varying levels of selectivity depending on the substitution. In addition, compounds 33 and 38 from this series have good in vitro ADME properties, good oral bioavailability, and efficacy after oral delivery in guinea pig LTB(4) and nonhuman primate allergen challenge models. Further profiling in a rat non-GLP toxicity experiment provided the rationale for differentiation and selection of one compound (33) for clinical development.

Although IL-12 has been reported to synergize with c-kit ligand (KL) in promoting hematopoietic stem cell proliferation in vitro, administration of recombinant mouse IL-12 (rIL-12) to normal mice caused a dose- and time-dependent anemia, leukopenia, and thrombocytopenia in vivo. Decreased numbers of bone marrow cells were recovered from the tibiae of IL-12-treated mice, and histologic examination of the marrow revealed a loss of mature neutrophils and red blood cell precursors. However, simultaneously with the suppression of hematopoiesis in the bone marrow, the IL-12-treated mice developed splenomegaly, which was largely caused by a marked enhancement of splenic extramedullary hematopoiesis of the erythroid, myeloid, and megakaryocytic lineages. These histologic observations were confirmed by colony-forming cell assays in which administration of IL-12 was shown to cause a time-dependent decrease in bone marrow CFU-GM, CFU-E, and BFU-E hematopoietic colony-forming cells while causing an increase in splenic CFU-GM and BFU-E colony-forming cells. All these effects were reversible upon cessation of IL-12 treatment. The observation that in IL-12-treated mice hematopoiesis was suppressed in the marrow but enhanced in the spleen suggests that myelosuppression was not caused by a direct effect of IL-12 on hematopoietic progenitors. It seems likely that myelosuppression was caused instead by an IL-12-induced alteration in the local environment of the marrow.

The polymerase chain reaction technique (PCR), a primer-mediated enzymatic amplification of specific target sequences, was used to monitor gene transfer into hematopoietic progenitor stem cells. A gene coding for human interleukin 1 alpha (IL-1 alpha) was cotransfected with the rous sarcoma virus (RSV) CAT plasmid into mouse bone marrow cells by electroporation. Individual chloramphenicol (CAM)-resistant bone marrow progenitor colonies (granulocyte-macrophage colony-forming units; CFU-GM) containing 50-100 cells were analyzed by PCR for the presence and expression of IL-1 alpha and CAT sequences. Amplified IL-1 alpha DNA sequences were detected from a 50-cell CFU-GM colony. CAT and IL-1 alpha RNA expression was demonstrated from the CAM-resistant CFU-GM colonies.

Adult male mice were treated with various doses of 6-hydroxydopamine in order to assess the effects of this drug upon thymic dependent immunity. A consistent decrease in primary antibody titers to sheep erythrocytes was observed following treatment with this drug. Serum levels of thymosin alpha 1 were increased by day three after 6-OHDA with a return to normal by day five. Thymocyte terminal deoxynucleotidyl transferase changes were biphasic with an initial decrease after 6-OHDA followed by an increase. Changes in mitogen responsiveness were observed but were not consistently reproducible. Involvement of both catecholamines and corticosteroids in bringing about these observed changes was discussed.

Human rIL-1 alpha was shown to be a potent stimulus of granulopoiesis in mice that have been myelosuppressed with cyclophosphamide. Stimulation of granulopoiesis was demonstrated in IL-1-treated mice by an accelerated recovery of granulocyte-macrophage colony-forming cells, bone marrow and splenic granulocytic hyperplasia, and a profound granulocytosis. Granulopoiesis was stimulated by IL-1 in a dose-dependent manner at doses ranging from 0.5 to 50 micrograms/kg. Maximal increases in granulocytes were observed after 4 days of IL-1 treatment. Mice treated with IL-1 also exhibited increased splenic megakaryopoiesis with a resultant increase in the number of peripheral blood platelets. In contrast to these positive effects on hemopoiesis, bone marrow, but not splenic, erythropoiesis was depressed in IL-1-treated mice. IL-1 effects were observed in mice treated with a wide dose range (50 to 300 mg/kg) of cyclophosphamide, with optimal effects occurring at a dose of 200 mg/kg. The doses of IL-1 leading to enhanced granulopoiesis caused only minor and transient changes in selected clinical chemistry parameters and caused no toxicities that were evident by histologic examination of tissues. The stimulation of granulopoiesis in the absence of overt toxicity suggests that IL-1 may be useful clinically to enhance the recovery of granulocytes in myelosuppressed patients.

Leukotrienes are vasoactive arachidonic acid metabolites which are released by mast cells during hypersensitivity reactions. The mechanisms for regulating leukotriene biosynthesis are not well understood. A murine mast/basophil cell line (PT-18) was used to investigate this problem. Exogenously supplied [14C]arachidonic acid is not appreciably converted to leukotrienes by untreated PT-18 cells. However, when the cells were preincubated with the lymphocyte product 15-hydroxyeicosatetraenoic acid (15-HETE), addition of [14C]arachidonic acid consistently resulted in a dose-dependent synthesis of large amounts of both [14C]leukotriene B4 and [14C]5-HETE. These metabolites were isolated by high pressure liquid chromatography, converted to the methyl ester trimethylsilyl ether derivatives, and the structures confirmed by gas chromatography-mass spectrometry. These findings indicate that 15-HETE induces a direct activation of a cryptic 5-lipoxygenase in these cells. The closely related 12-HETE was ineffective. The activation phenomenon occurs rapidly and is reversible. Furthermore, the activation appears to be highly cell- and enzyme-specific, since lipoxygenases in three primary cell types including one that contains a 5-lipoxygenase and six other cell lines did not show this specific induction of leukotriene biosynthesis by 15-HETE. This report is the first evidence that 15-HETE, a major arachidonate metabolite in lymphocytes, can act as a signal to activate leukotriene production by susceptible mast cells.

The beneficial effects of IL-1 and other cytokines on hematopoiesis and on resistance to infection are profound. IL-1 stimulates proliferation of bone marrow cells in normal mice and potentiates the recovery of peripheral blood neutrophils in mice with drug-induced neutropenia. Prophylactic cytokine administration provides an elevated level of natural resistance to infections which is correlated with increased numbers of phagocytic leukocytes. These studies suggest that IL-1 has potential clinical application as a therapy to limit bone marrow dysfunction and immunosuppression and to augment hematopoiesis and natural immunity. Further research will continue to elucidate the mechanisms whereby interleukins and colony-stimulating factors act, and interact, to promote restoration of leukocyte production and to enhance host resistance.

There is developing evidence that certain thymosin peptides and lymphokines produce a transient increase in steroid hormones when introduced systemically. Conversely, the repressive effect of adrenocortical steroids on the immune system is well documented. In the present study, the direct effect of certain components of the immune system on steroid output by rat adrenal fasciculata cells was tested. With this system, there was no direct steroidogenic effect of either the partially purified thymosin fraction 5, or any of the purified peptide components tested (thymosin alpha 1, alpha 7, or beta 4). These peptides also did not synergize the cellular response to ACTH, nor did they induce cAMP production by a ACTH- and NaF-responsive adrenal membrane preparation. Supernatants from Con A-stimulated spleen cells, which were demonstrated to contain lymphokine activity, and partially purified mouse interferon were also without a significant direct or synergistic effect on steroidogenesis by adrenocortical cells. These results suggest that the steroidogenic response to these peptides observed in vivo may be mediated by the central nervous system.