Assessment of chemokine receptor function on monocytes in whole blood: In vitro and ex vivo evaluations of a CCR2 antagonist.

Inhibition of monocyte and macrophage function by targeting chemokine receptors represents an attractive strategy for therapeutic intervention in inflammatory diseases. We describe an assay to assess chemokine receptor function on whole blood monocytes by measuring chemokine stimulated change in cell shape as measured by flow cytometry. The relative potential of the chemokine receptors CCR1, CCR2, CCR5, CX(3)CR1, and CXCR4 to activate monocytes in whole blood was evaluated and compared. Analysis of MCP-1 response for monocytes in blood from numerous donors revealed that the assay method had excellent intra-donor reproducibility and sensitivity. Further, the utility of this assay to determine target engagement by chemokine receptor antagonists was demonstrated using a CCR2 antagonist in rhesus monkeys. Blockade of CCR2 on whole blood monocytes was demonstrated ex vivo on blood samples collected from rhesus monkeys administered a small molecule CCR2 antagonist (MK-0812). Using a delayed-type hypersensitivity reaction to elicit monocyte recruitment to the skin of rhesus monkeys, we also evaluated the ability of MK-0812 to block monocyte migration in vivo. Blockade of CCR2 stimulation of whole blood monocytes was correlated with the inhibition of monocyte recruitment to the skin, validating the potential to use this approach in the evaluation of dose selection for chemokine receptor antagonists clinically.

Maternal exposure to benzo[a]pyrene alters development of T lymphocytes in offspring.

Childhood cancer has been increasing significantly over the past two decades in the United States, suggesting that environmental exposures may be playing a causative role. One such cause may be maternal smoking during pregnancy. Suspected carcinogens in cigarette smoke and environmental pollution include N-nitrosamines and polycyclic aromatic hydrocarbons, which may be several micrograms per exposure. Previously, we have shown that mouse progeny of mothers exposed to benzo[a]pyrene (B[a]P) during midpregnancy had abnormalities in their humoral and cell-mediated immune response. Immunodeficiency was detectable during gestation, at one week after birth and persisted for 18 months. Tumor incidences in progeny were eight to 10-fold higher than in controls. The present study compared frequencies of CD4+, CD8+, V gamma 2+, and V beta 8+ T cells in progeny following in utero exposure to B[a]P. The significant reduction in newborn CD4+CD8+, CD4+CD8+V beta 8+ thymocytes and CD4+ splenocytes from 1-week-old progeny, suggests that B[a]P induces abnormal changes in developing T cells. These early alterations may lead to postnatal T cell suppression, thus providing a more suitable environment for the growth of tumors later in life. These results suggest that developmental immunosuppression mediated by B[a]P may play a critical role in the relationship between maternal exposures and childhood carcinogenesis.

Gastric H+-K+-ATPase in situ: evidence for compartmentalization.

Gastric glands, isolated from rabbit, were permeabilized with digitonin to permit measurement of H+-K+-adenosinetriphosphatase (ATPase) activity and proton transport in situ. Measurement of proton gradient formation using acridine orange fluorescence showed two phases of ATP-driven proton accumulation; one phase occurs spontaneously in KCl medium and one phase requires the K+ ionophore valinomycin. Valinomycin was found to increase H+-K+-ATPase activity, indicating that the second phase is because of increased proton transport rather than a decrease in proton leak rate. The acid-activated, irreversible inhibitor, omeprazole, was used to selectively eliminate the H+-K+-ATPase molecules associated with the spontaneous component of proton transport. After omeprazole treatment a residual, valinomycin-dependent component of proton transport could be demonstrated. These results are interpreted as evidence for two compartments of H+-K+-ATPase, separated by a barrier that prevents K+ diffusion and pH equilibration. The two compartments may be separated also on the basis of anion selectivity. The spontaneously active compartment was found to be functional with various anions, including sulfate and isethionate, whereas the valinomycin-dependent component is highly selective for chloride. The proportion of H+-K+-ATPase that exists in each compartment was quantitated by measuring the fraction of total ATPase activity that could be inhibited by omeprazole in the absence and presence of valinomycin. For glands that were preconditioned with cimetidine, approximately 30% of the inhibitable enzyme was found associated with the spontaneous compartment, and this fraction increased to approximately 70% with histamine preconditioning.(ABSTRACT TRUNCATED AT 250 WORDS)

Gastric H+-K+-ATPase in situ: relation to secretory state.

Isolated gastric glands from rabbit were used to measure the gastric H+-K+-adenosinetriphosphatase (ATPase) and its partial reaction, a K+ p-nitrophenyl phosphatase (pNPPase), in situ. Measurement of the enzyme activities required permeabilization of the cells with digitonin and the use of several ATPase inhibitors to reduce nonspecific activity. The enzyme activities were identified as the H+-K+-ATPase according to the following criteria: dependence on K+, association with parietal cells, insensitivity to ouabain and ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid, and inhibition by the specific inhibitors omeprazole and Sch 28080. K+-stimulated ATPase, but not K+-pNPPase, was enhanced by K+ ionophores, valinomycin and nigericin, with nigericin resulting in the greatest activity. Comparison of tissues that were preincubated to establish resting and stimulated states showed that prestimulation results in an increase in K+-stimulated ATPase activity with no change in the total activity. With the use of a two-step assay procedure, it could be shown that stimulation also results in an increase in the omeprazole-sensitive maximal ATPase activity. These results indicate that the major effects of stimulation are to enhance KCl activation of the enzyme and to increase the number of active enzyme molecules.