Chemokines are cytokines that induce chemotaxis of inflammatory cells. We studied the presence of chemokines in bronchoalveolar lavage fluid (BALF) obtained from nine allergic asthmatic patients and six nonsmoking normal individuals. The cells were pelleted, and ribonucleic acid (RNA) was extracted by using RNAzol B. BALF was assayed for monocyte chemoattractant protein-1 (MCP-1), regulated upon activation in normal T cells, expressed, probably secreted (RANTES), macrophage inflammatory protein-1alpha (MIP-1alpha) and interleukin-8 (IL-8) by enzyme-linked immunosorbent assay (ELISA). The levels of MCP-1, RANTES, and MIP-1alpha were significantly higher in the asthma patients than in the control subjects (p<0.04). The concentrations of RANTES and MCP-1 correlated with the lymphocyte count in the BAL specimens (r = 0.61 and 0.68, respectively). BALF showed eosinophil chemotactic activity in vitro that was blocked by anti-RANTES and anti-MCP-3 antibodies. The total cellular RNA was reverse-transcribed and the complementary deoxyribonucleic acid (cDNA) was amplified with the polymerase chain reaction (PCR) for MCP-1, MCP-3, RANTES, MIP-1alpha, IL-8, and beta-actin. We found that messenger ribonucleic acids (mRNAs) for MCP-1, MCP-3, RANTES, MIP-1alpha, and IL-8 were produced by BAL cells from most asthmatic and normal subjects. We conclude that chemokines are produced in the airways, and that an increased recovery of MCP-1, RANTES, and MIP-1alpha is observed in allergic asthmatic patients.

SummaryInterleukin 5 (IL-5) regulates the growth and function of eosinophils. The objective of this study was to investigate the intraceUular signal transduction mechanism of IL-5 in eosinophils. Purified eosinophils were stimulated with IL-5, and the involvement of various kinases was investigated by immunoblotting, immune complex kinase assay, and in situ denatured/renatured kinase assay. We found that IL-5 induced tyrosine phosphorylation and activation of a number of kinases. Two species of lyn kinases (53 and 56 kD) were present in eosinophils. Both forms were Tyrphosphorylated and activated rapidly within 1 min. Further, lyn kinase was physically associated with the IL-53 receptor in eosinophils. Ras was studied by immunoprecipitation followed by thin-layer chromatography. Ras bound higher quantities of [ot-32p]guanosine 5'-triphosphate upon stimulation with IL-5. Raf-1 kinase showed increased Tyr phosphorylation on immunoblotting and increased activity in the immune complex kinase assay. Two species of MEK (MAP or Erk kinase) (41 and 45 kD) were identified in eosinophils, which underwent autophosphorylation upon stimulation. Microtubule-associated protein (MAP) kinase (p44) was Tyr-phosphorylated on immunoblotting and had increased activity in the immune-complex kinase assay. MAP kinases were also studied after metabolic radiolabeling of the cells with [32p]orthophosphates. IL-5 stimulated phosphorylation of MAP kinases in situ. Thus, we have delineated major components of an important signaling pathway in eosinophils. We believe that one of the signals generated by IL-5 receptor activation is propagated through the lyn-Ras-Raf-I-MEK-MAP kinase pathway.

Hematopoietins, interleukin (IL)-3, IL-5, and granulocyte/macrophage colony-stimulating factor (GM-CSF) have previously been shown to prolong eosinophil survival and abrogate apoptosis. The objective of this study was to investigate the effect of transforming growth factor beta (TGF-beta) on eosinophil survival and apoptosis. Eosinophils from peripheral blood of mildly eosinophilic donors were isolated to &gt; 97% purity using discontinuous Percoll density gradient. Eosinophils were cultured with hematopoietins with or without TGF-beta for 4 d and their viability was assessed. We confirmed previous observations that hematopoietins prolonged eosinophil survival and inhibited apoptosis. TGF-beta at concentrations &gt; or = 10(-12) M abrogated the survival-prolonging effects of hematopoietins in a dose-dependent manner and induced apoptosis as determined by DNA fragmentation in agarose gels. The effect of TGF-beta was blocked by an anti-TGF-beta antibody. The anti-TGF-beta antibody also prolonged eosinophil survival on its own. The culture of eosinophils with IL-3 and GM-CSF stimulated the synthesis of GM-CSF and IL-5, respectively, suggesting an autocrine mechanism of growth factor production. TGF-beta inhibited the synthesis of GM-CSF and IL-5 by eosinophils. TGF-beta did not have any effect on the expression of GM-CSF receptors on eosinophils. We also studied the effect of TGF-beta on eosinophil function and found that TGF-beta inhibited the release of eosinophil peroxidase. Thus, TGF-beta seems to inhibit eosinophil survival and function. The inhibition of endogenous synthesis of hematopoietins may be one mechanism by which TGF-beta blocks eosinophil survival and induces apoptosis.

SummaryMacrophage inflammatory protein-1 (MIP) is a recently cloned cytokine that causes neutrophilic infiltration and induces an inflammatory response. We studied the effect of MIP-lo~ on histamine secretion from basophils and mast cells. Leukocytes from allergic and normal subjects were studied. MIP-loe caused dose-dependent release of histamine from basophils of 14 of 20 allergic donors at concentrations of 10-9-10 -7 M, and the mean release was 13.50 + 2.9% at the highest concentration. In the same experiments, the mean histamine release by anti-immunoglobulin E and monocyte chemotactic and activating factor (MCAF) (10 -7 M) was 32 + 7% and 31 _+ 3%, respectively. The cells from only 2 of 10 normal subjects released histamine in response to MIP-loe. Histamine release by MIP-lo~ was rapid, and almost complete within the first 3 rain. MIP-lce-induced degranulation was a calcium-dependent noncytotoxic process. MIP-lo~ showed chemotactic activity for purified basophils that was comparable to MCAF. Both MIP-lo~ and MCAF at 10-7 M concentration elicited a chemotactic response that was 40% of the maximal response to CSa (1/zg/ml). Murine MIP-lo~ induced histamine release from mouse peritoneal mast cells in a dose-dependent manner. Thus, we have established that MIP-lo~ is a novel activator of basophils and mast cells.

Monocyte chemotactic and activating factor (MCAF) is a recently cloned cytokine that causes chemotaxis of basophils. In our pursuit of cytokines affecting basophil function, we studied the effect of MCAF on histamine secretion from basophils. Leukocytes from 20 donors, 10 allergic and 10 normal subjects, were studied. MCAF caused dose-dependent release of histamine at concentrations of i0' and 10-7 M, and the mean release was 31.25±2.9% at the highest concentration. In the same experiments the mean histamine release by anti-IgE and histamine releasing factor (HRF) was 27.05±4% and 32.70±2.7%, respectively. All 20 subjects responded to MCAF with significant histamine release. Allergic subjects released significantly more histamine than normals in response to anti-IgE (P < 0.01) but not to MCAF (P = 0.2) and HRF (P = 0.1). The histamine release was significantly correlated between MCAF and HRF (P < 0.01), but not between MCAF and anti-IgE (P > 0.05). The histamine release by MCAF was complete within the first 3 min. MCAF-induced degranulation was a calcium-dependent process. Leukocytes depleted of monocytes responded equally well to MCAF. Using an anti-MCAF affinity column we determined that > 50% of HRF activity of crude PBMC supernatant could be attributed to MCAF. Thus, we established that MCAF is a potent secretagogue for basophils. (J. Clin. Invest.