One of the major inducible cytokines secreted by mononuclear phagocytes is macrophage inflammatory protein 1 (MIP-1), which consists of two homologous polypeptides, MIP-1 alpha and MIP-1 beta. MIP-1 alpha possesses chemotactic and stimulatory activities for lymphocytes, eosinophils, and monocytes and may play a role in various pulmonary inflammatory conditions. We investigated the expression and release of MIP-1 alpha from human peripheral blood monocytes (PBM) and alveolar macrophages (AM) after stimulation with lipopolysaccharide (LPS), interleukin-1 beta (IL-1 beta), tumor necrosis factor-alpha, and interferon-gamma and the inhibitory effects of corticosteroids. LPS and IL-1 beta only enhanced MIP-1 alpha mRNA and protein in a dose- and time-dependent fashion. Dexamethasone (10(-9) to 10(-4) M) inhibited the basal and induced production and expression of MIP-1 alpha. In PBM, dexamethasone (10(-6) M) reduced LPS- and IL-1 beta-stimulated production of MIP-1 alpha protein by 50 and 63%, respectively, maximally at 24 h, whereas the inhibition of mRNA expression occurred maximally at 4 h. Similar trends were observed for AM. MIP-1 alpha mRNA decay was only slightly decreased in the presence of dexamethasone. Inhibition of LPS-induced MIP-1 alpha mRNA by dexamethasone was attenuated by the protein synthesis inhibitor cycloheximide, indicating the involvement of a protein intermediate. Corticosteroids are a potent inhibitor of IL-1 beta- and LPS-induced expression of MIP-1 alpha through mechanisms involving mainly inhibition of transcription and to a minor degree by reducing mRNA stability. Corticosteroids may be effective anti-inflammatory agents by preventing the expression of chemokines such as MIP-1 alpha.

An influx of eosinophils into the lungs occurs in several pulmonary disorders. However, the mechanisms involved remain unknown. Lung epithelial cell release of eosinophil chemotactic factors such as RANTES or macrophage inflammatory protein-1 alpha (MIP-1 alpha) could account for the influx of eosinophils into the lungs. In order to demonstrate the potential role for lung epithelial cells to release RANTES and/or MIP-1 alpha, we investigated the mRNA expression and protein release in cultured A549 cells. Tumor necrosis factor-alpha (TNF alpha) and interleukin-1 beta (IL-1 beta) induced a time- and dose-dependent increase in RANTES mRNA expression and protein release. In contrast, MIP-alpha protein release was not detectable in these cells. As corticosteroids decrease the influx of eosinophils into the lungs in vivo, we also investigated the capacity of dexamethasone to decrease the TNF alpha-induced RANTES release and mRNA expression; both were decreased in a time- and concentration-dependent manner. Dexamethasone did not affect the TNF alpha-induced RANTES mRNA half-life and did not require protein synthesis to manifest an inhibitory effect. Supernatant from cells stimulated with TNF alpha and IL-1 beta increased eosinophil chemotaxis and this was also inhibited by dexamethasone. These findings suggest a role for RANTES release by lung epithelial cells in the recruitment of eosinophils into the lungs in pulmonary disorders such as interstitial lung diseases, idiopathic pulmonary fibrosis, or asthma and suggest that one beneficial effect of corticosteroids may be inhibition of lung epithelial cell RANTES mRNA expression and protein release.

Eotaxin is an eosinophil-specific chemoattractant originally identified in bronchoalveolar lavage fluid after allergen challenge of sensitized guineapigs. We have determined and quantified for the first time the cellular sources of guinea-pig lung eotaxin and localized gene expression in structural cells of large and small airways and in alveolar macrophages.We used anti-guinea-pig eotaxin monoclonal and polyclonal antibodies and a complementary ribonucleic acid (cRNA) probe to detect eotaxin protein and cytoplasmic messenger ribonucleic acid (mRNA) transcripts by the techniques of immunohistochemistry and in situ hybridization in: 1) naive; 2) ovalbumin-sensitized/ saline-exposed; and 3) ovalbumin-sensitized/ovalbumin-challenged animals (n=5 for each group).Compared with the naive animals, there was a fivefold increase of eotaxin protein and a 25 fold upregulation of eotaxin gene expression in the airway epithelium 3 h after ovalbumin challenge of sensitized animals (p<0.001). The average percentages of alveolar macrophages staining for eotaxin protein and mRNA in the naive animals were approximately 30 and 10% respectively: both increased significantly in the sensitized/ovalbumin-challenged animals to 78 and 57%, respectively (p<0.0001). Compared with the naive animals, the procedure of sensitization and saline exposure significantly increased eotaxin gene expression in both bronchial epithelium and alveolar macrophages (p<0.01): the upregulation at these two sites showed a strong positive association (rcorr=0.95; p<0.0001).The results indicate that there are multiple cellular sources of guinea-pig lungderived eotaxin, including bronchial and bronchiolar epithelial cells, airway smooth muscle, bronchial vascular endothelium, and chondrocytes and alveolar macrophages, and that there are relatively rapid and marked increases of eotaxin protein and gene expression in airway epithelium and alveolar macrophages following allergen challenge.

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The guinea pig C-C chemokine, eotaxin, is a potent and selective eosinophil chemoattractant in guinea pig airways and skin in vivo, and stimulates both guinea pig and human eosinophils in vitro. The human C-C chemokine RANTES (30% homology with guinea pig eotaxin) stimulates human eosinophils in vitro, but does not stimulate guinea pig eosinophils, even though these cells bind 125I-RANTES. Similar concentrations of eotaxin and unlabeled RANTES competitively inhibit the binding of 125I-RANTES to guinea pig eosinophils, suggesting that eotaxin and RANTES share a common binding site on these cells. In the present study, we investigated the possibility that human RANTES, binding to a putative eotaxin receptor on guinea pig eosinophils, might block functional responses to eotaxin. When fura-2-loaded cells were first exposed to RANTES, which failed to elevate the intracellular calcium concentration, the response to a subsequent challenge with eotaxin was inhibited in a dose-dependent manner. Inhibition was also demonstrated when the two chemokines were added simultaneously. Another human C-C chemokine, MCP-3 (52% homology with guinea pig eotaxin), had similar inhibitory effects on the eotaxin-induced activation of guinea pig eosinophils in vitro. RANTES inhibited (111)In-eosinophil accumulation in response to intradermal eotaxin in vivo. In contrast, RANTES had no significant effect on responses to leukotriene B4 in vitro or in vivo. Thus, these experiments in the guinea pig demonstrate that human RANTES is the first prototypic antagonist of an eotaxin receptor.