Citations

Abstract Pneumocystis carinii is an opportunistic organism that causes severe lung injury in immunocompromised hosts. Macrophage responses to P. carinii are poorly defined. Arachidonic acid (AA) and its metabolites are potent mediators of inflammation and have been implicated in host response to microorganisms. We therefore examined the production of eicosanoids from rat and rabbit alveolar macrophages stimulated with purified P. carinii. [14C]AA-labeled rabbit macrophages released 8.50 ± 1.33% of the incorporated [14C]AA after 90 min in response to P. carinii (P = 0.0001 compared with unstimulated controls). In contrast, a similar number of rat alveolar macrophages exhibited a smaller but significant response to P. carinii, releasing 3.84 ± 1.54% of their [14C]AA after 90 min (P = 0.001 compared with control). We further determined that P. carinii stimulated substantial production of prostaglandin E2 and concurrently a small amount of leukotriene B4 release from alveolar macrophages. To further investigate whether serum opsonization of P. carinii enhances these alterations in AA metabolism, we assessed the effect of P. carinii immune serum on P. carinii-induced AA release. P. carinii opsonized with this antiserum caused significantly greater AA release from rat alveolar macrophages than either unopsonized P. carinii or organisms opsonized with nonimmune serum. Previous studies suggest that P. carinii interacts with macrophage β-glucan and mannose receptors. However, incubation of macrophages with P. carinii in the presence of either soluble β-glucan or α-mannan failed to alter the release of AA from macrophages in response to P. carinii. Macrophage release of eicosanoids represents a potentially important host inflammatory response to P. carinii infection.

Abstract Fibulin is a newly described extracellular matrix (ECM) glycoprotein whose function has not been elucidated. We have observed that cultured fetal lung fibroblasts produce fibulin and have postulated that its expression may be important during lung development. To begin to understand the potential function of fibulin in lung development, we examined its expression and distribution in cultured fetal lung fibroblasts. Immunofluorescence staining of cultured fibroblasts revealed that fibulin was distributed upon their surface in a fibrillar array resembling fibronectin (FN), another ECM glycoprotein expressed by fetal lung fibroblasts and implicated in lung and heart development. Detection of fibulin by immunofluorescence staining of nonpermeabilized cells, its immunoprecipitation from 125I-cell surface-labeled fibroblasts, pulse-chase analysis, and temperature-induced phase separation studies revealed that fibulin is an ECM peripheral membrane protein that is synthesized and secreted by cultured fetal lung fibroblasts shortly after plating and incorporated into their matrix in a divalent cation-dependent manner. Because fibulin co-distributes with both FN and the FN receptor, the integrin α5β1, we examined the possibility that fibulin was interacting with both components. Dissociation of FN receptors from FN fibers with anti-FN receptor antibodies did not affect fibulin's distribution, suggesting that fibulin binds FN and that this interaction is not affected by the state of FN receptor binding. Finally, inhibition of FN matrix assembly prevented the deposition of fibulin, providing further support for FN-fibulin interactions and suggesting that fibulin deposition is dependent on FN matrix assembly.

Abstract Migration of eosinophils through the basement membrane barrier is an important step for their infiltration into tissues. To investigate the mechanism of transmigration, we used a chamber fitted with a Matrigel membrane as a model of the basement membrane. In this model, eosinophils treated with cytokines or chemotactic factors alone did not transmigrate from the upper to the lower chamber. However, platelet-activating factor (PAF) strongly induced transmigration of eosinophils stimulated by interleukin (IL)-5, indicating that both a cytokine and a chemotactic factor are required for eosinophil migration through Matrigel. Granulocyte-macrophage colony-stimulating factor (GM-CSF) and IL-3 also stimulated eosinophil transmigration in the presence of PAF. Of seven eosinophil chemotactic factors tested, leukotriene B4, C5a, RANTES, macrophage inflammatory protein-1α, and IL-8 did not induce significant eosinophil transmigration. Only PAF and eotaxin induced transmigration of eosinophils through Matrigel in the presence of IL-5; PAF was more potent than eotaxin at the optimal concentration. In contrast, PAF, eotaxin, and RANTES all potently induced migration of eosinophils through bare membrane in the absence of IL-5. Finally, eosinophil migration through Matrigel was markedly reduced by a combination of anti-CD18 and anti-CD29 monoclonal antibodies, suggesting that it is mediated by β1- and β2-integrin adhesion molecules. Our findings demonstrate that eosinophil transmigration through a basement membrane model requires both a specific chemoattractant, such as PAF, and an eosinophil-activating cytokine, such as IL-5. This synergistic effect is likely important in the tissue accumulation of activated eosinophils in allergic and other eosinophil-associated diseases.

Abstract In general, inflammatory cells cross basement membranes by producing proteinases. To investigate the role of proteinases in eosinophil basement membrane migration, we studied peripheral blood eosinophils in Matrigel®-coated chemotaxis chambers. Electron microscopy showed degradation of the Matrigel® layer when eosinophils, added to the upper chamber, transmigrated the membrane in the presence of both platelet-activating factor (PAF) in the lower chamber and interleukin (IL)-5 in both chambers. In the absence of either or both PAF and IL-5, no changes occurred in the Matrigel® layer. Matrigel® transmigration of eosinophils induced by PAF and IL-5 was inhibited by 1,10-phenanthroline, batimastat, 3,4-dichloroisocoumarin, chymostatin, and a neutralizing antibody for the matrix metalloproteinase (MMP)-9, indicating that serine proteinase(s) and MMP, specifically MMP-9, were involved in the transmigration of eosinophils through Matrigel®. In contrast, eosinophil migration through a bare membrane was not affected by batimastat. Using gelatin zymography and immunoblotting, MMP-9 was detected in the migration upper chamber supernatant of the eosinophil transmigration assay and in the conditioned medium of eosinophils. Release of MMP-9 by eosinophils was increased by IL-5, PAF, or both, but the substrate-degrading activity of MMP-9 was increased only in the presence of both IL-5 and PAF, indicating that the releasing and activating mechanisms of MMP-9 are involved in eosinophil basement membrane migration. This study implicates MMP-9 in basement membrane migration of eosinophils and suggests its involvement in inflammatory diseases where tissue eosinophilia plays a role.

Abstract A previous lectin binding study demonstrated the presence of high molecular-mass mucin-like glycoproteins (HMGP) on the surface of hamster tracheal surface epithelial (HTSE) secretory cells (Proc. Natl. Acad. Sci. USA 1987;84:9304). In the present study, we intended to isolate and characterize these HMGP from the plasma membrane of the primary HTSE cells and then to determine whether or not these membrane HMGP are Muc-1 mucins, a type of mucins originally discovered on the surface of some carcinomas. A subcellular fraction enriched with the plasma membrane was obtained using a sucrose density gradient centrifugation. This fraction contained high molecular-mass glycoconjugates which were excluded from Sepharose CL-4B gel. Biochemical characterization of these glycoconjugates revealed the following characteristics: (1) susceptibility to both pronase and mild alkaline treatments, but totally resistant to proteoglycan-digesting enzymes; (2) partitioning in the detergent phase of Triton X-114 and resistance to digestion by phosphatidylinositol phospholipase C or D; (3) a buoyant density of 1.5 g/ml based on CsCl density gradient centrifugation; (4) polydispersity in terms of both size and charge density; and (5) lack of immunoreactivity with an anti-Muc-1 mucin antibody. We conclude that the plasma membrane of HTSE cells at confluence contains HMGP, which seem to be the integral membrane proteins but different from Muc-1 mucins, and that these membrane HMGP appear to share some similarities with secreted mucins in terms of size and charge.

Abstract Intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) are members of the immunoglobulin superfamily adhesion molecules on vascular endothelium and important in the development of eosinophil (EOS) accumulation in allergic inflammation. To define the role of these adhesion proteins in EOS inflammation, peripheral blood EOS from allergic donors were incubated in either buffer (control)-, recombinant human (rh)-VCAM-1-, or rh-ICAM-1-coated plates, and the effects of these adhesion proteins on EOS effector functions were determined. VCAM-1 induced spontaneous EOS adhesion whereas EOS adhesion to ICAM-1 required a second signal, such as granulocyte macrophage colony-stimulating factor (GM-CSF). Although only VCAM-1 stimulated EOS superoxide anion (O2−) generation, the addition of GM-CSF (100 pM) to the reactions resulted in a greater and equivalent production of O2− with VCAM-1 and ICAM-1. In the presence of GM-CSF, ICAM-1 and VCAM-1 caused significant release of EOS-derived neurotoxin (EDN). Moreover, only ICAM-1 (no GM-CSF) promoted calcium ionophore A23187 (0.2 μM)-induced EOS leukotriene C4 (LTC4). Enhanced O2− generation, EDN release, and LTC4 generation observed with ICAM-1 and VCAM-1 were significantly inhibited by anti-β2-integrin antibody. These results suggest that ICAM-1 and VCAM-1 are important in determining the eventual function of airway EOS.

Abstract Evidence suggests that cellular adhesion is critical for eosinophil effector functions. Here, we tested the hypothesis that an adhesion molecule, specifically β2 integrin, participates in intracellular signaling events of eosinophils. Eosinophils stimulated with interleukin (IL)-5 and adherent to protein-coated tissue culture plates via β2 integrin (CD18) showed tyrosine phosphorylation of a number of proteins. Among these proteins, tyrosine phosphorylation of the 105 kD and 115 kD proteins and the product of the c-cbl protooncogene, Cbl, was specifically inhibited using soluble anti-CD18 monoclonal antibody (mAb) to block eosinophil cell adhesion. Furthermore, phosphoinositide turnover of IL-5-stimulated adherent eosinophils was also inhibited by anti-CD18 mAb, suggesting that cellular adhesion plays important roles in eosinophil signal transduction. αMβ2 (Mac-1, CD11b/18) was one of the β2 integrins involved in eosinophil adhesion to protein-coated plates. We found that direct ligation of eosinophil αMβ2 with anti-CD11b mAb coupled to polystyrene microbeads induced tyrosine phosphorylation of a 115 kD protein and Cbl. Furthermore, anti-CD11b mAb microbeads induced increases in both phosphoinositide hydrolysis and the eosinophil degranulation response. Control antibodies, such as mouse myeloma IgG1 and anti-HLA class I antigen mAb, did not induce these cellular responses. These results suggest that engagement of β2 integrin either by cell adhesion or by anti-CD11b mAb triggers activation of an intracellular signaling cascade, including protein tyrosine phosphorylation and phosphoinositide turnover, and subsequent cellular degranulation in human eosinophils. Tyrosine phosphorylation of a 115 kD protein and Cbl may play important roles in adhesion-dependent cellular functions of eosinophils.