Monocytes (m phi s) come into intimate contact with basement membranes and extracellular matrix proteins as they extravasate from the blood to the interstitium or to sites of tissue injury. We examined the in vitro effects of m phi adherence to an endothelial cell-derived basement membrane or to purified extracellular matrix proteins on phorbol myristate acetate (PMA)-stimulated superoxide production and prostanoid secretion. Elutriation-purified human peripheral blood m phi s were adhered to tissue culture wells that were precoated with the following purified proteins: bovine serum albumin (BSA), collagen type I (COL I), collagen type IV (COL IV), fibronectin (FN), or laminin (LM). To model the provisional matrix at sites of tissue injury, m phi s were also adhered to wells coated with either denatured collagen type I or gelatin (GEL) or coated with basement membrane (BM) derived from endothelial cell monolayers. The m phi s were adhered to the protein-coated surfaces for 1 h at 37 degrees C in serum-free medium and washed to remove nonadherent cells, and the number of adherent m phi s was measured. Monolayers of m phi s were also incubated for an additional 18 h, at which time both adherence and cell spreading were measured. PMA-stimulated superoxide production by adherent m phi s was determined after 1 and 18 h of adherence to the protein-coated surfaces. PMA-stimulated release of two prostanoids, prostaglandin E2 (PGE2) and thromboxane B2 (TxB2) was measured after 18 h of m phi adherence to the surfaces. Following 18 h of adherence, PMA-stimulated superoxide anion secretion and secretion of PGE2 and TxB2 were augmented of primed by m phi s adherent to COL I, GEL, or BM. In contrast, no such priming effects were observed by m phi s adherent to COL IV, FN, or LM. The results suggest that adherence to basement membranes, collagen type I-containing surfaces in the interstitium, or denatured collagen at sites of tissue injury primes m phi respiratory burst and arachidonate metabolism to inflammatory agonists. Induction of priming events by substrate-specific adherence may be an important factor regulating host defense functions of m phi s in the extracellular matrix.

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The emigration of peripheral blood monocytes into the interstitium allows for contact with a variety of surfaces which may provide signals important for monocyte function in both normal and inflammatory states. In the present study, we examined the effect of adherence to an endothelial cell-derived basement membrane and to collagen I, the major collagen of the interstitium, on monocyte release and gene expression of the potent chemotactic cytokine Interleukin-8 (IL-8). We further evaluated neutrophil chemotactic activity of the conditioned media containing antigenic IL-8 from monocytes adherent to these same surfaces. Elutriation-purified monocytes were adhered for 1 hour to plastic tissue culture wells either uncoated (PL) or coated with bovine serum albumin (BSA), collagen type I (C-I), or endothelial cell-derived basement membrane (BM). Following removal of nonadherent cells, monocytes were further incubated in a serum-free media for 18 hours in the presence or absence of lipopolysaccharide (IPS). Following 18 hrs of incubation there were significantly less monocytes remaining adherent to BM when compared to other surfaces tested. In the absence of LPS, adherent monocytes released significant amounts of IL-8 that was not surface specific. In the presence of LPS, monocytes adherent to BM released significantly more IL-8, when corrected for adherent cell number, than monocytes adherent to PL, BSA, or C-I. Conditioned media from adherent monocytes expressed IL-8 dependent neutrophil chemotactic activity that was not influenced by the surfaces tested. Northern blot analysis indicated greater induction for IL-8 mRNA by monocytes adhered to BM after 18 hrs in the presence of LPS. These results suggest that monocyte adherence to the subendothelial basement membrane provides a priming signal for the induction and secretion of the chemotactic cytokine IL-8 in response to inflammatory stimuli.

Chemokine and integrin receptors must work in concert when circulating leukocytes mobilize toward a site of tissue inflammation or infection. In a previous study, we reported that ligation of the alpha5beta1 integrin with a 120-kDa cell-binding fibronectin fragment (120-kDa FN) in suspensions of human polymorphonuclear leukocytes (PMNLs) inhibited chemotaxis toward the chemokine called interleukin-8 (IL-8). Binding of chemokines to their receptors on leukocytes leads to the activation of heterotrimeric G proteins that initiate multiple signaling cascades, including p38 and p42/p44 mitogen-activated protein kinase (MAPK) pathways. In the present study, we examine the potential interaction of beta1 integrin ligation on chemokine-mediated MAPK signaling in human PMNLs. We demonstrate that blockade of the p42/p44 MAPK signaling pathway by the inhibitor PD98059 suppresses IL-8-mediated PMNL chemotaxis. Furthermore, when PMNLs are pretreated with 120-kDa FN or an activating antibody to beta1 integrins (TS2/16), IL-8-mediated phosphorylation of p42/p44 MAPK is also inhibited. In contrast, pretreating PMNL with a specific ligand (laminin-1) for the alpha6beta1 integrin does not suppress IL-8-mediated phosphorylation of p42/p44 MAPK. These observations demonstrate a desensitization of IL-8-mediated p42/p44 MAPK signaling in response to ligation of the alpha5beta1 integrin in PMNL. Also, they suggest an interplay between integrin and chemokine signaling during PMNL migration through the extracellular matrix.