Paul W. Gudewicz

Human urokinase-type plasminogen activator stimulates chemotaxis of human neutrophils

Human purified urokinase-type plasminogen activator (u-PA) stimulates chemoattractant activity for human neutrophils using modified Boyden chambers. Checkerboard analysis performed by adding different concentrations of u-PA above and below the polycarbonate filters revealed maximum migration required a positive concentration gradient. These results suggest that uPA was in fact stimulating neutrophil chemotaxis. Incubation of u-PA with an anti-u-PA goat antibody completely abolished the chemotactic activity of u-PA while incubation with the serine protease inhibitor, diisopropyl fluorophosphate, did not reduce chemotactic activity. Purified human tissue-type plasminogen activator demonstrated no chemotactic activity for human neutrophils when tested at concentrations similar to u-PA. These results suggests that the expression of chemotactic activity of u-PA may serve to recruit circulating leukocytes to the inflammatory site.

Effect of phagocytosis of erythrocytes and erythrocyte ghosts on macrophage phagocytic function and hydrogen peroxide production

Our previous studies have shown that an in vivo phagocytic challenge with IgG-coated erythrocytes can depress Kupffer cell complement and Fc receptor function, as well as decrease the survival rate following endotoxemia and bacteremia. In an effort to better understand the mechanism underlying these in vivo findings, the present study evaluated the in vitro effects of a phagocytic challenge with either IgG-coated erythrocytes (EIgG) or erythrocyte ghosts (GIgG) on macrophage phagocytic and respiratory burst activity. Elicited rat peritoneal macrophage (PM) monolayers were challenged with varying doses of EIgG, then the noninternalized EIgG were lysed hypotonically and the monolayers incubated for an additional hour prior to determining phagocytic function and PMA-stimulated hydrogen peroxide production. Challenge of PM with 1×106 EIgG per well had no effect, but challenge with 1×107 or 1×108 EIgG per well caused a dose-dependent depression of phagocytic function or hydrogen peroxide production. GIgG were formed by hypotonically lysing EIgG bound to PM at 4°C. The bound GIgG were phagocytized during a subsequent incubation at 37°C. Challenge with GIgG depressed phagocytic function only with the highest challenge dose tested (1×108 per well) and did not depress hydrogen peroxide production. The observation that prior phagocytic challenge with EIgG depressed macrophage function to a greater extent than challenge with GIgG supports our previous in vivo observations. Furthermore, these studies suggest that the internalization of erythrocyte contents, and not phagocytosis per se, plays an important role in determining macrophage host defense function.

Priming of human monocyte superoxide production and arachidonic acid metabolism by adherence to collagen- and basement membrane-coated surfaces.

Monocytes (mφs) 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 mφ 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 mφs 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, mφs 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 mφs were adhered to the protein‐coated surfaces for 1 h at 37° in serum‐free medium and washed to remove nonadherent cells, and the number of adherent mφs was measured. Monolayers of mφs were also incubated for an additional 18 h, at which time both adherence and cell spreading were measured. PMA‐stimulated superoxide production by adherent mφs 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 mφ adherence to the surfaces. Following 18 h of adherence, PMA‐stimulated superoxide anion secretion and secretion of PGE2 and TxB2 were augmented or primed by mφs adherent to COL I, GEL, or BM. In contrast, no such priming effects were observed by mφs 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 mφ 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 mφs in the extracellular matrix. J. Leukoc. Biol. 55: 423–429; 1994.

Heterologous desensitization of IL-8-mediated chemotaxis in human neutrophils by a cell-binding fragment of fibronectin

In this study, we have explored the mechanism for the desensitization of IL‐8‐mediated neutrophil chemotaxis by a cell‐binding fragment of fibronectin (120‐kDa FN). Preincubation of neutrophil suspensions with the 120‐kDa FN fragment resulted in a heterologous desensitization of IL‐8‐mediated chemotaxis while not affecting neutrophil chemotaxis to either fMLP or zymosan‐activated serum. Preincubation of neutrophils with the βx‐integrin‐activating antibody (TS2/16) mimicked the effects of the 120‐kDa FN fragment while preincubating neutrophils with the β1‐integrin blocking antibody (mAb13) abrogated the inhibitory effects of the 120‐kDa FN fragment on IL‐8‐mediated chemotaxis. Furthermore, we also demonstrated that the 120‐kDa FN fragment did not inhibit chemotaxis to the CXC chemokine MGSA/GROα which interacts with high affinity to the IL‐8 receptor B (CXCR2). By in vivo phosphorylation of neutrophils and probing lysates with an anti‐CXCR1 antibody, we demonstrated that the addition of the cell‐binding fragment of fibronectin resulted in a time‐dependent phosphorylation of CXCR1. These findings suggest that the mechanism of heterologous desensitization of IL‐8‐mediated chemotaxis following ligation of FN‐dependent integrins is the result of phosphorylation of the CXCR1 receptor. J. Leukoc. Biol. 65: 515–522; 1999.

Inhibition of IL-8-mediated MAPK activation in human neutrophils by beta1 integrin ligands.

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 α5β1 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 β1 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 β1 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 α6β1 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 α5β1 integrin in PMNL. Also, they suggest an interplay between integrin and chemokine signaling during PMNL migration through the extracellular matrix.

Proteolysis of gelatin-bound fibronectin by activated leukocytes: a role for leukocyte elastase.

Fragmentation of subendothelial matrix‐bound fibronectin by proteases released from stimulated leukocytes has been implicated in lung vascular injury. We studied the degradation of fibronectin bound to denatured collagen by inflammatory polymorphonuclear leukocytes (PMNL). Tissue culture wells coated with denatured collagen (gelatin) were pretreated with 125I rat plasma fibronectin to allow for fibronectin binding prior to the addition of rat inflammatory PMNL. The release of both intact and fragmented fibronectin from the 125l‐labelled artificial matrix was quantified following the addition of PMNL stimulated by the phagocytosis of opsonized zymosan as well as leukocyte elastase. Stimulated PMNL released three times more radiolabelled fibronectin from the denatured collagen surface during a 4 h incubation as compared with unstimulated PMNL. This pattern of 125I‐fibronectin release could also be elicited by the addition of purified leukocyte elastase alone, in the absence of PMNL. The release of radiolabelled fibronectin by stimulated PMNL was blocked in a dose‐dependent manner by the addition of both methoxysuccinyl‐alanine‐alanine‐valine chloromethyl ketone (AAPVCK), a leukocyte elastase specific inhibitor as well as phenylmethylsulfonylfluoride (PMSF), a non‐specific serine protease inhibitor. Western blot analysis coupled with autoradiography confirmed the presence of fibronectin fragments in the medium after addition of PMNL or leukocyte elastase. The large molecular weight fragments (60–200 kD) were not labelled, but the smaller molecular weight fragments (<45 kD), derived from the artifical matrix, were labelled. Thus, fibronectin complexed with denatured collagen is susceptible to proteolytic degradation by stimulated inflammatory PMNL. Such a process may have a role in the pathogenesis of acute vascular injury following microvascular margination of activated blood leukocytes.

Evidence for regulation of endothelial plasminogen-activating system by polymorphonuclear leukocyte elastase

Incubation of unstimulated polymorphonuclear leukocytes with cultured bovine pulmonary artery endothelial cells increased the activity of endothelial plasminogen activator. On the other hand, polymorphonuclear leukocytes stimulated by serum-opsonized zymosan decreased the plasminogen activator activity. A specific elastase inhibitor increased the enhancing effect of the unstimulated polymorphonuclear leukocytes and reversed the suppressing effect of the stimulated polymorphonuclear leukocytes. Catalytically active elastase suppressed the plasminogen activator activity and increased the activity of plasminogen activator inhibitor. In contrast, inactivated elastase enhanced the activity of plasminogen activator. Both, active and inactive forms of elastase bound to the endothelial cells. These findings suggest that elastase modulates the endothelial plasminogen-activating system, apparently by binding to the endothelial cells.

Monocyte adherence to the subendothelial basement membrane increases interleukin-8 gene expression and antigen release.

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 antigenie 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 cellderived 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.

Generation of Neutrophil Chemotactic Activity by Phorbol Ester-Stimulated Calf Pulmonary Artery Endothelial Cells

Chemotactic activity for human polymorphonuclear leukocytes (PMNL) was detected in serum‐free conditioned media 1 to 4 hr after monolayers of calf pulmonary artery endothelial cells were pretreated with phorbol myristate acetate (PMA). Chemotactic activity was increased in conditioned media following pretreatment with either PMA or the less lipophilic active phorbol ester, 4‐β‐phorbol‐12,13‐dibutyrate (P(Bu)2) in a dose‐dependent manner. Chemotactic activity of conditioned media from PMA‐treated endothelial cells was confirmed by checkerboard analysis. The chemotactic activity in conditioned media from PMA‐pretreated endothelial cells was completely inhibited by pretreating endothelial cells with either cycloheximide, actinomycin D, or the lipooxygenase inhibitor, diethylcarbamazine. Furthermore, the chemotactic activity was heat‐stable, inhibited by trypsin treatment, and present in both aqueous and lipid phases after ether extraction. The data demonstrate that pulmonary artery endothelial cells exposed to active phorbol esters release potent chemotactic factor(s) for PMNL. These findings suggest a role for activators of protein kinase C in mediating endothelial cell release of chemotactic factor(s) that may be important in the directed migration of circulating leukocytes to sites of vascular injury.

IgG-coated erythrocytes augment the lipopolysaccharidestimulated increase in serum tumor necrosis factor-α

Previous studies have shown that the injection of IgG-coated erythrocytes (EIgG) caused an increase in the mortality rate due to bacterial lipopolysaccharide (LPS). This observation led to the present evaluation of the effect of EIgG on the LPS-stimulated increase in serum tumor necrosis factor-α (TNF-α) levels and TNF-α secretion by macrophages. The prior injection of EIgG augmented the increase in LPS-stimulated serum TNF-α levels ninefold at 1 h after LPS. Serum TNF-α levels were augmented when LPS was injected 2 or 6 h after EIgG but not at 0.5 or 12 h after EIgG. Complement activation caused by EIgG may contribute to the priming for TNF-α, because activation of complement with cobra venom factor caused a threefold augmentation of the LPS-stimulated serum TNF-α levels. Isolated macrophages that had ingested EIgG or were adherent to immobilized IgG showed augmented TNF-α secretion in response to LPS. Thus clearance of immune complexes from the blood can augment the LPS-stimulated increase in serum TNF-α levels that is due, in part, to complement activation and signaling via FcγR.