Role of 5-lipoxygenase products in the local accumulation of neutrophils in dermal inflammation in the rabbit.

Studies were undertaken to define the role of 5-lipoxygenase (5-LO) products and, in particular, of leukotriene (LT) B4 in the polymorphonuclear leukocyte (PMN) emigration process using a rabbit model of dermal inflammation. Our results show that i.v. administration to rabbits of MK-0591, a compound that inhibits LT biosynthesis in blood and tissues when administered in vivo, significantly reduced 51Cr-labeled PMN accumulation in response to intradermally injected chemotactic agonists, including IL-8, FMLP, C5a, and LTB4 itself. In addition, pretreatment of the labeled PMN with MK-0591 ex vivo before their injection in recipient animals was equally effective in reducing 51Cr-labeled PMN emigration to dermal inflammatory sites. These results support a role for de novo synthesis of 5-LO metabolites by PMN for their chemotactic response to inflammatory mediators. Other studies demonstrated that elevated intravascular concentration of LTB4 interferes with PMN extravasation inasmuch as a continuous i.v. infusion of LTB4, in the range of 5-300 ng/min/kg, dose-dependently inhibited extravascular PMN accumulation to acute inflammatory skin sites elicited by the chemoattractants LTB4, FMLP, C5a, and IL-8 and by TNF-alpha, IL-1beta, and LPS; such phenomena may constitute a natural protective mechanism from massive tissue invasion by activated PMN in specific pathologic conditions such as ischemia (and reperfusion). These studies demonstrate additional functions of 5-LO products in the regulation of PMN trafficking, distinct from the well-characterized chemotactic activity of LTB4 present in the extravascular compartment.     

Endothelial and leukocyte forms of IL-8. Conversion by thrombin and interactions with neutrophils

We have recently shown that endothelial cell-derived IL-8 inhibits neutrophil adhesion to IL1-beta-activated human umbilical vein endothelial cell monolayers. IL-8 secreted by T lymphocytes or monocytes has been characterized as a promoter of neutrophil degranulation and chemotaxis. The IL-8 isolated from each of these cell types is a mixture of two IL-8 polypeptides, one consisting of 72 amino acids (herein called [ser-IL-8]72) and the other 77 amino acids (an N-terminal extended form herein called [ala-IL-8]77). IL-8 derived from T lymphocytes and monocytes is predominantly [ser-IL-8]72, whereas endothelial-derived IL-8 is highly enriched (greater than 80%) in [ala-IL-8]77. We address the relationship and activities of these two forms of IL-8 using recombinant proteins expressed by both mammalian cells and Escherichia coli. Thrombin was found to efficiently convert [ala-IL-8]77 to [ser-IL-8]72. In contrast, urokinase and tissue-type plasminogen activator were unable to cleave [ala-IL-8]77, and trypsin generated multiple IL-8 cleavage fragments. In competitive binding assays using 125I[ala-IL-8]77 neutrophils exhibited a twofold preference for [ser-IL-8]72 over [ala-IL-8]77. Both forms of IL-8 inhibited neutrophil adhesion to IL-1-beta-activated HUVEC monolayers by up to 90%. However, [ser-IL-8]72 was approximately 10-fold more potent than [ala-IL-8]77 in these assays (ED50 approximately 0.3 nM for [ser-IL-8]72 vs approximately 3 nM for [ala-IL-8]77. Both forms of IL-8 promoted degranulation of cytochalasin B-treated neutrophils [[ser-IL-8]72 (ED50 greater than 10 nM) was two- to three-fold more potent than [ala-IL-8]77], although in this regard they were less active than FMLP. Our data suggest that [ala-IL-8]77 and [ser-IL-8]72 have qualitatively similar and potentially complex biological activities, and that full activation of IL-8 requires cleavage to the [ser-IL-8]72 form. In the case of inflamed endothelial cells this activation could be mediated by thrombin generated in the procoagulant environment associated with these cells.     

Differential effects of neutrophil-activating peptide 1/IL-8 and its homologues on leukocyte adhesion and phagocytosis.

Several structural homologues of the chemotactic peptide neutrophil-activating peptide 1/IL-8 (NAP-1/IL-8) were tested for their ability to influence the expression and function of adhesion-promoting receptors on human polymorphonuclear leukocytes (PMN). NAP-2, melanoma growth stimulatory activity, and two forms of NAP-1/IL-8 (ser-NAP-1/IL-8 and ala-NAP-1/IL-8, consisting of 72 and 77 amino acids, respectively), each caused an increase in the expression of CD11b/CD18 (CR3) and CR1, which was accompanied by a decrease in the expression of leukocyte adhesion molecule-1 (LAM-1, LECAM-1). The binding activity of CD11b/CD18 was also enhanced 3- to 10-fold by these peptides, but enhanced function was transient: binding of erythrocytes coated with C3bi reached a maximum by 30 min and declined thereafter. Ser-NAP-1/IL-8, ala-NAP-1/IL-8, NAP-2, and melanoma growth stimulatory activity also caused a two- to threefold enhancement of the phagocytosis of IgG-coated erythrocytes (EIgG) by PMN without causing a large increase in the expression of Fc gamma receptors. Enhanced phagocytosis of EIgG appeared to be mediated through CD11b/CD18, because F(ab')2 fragments of an antibody directed against CD18 inhibited NAP-1/IL-8-stimulated ingestion of EIgG. The four active peptides caused a rapid, transient increase in the amount of F-actin within PMN, indicating that they are capable of influencing the structure of the microfilamentous cytoskeleton, which participates in phagocytosis. Two other NAP-1/IL-8-related peptides, platelet factor 4 and connective tissue-activating peptide III, were without effect on expression of CD11b/CD18, CR1, and LAM-1, binding activity of CD11b/CD18, or Fc-mediated phagocytosis, and increased actin polymerization only slightly. Our observations indicate that several members of the NAP-1/IL-8 family of peptides were capable of promoting integrin-mediated adhesion and Fc-mediated phagocytosis, processes important in the recruitment of PMN to sites of inflammation and antimicrobial responses of PMN.     

In vitro inhibitory effect of IL-8 and other chemoattractants on neutrophil-endothelial adhesive interactions.

We have previously reported that cytokine- or LPS-activated human umbilical vein endothelial cell (HUVEC) monolayers secrete IL-8 that can act as a neutrophil-selective adhesion inhibitor. In our study we investigated the mechanisms involved in the leukocyte adhesion inhibitory action of IL-8. The leukocyte adhesion inhibitory effect appears to be mediated by the action of IL-8 on the neutrophil, does not involve down-regulation of relevant endothelial adhesion molecules such as endothelial-leukocyte adhesion molecule-1 or intercellular adhesion molecule-1, and is quantitatively similar in different endothelial activation states that are predominantly endothelial-leukocyte adhesion molecule-1 dependent or intercellular adhesion molecule-1 dependent. In addition to inhibiting the attachment of freshly isolated peripheral blood neutrophils to cytokine-activated HUVEC monolayers, IL-8 also promoted a rapid detachment of tightly adherent neutrophils from activated HUVEC, and abolished neutrophil transendothelial migration. Certain other chemoattractants, including FMLP and C5a, had similar inhibitory actions, indicating IL-8 was not unique in its ability to inhibit various neutrophil-endothelial interactions. In contrast, two other neutrophil agonists 1-0-alkyl-2-acetyl sn-glycero-3-phosphocholine and granulocyte-macrophage-CSF, which, like IL-8, are produced by activated HUVEC, as well as the leukocyte-derived chemoattractant leukotriene B4, exerted minimal inhibitory effects on adhesion. Regardless of their ability to modulate neutrophil-endothelial cell adhesion, all these agents induced altered leukocyte surface expression of functionally important adhesion molecules, including loss of L-selectin (leukocyte adhesion molecule-1, LECAM-1) and increase in CD11b/CD18. Thus, although the above agonists have been characterized primarily as chemoattractants, our findings demonstrate that these agents can exert a wide range of modulatory effects on neutrophil-endothelial adhesive interactions.     

Differential inhibition of polymorphonuclear leukocyte recruitment in vivo by dextran sulphate and fucoidan

The selectin-mediated rolling of leukocytes along the endothelial cells is a prerequisite step followed by firm adhesion and extravasation into the inflamed tissue. This initial contact can be suppressed by sulphated polysaccharides. We have studied the effect of sulphated polysaccharides on the ultimate polymorphonuclear leukocyte (PMN) recruitment and plasma leakage in rabbit skin in response to intradermal injection of various inflammatory mediators. PMN infiltration evoked by various PMN chemoattractants (FMLP, C5a desArg, LTB(4) and IL-8) was significantly inhibited after intravenous injection of dextran sulphate (25 mg/kg), heparin (2 x 90 mg/kg) or fucoidan (1 mg/kg). PMN-dependent plasma leakage was equally well reduced by the different sulphated polymers. Vascular permeability induced by histamine or thrombin acting via a PMN-independent mechanism was not reduced. Fucoidan was the only polysaccharide able to suppress IL-1-induced PMN infiltration for 60-70%. Local administration of dextran sulphate had no effect on PMN-dependent plasma leakage. Differential inhibition of PMN recruitment was determined after injection of dextran sulphate or fucoidan depending on the type of insult. Therefore, these results suggest that different adhesion pathways are utilized during PMN recruitment in vivo in response to chemoattractants and IL-1.     

Tumor necrosis factor combines with IL-4 or IFN-gamma to selectively enhance endothelial cell adhesiveness for T cells. The contribution of vascular cell adhesion molecule-1-dependent and -independent binding mechanisms.

The adhesion of lymphocytes to vascular endothelium is the first step in their passage from the blood into inflammatory tissues. By modulating endothelial cell (EC) adhesiveness for lymphocytes, cytokines may regulate lymphocyte accumulation and hence the nature and progression of inflammatory responses. IL-1, TNF, IFN-gamma, and IL-4 each increase EC adhesiveness for T cells when used alone in adhesion assays in vitro. As cytokines are more likely to act in combination at sites of inflammation in vivo, we have studied the stimulating effect of different combinations of cytokines on EC adhesiveness for T cells and polymorphonuclear leukocytes (PMN). Acting alone IL-1, TNF, IFN-gamma, and IL-4 each significantly enhanced EC adhesiveness for T cells (p less than 0.005), whereas only IL-1 (p less than 0.005) and TNF (p less than 0.005) but not IFN-gamma or IL-4 significantly enhanced adhesiveness for PMN. When EC were stimulated with optimal concentrations of TNF in combination with IL-4 or IFN-gamma, there was a significant further increase in adhesiveness for T cells (p less than 0.003), but not PMN, over that seen with TNF alone. The additive effect of TNF and IL-4 was more marked than that of TNF and IFN-gamma. Although approximately equal proportions of T cells and PMN bound to TNF-stimulated EC, nearly double the proportion of T cells compared with PMN bound EC preincubated with TNF and IL-4 together. A similar interaction with IL-4 or IFN-gamma was exhibited by lymphotoxin. mAb-inhibition studies indicated that the extra increase in binding caused by stimulating EC with TNF and IL-4 in combination was mediated by VCAM-1 whereas that caused by stimulating with TNF and IFN-gamma in combination was substantially mediated through leukocyte function-associated Ag-1- and VCAM-1-independent mechanisms. These observations suggest that whereas IL-1 and TNF alone are unselective in terms of leukocyte adhesion to EC, the combination of TNF (or LT) with IL-4 or IFN-gamma may be of key importance in determining the recruitment of a lymphocyte-predominant infiltrate in immune mediated inflammation, and in initiating the transition from acute to chronic inflammation.     

Emigration and accumulation of PMN-leukocytes induced by endotoxin, interleukin 1 and other chemotactic substances

This publication describes polymorphonuclear leukocyte (PMN) emigration and accumulation, which is prerequisite for their defensive function in infected tissues. The extravasated PMNs can kill microorganisms, but in this process they also release proteolytic enzymes and other cell constituents which can alter and even injure the tissues, primarily the microcirculation. In the first part of the paper in vivo quantitation of the acute inflammatory reaction is described with emphasis on PMN emigration and accumulation. With 51Cr-labeled PMNs the kinetics of their emigration induced by a number of chemotaxins and chemotaxinigens was found to be similar, peaking in 1-4 hour old lesions and returning to baseline values thereafter. The most potent substance tested was endotoxin, which induced a PMN influx at a molar concentration a least 3 orders of magnitude lower than the other substances tested, implying the these substances are not the primary endogenous mediators of endotoxin induced inflammation. Next we describe an observation which shed considerable light on the mechanisms underlying PMN emigration. When a chemotaxin or endotoxin was injected intradermally and after varying periods of time reinjected into the same site, the PMN influx into those sites was diminished, compared to sites not previously injected, i. e. injected for the first time. This tachyphylaxis or diminished responsiveness was attributed to a downregulation of receptors, presumably on endothelial cells, coupled to a facilitatory mechanism. Other mechanism proposed to terminate emigration of PMNs during inflammatory reaction were unlikely, based on our experimental findings. Endotoxin is not chemotactic in vitro but it induces PMN emigration when injected intradermally. Hence the third part of the publication deals with PMN emigration induced by interleukin 1 and its significance for endotoxin-induced inflammation. IL 1 is the only chemotaxin which induces PMN accumulation at a concentration comparable to that of endotoxin and considerably lower than the other chemotaxins. There was cross tachyphylaxis between endotoxin and IL 1 and vice versa. The PMN influx into IL 1 sites injected 6 hours earlier with IL 1 or with endotoxin was diminished compared to IL 1 sites injected into normal skin. Sites injected first with IL 1 and then with a low dose of endotoxin also exhibited cross tachyphylaxis. FMLP or LTB4 injected into sites pretreated with endotoxin did not exhibit cross tachyphylaxis, i. e. the PMN influx was similar to sites injected for the first time with these chemotaxins.(ABSTRACT TRUNCATED AT 400 WORDS)     

Rapid accumulation of polymorphonuclear neutrophils in the Corpus luteum during prostaglandin F(2α)-induced luteolysis in the cow

Prostaglandin F(2α) (PGF(2α)) induces luteolysis within a few days in cows, and immune cells increase in number in the regressing corpus luteum (CL), implying that luteolysis is an inflammatory-like immune response. We investigated the rapid change in polymorphonuclear neutrophil (PMN) numbers in response to PGF(2α) administration as the first cells recruited to inflammatory sites, together with mRNA of interleukin-8 (IL-8: neutrophil chemoattractant) and P-selectin (leukocyte adhesion molecule) in the bovine CL. CLs were collected by ovariectomy at various times after PGF(2α) injection. The number of PMNs was increased at 5 min after PGF(2α) administration, whereas IL-8 and P-selectin mRNA increased at 30 min and 2 h, respectively. PGF(2α) directly stimulated P-selectin protein expression at 5-30 min in luteal endothelial cells (LECs). Moreover, PGF(2α) enhanced PMN adhesion to LECs, and this enhancement by PGF(2α) was inhibited by anti-P-selectin antibody, suggesting that P-selectin expression by PGF(2α) is crucial in PMN migration. In conclusion, PGF(2α) rapidly induces the accumulation of PMNs into the bovine CL at 5 min and enhances PMN adhesion via P-selectin expression in LECs. It is suggested that luteolytic cascade by PGF(2α) may involve an acute inflammatory-like response due to rapidly infiltrated PMNs.     

A role for beta2 integrin (CD11/CD18)-mediated tyrosine signaling in extravasation of human polymorphonuclear neutrophils

During the recruitment of human polymorphonuclear neutrophils (PMN) to sites of inflammation, leukocyte adhesion molecules of the beta2 integrin (CD11/CD18) family mediate firm adhesion of these cells to the endothelial cell monolayer lining the vessel wall. This process is a prerequisite for shape change and spreading of PMN on the endothelium which eventually allows PMN emigration into the extravascular space. In order to elucidate the molecular mechanisms which mediate this sequence of events, intracellular protein tyrosine signaling was studied subsequent to beta2 integrin-mediated ligand binding. Using western blotting technique, beta2 integrin-mediated adhesion was found to induce tyrosine phosphorylation of different proteins. The effect was absent in PMN derived from CD18-deficient mice which lack any beta2 integrin expression on the cell surface demonstrating the specificity of the observed response. Inhibition of beta2 integrin-mediated tyrosine signaling by herbimycin A almost completely inhibited adhesion, shape change, and subsequent spreading of PMN. Herbimycin A also diminished chemotactic migration of these cells in response to the soluble mediator N-formyl-Met-Leu-Phe (fMLP). In contrast, treatment of PMN with cytochalasin D had no substantial effect on beta2 integrin-mediated signaling or adhesion but inhibited shape change, spreading, and chemotactic migration of PMN. This suggests that the signaling capacity exerted by beta2 integrins upon ligand binding was independent of an intact cytoskeleton. Moreover, the beta2 integrin-mediated activation of intracellular signal transduction pathways was critical for firm adhesion of PMN, the prerequisite subsequent shape change and spreading, which allows emigration of PMN into the extravascular space.     

Effects of human neutrophil chemotaxis across human endothelial cell monolayers on the permeability of these monolayers to ions and macromolecules

We have developed a method for studying the permeability properties of human endothelia in vitro. Human umbilical vein endothelial cells (HUVEC) were cultured on a substrate of human amnion. Confluent monolayers of these cells demonstrated 6-12 delta.cm2 of electrical resistance (a measure of their permeability to ions) and restricted the transendothelial passage of albumin from their apical to their basal surface. To determine whether leukocyte emigration alters endothelial permeability in this model, we examined the effects of migrating human polymorphonuclear leukocytes (PMN) on these two parameters. Few PMN migrated across the HUVEC monolayers in the absence of chemoattractants. In response to chemoattractants, PMN migration through HUVEC monolayers was virtually complete within 10 minutes and occurred at random locations throughout the monolayer. PMN migrated across the monolayer via the paracellular pathway. Although one PMN migrated across the monolayer for each HUVEC, PMN migration induced no change in electrical resistance or albumin permeability of these monolayers. At this PMN:HUVEC ratio, these permeability findings were correlated morphologically to measurements that HUVEC paracellular pathway size increases by less than 0.22% with PMN migration. This increase is insufficient to effect a measurable change in the electrical resistance of the endothelial cell monolayer. These findings demonstrate that increased permeability of cultured endothelial cell monolayers is not a necessary consequence of PMN emigration.     

Effects of intravenous IL-8 administration in nonhuman primates.

IL-8, a cytokine known for its potent and specific neutrophil activation and chemoattractant properties, has been recently detected in the circulation during septic shock, endotoxemia, and after IL-1 alpha administration. Because of its observed in vitro actions, it has been hypothesized that IL-8 may contribute to the dynamics of circulating granulocytes and to the pathologic sequelae seen in sepsis. Here, human rIL-8 is administered to healthy nonhuman primates as a single i.v. injection or as a continuous 8-h i.v. infusion. We demonstrate that both methods of i.v. administration result in a rapid but transient, severe granulocytopenia, followed by a granulocytosis that persists as long as IL-8 levels are detectable in the circulation. There were no hemodynamic changes after IL-8 administration, and animals remained clinically stable during the 24-h observation period. No detectable circulating TNF-alpha, IL-1 beta, or IL-6 response was induced by either IL-8 administration regimen. Histopathologic examination revealed mild to moderate neutrophilic margination in lung, liver, and spleen, of greater severity in baboons receiving the 8-h infusion. There was no associated neutrophilic infiltration or tissue injury. Thus, IL-8 modulates circulating granulocyte dynamics and likely directs their actions, but when administered i.v. to healthy animals, either as a bolus dose or as a continuous infusion for up to 8 h, does not induce the hemodynamic and metabolic aberrations or the acute organ damage seen during sepsis.     

Blockade of alpha 5 beta 1 integrins reverses the inhibitory effect of tenascin on chemotaxis of human monocytes and polymorphonuclear leukocytes through three-dimensional gels of extracellular matrix proteins

Tenascin is an extracellular matrix protein found in adults in T cell-dependent areas of lymphoid tissues, sites of inflammation, and tumors. We report here that it inhibited chemotaxis of chemoattractant-stimulated human monocytes and chemoattractant-stimulated polymorphonuclear leukocytes (PMN) through three-dimensional gels composed of collagen I or Matrigel, and chemotaxis of leukotriene B4-stimulated PMN through fibrin gels. The inhibitory effect of tenascin on monocyte or PMN chemotaxis through these matrices was reversed by Abs directed against alpha5beta1 integrins or by a peptide (GRGDSP) that binds to beta1 integrins. Tenascin did not affect leukotriene B4- or fMLP-stimulated expression of beta1 or beta2 integrins, but did exert a small inhibitory effect on PMN adhesion and closeness of apposition to fibrin(ogen)-containing surfaces. Thus, alpha5beta1 integrins mediate the inhibitory effect of tenascin on monocyte and PMN chemotaxis, without promoting close apposition between these leukocytes and surfaces coated with tenascin alone or with tenascin bound to other matrix proteins. This contrasts with the role played by alpha5beta1 integrins in promoting close apposition between fMLP-stimulated PMN and fibrin containing surfaces, thereby inhibiting chemotaxis of fMLP-stimulated PMN through fibrin gels. Thus, chemoattractants and matrix proteins regulate chemotaxis of phagocytic leukocytes by at least two different mechanisms: one in which specific chemoattractants promote very tight adhesion of leukocytes to specific matrix proteins and another in which specific matrix proteins signal cessation of migration without markedly affecting strength of leukocyte adhesion.     

CD18-dependent and -independent mechanisms of neutrophil emigration in the pulmonary and systemic microcirculation of rabbits

Neutrophil (PMN) migration in the systemic and pulmonary circulation of rabbits was compared by using different inflammatory stimuli to determine the role of the leukocyte adhesion complex, CD11/CD18, in each of these vascular beds. The adhesion complex was blocked by administering the anti-CD18 mAb 60.3. The data show that mAb 60.3 blocks PMN emigration into inflammatory foci in the abdominal wall produced by implanting sponges containing either hydrochloric acid, Streptococcus pneumoniae, Escherichia coli endotoxin, or PMA. mAb 60.3 also inhibited PMN emigration in response to peritoneal instillation of S. pneumoniae. The effect of mAb 60.3 on PMN emigration in the lungs varied depending upon the stimulus. PMN failed to migrate into the PMA-induced pneumonia; however, mAb 60.3 pretreatment only partially inhibited endotoxin-induced pneumonia and did not inhibit S. pneumoniae or hydrochloric acid-induced pneumonias. PMN lavaged from the alveolar spaces in the Streptococcal pneumonia had similar quantities of mAb 60.3 bound to their surfaces as the circulating PMN. We conclude that the CD11/CD18 complex mediates PMN adherence in the systemic circulation. However, PMN adherence in the pulmonary circulation may occur by either CD18-dependent or -independent mechanisms that are specific to the inciting stimulus.     

S100 protein CP-10 stimulates myeloid cell chemotaxis without activation

Leukocyte recruitment to inflammatory foci is generally associated with cellular activation. Recent evidence suggests that chemotactic agents can be divided into two classes, “classical chemoattractants” such as FMLP, C5a, and IL-8, which stimulate directed migration and activation events and “pure chemoattractants” such as TGF-β1 which influence actin polymerisation and movement but not oxidative burst and associated granular enzyme release. The studies reported here demonstrate that the murine S100 chemoattractant protein, CP-10, belongs to the “non-classical” group. Despite its potent chemotactic activity for neutrophils and monocytes/macrophages, CP-10 failed to increase [Ca²⁺]_i in human or mouse PMN, although chemotaxis was inhibited by pertussis toxin, confirming the suggestion of a novel Ca²⁺-independent G-protein-coupled pathway for post-receptor signal transduction triggered by “pure chemoattractants.” The co-ordinated up-regulation of Mac-1 and down-regulation of L-selectin induced by FMLP on human PMN in vitro was not observed with CP-10. Quantitative changes in immediate (30 s) actin polymerisation occurred with FMLP and CP-10-treated human PMN. The relative F-actin increases induced in WEHI 265 monocytoid cells by FMLP and CP-10 was optimal at 60 s and declined over 120 s. F-actin changes reflected the concentration and potencies of the agonists required to provoke chemotaxis. After 90 min, CP-10 profoundly altered cell shape and increased both cell size and F-actin within pseudopodia. These changes are typical of those mediating leukocyte deformability, and CP-10 may mediate leukocyte retention within microcapillaries and thereby contribute to the initiation of inflammation in vascular beds. © 1996 Wiley-Liss, Inc.     

Transgenic mice expressing different levels of soluble platelet/endothelial cell adhesion molecule-IgG display distinct inflammatory phenotypes.

Platelet/endothelial cell adhesion molecule-1 (PECAM-1, CD31), expressed on the surfaces of leukocytes and concentrated in the junctions between endothelial cells plays an important role in transendothelial migration of neutrophils and monocytes. Soluble recombinant PECAM-IgG injected i.v. into mice blocks acute leukocyte emigration by 80%. To study the role of PECAM in models of chronic inflammation, we generated transgenic mice constitutively expressing soluble full-length murine PECAM as an IgG chimera. Three founder lines expressed this transgene and constitutively secreted murine PECAM-IgG into the plasma where it was maintained at characteristic concentrations for each line. All mice had similar hematologic profiles to wild-type littermates and were healthy when maintained in the standard laboratory animal facility. Both the leukocytes and the endothelium of mice of all transgenic lines expressed the same levels of endogenous PECAM-1 as wild-type littermates. Similarly, there were no detectable differences in the expression of several other common leukocyte and endothelial cell adhesion molecules. Mice that produced moderate (10-20 microg/ml) concentrations of PECAM-IgG demonstrated a severely blunted acute inflammatory response, despite mobilizing appropriate numbers of circulating leukocytes. Surprisingly, mice that constitutively produced high (400-1,000 microg/ml) concentrations of PECAM-IgG were unresponsive to its anti-inflammatory effects. This is the first demonstration that a soluble form of a cell adhesion molecule can be stably expressed and retain efficacy in vivo over prolonged periods. This approach is applicable to many other extracellular molecules. However, the plasma concentrations of such constitutively produced inhibitors may greatly influence the resulting phenotype.     

Platelets enhance neutrophil transendothelial migration via P-selectin glycoprotein ligand-1

Platelets are increasingly recognized as important for inflammation in addition to thrombosis. Platelets promote the adhesion of neutrophils [polymorphonuclear neutrophils (PMNs)] to the endothelium; P-selectin and P-selectin glycoprotein ligand (PSGL)-1 have been suggested to participate in these interactions. Whether platelets also promote PMN transmigration across the endothelium is less clear. We tested the hypothesis that platelets enhance PMN transmigration across the inflamed endothelium and that PSGL-1 is involved. We studied the effects of platelets on PMN transmigration in vivo and in vitro using a well-characterized corneal injury model in C57BL/6 mice and IL-1β-stimulated human umbilical vein endothelial cells (HUVECs) under static and dynamic conditions. In vivo, platelet depletion altered PMN emigration from limbal microvessels after injury, with decreased emigration 6 and 12 h after injury. Both PSGL-1-/- and P-selectin-/- mice, but not Mac-1-/- mice, also had reduced PMN emigration at 12 h after injury relative to wild-type control mice. In the in vitro HUVEC model, platelets enhanced PMN transendothelial migration under static and dynamic conditions independent of firm adhesion. Anti-PSGL-1 antibodies markedly inhibited platelet-PMN aggregates, as assessed by flow cytometry, and attenuated the effect of platelets on PMN transmigration under static conditions without affecting firm adhesion. These data support the notion that platelets enhance neutrophil transmigration across the inflamed endothelium both in vivo and in vitro, via a PSGL-1-dependent mechanism.     

A novel inflammatory pathway involved in leukocyte recruitment: role for the kinin B1 receptor and the chemokine CXCL5

The kinin B1 receptor is an inducible receptor not normally expressed but induced by inflammatory stimuli and plays a major role in neutrophil recruitment, particularly in response to the cytokine IL-1beta. However, the exact mechanism involved in this response is unclear. The aim of this study was to dissect the molecular mechanism involved, in particular to determine whether specific ELR-CXCL chemokines (specific neutrophil chemoattractants) played a role. Using intravital microscopy, we demonstrated that IL-1beta-induced leukocyte rolling, adherence, and emigration in mesenteric venules of wild-type (WT) mice, associated with an increase in B1 receptor mRNA expression, were substantially attenuated (>80%) in B1 receptor knockout mice (B1KO). This effect in B1KO mice was correlated with a selective down-regulation of IL-1beta-induced CXCL5 mRNA and protein expression compared with WT mice. Furthermore a selective neutralizing CXCL5 Ab caused profound suppression of leukocyte emigration in IL-1beta-treated WT mice. Finally, treatment of human endothelial cells with IL-1beta enhanced mRNA expression of the B1 receptor and the human (h) CXCL5 homologues (hCXCL5 and hCXCL6). This response was suppressed by approximately 50% when cells were pretreated with the B1 receptor antagonist des-Arg9-[Leu8]-bradykinin while treatment with des-Arg9-bradykinin, the B1 receptor agonist, caused a concentration-dependent increase in hCXCL5 and hCXCL6 mRNA expression. This study unveils a proinflammatory pathway centered on kinin B1 receptor activation of CXCL5 leading to leukocyte trafficking and highlights the B1 receptor as a potential target in the therapeutics of inflammatory disease.     

Stroma cell-derived factor 1alpha mediates desensitization of human neutrophil respiratory burst in synovial fluid from rheumatoid arthritic patients

Classical chemoattractants such as fMLP or the complement factor C5a use G protein (Gi)-coupled receptors to stimulate both chemotaxis and production of reactive oxygen species (respiratory burst, RB) by polymorphonuclear leukocytes (PMN). The chemokine stroma cell-derived factor 1alpha (SDF1alpha) and its Gi-coupled receptor, CXCR4, regulate leukocyte trafficking and recruitment to the synovial fluid of rheumatoid arthritic patients (RA-SF). However, the role of SDF1alpha in the RB is unknown and was studied in this work in vitro with healthy PMN in the absence and presence of RA-SF. In healthy PMN, SDF1alpha failed to stimulate the RB, even though the p38 mitogen-activated protein kinase was activated to a similar level as in fMLP-stimulated PMN. In contrast, the SDF1alpha-mediated calcium transients and activation of phosphatidylinositol 3-kinase/Akt were partially deficient, while p44/42 mitogen-activated protein kinases were not activated. SDF1alpha actually desensitized weakly the fMLP-mediated RB of healthy PMN. This cross-inhibitory effect was amplified in PMN treated with RA-SF, providing a protection against the exacerbation of RB induced by C5a or fMLP. This SDF1alpha beneficial effect, which was prevented by the CXCR4 antagonist AMD3100, was associated with impairment of C5a- and fMLP-mediated early signaling events. Thus, although SDF1alpha promotes leukocyte emigration into rheumatoid synovium, our data suggest it cross-desensitizes the production of oxidant by primed PMN, a property that may be beneficial in the context of arthritis.     

Abundant expression of chemokines in malignant and infective human lymphadenopathies.

Lymph nodes can be the primary target of infection or malignant transformation and may exhibit characteristic patterns of leukocyte infiltration analogous to those seen in inflammation of other tissues. Leukocyte migration to lymph nodes in vivo is a highly regulated, multi-step process that depends upon adhesion molecules and as yet, uncharacterized chemotactic signals. Chemokines are a key part of the orchestrated code of signals that directs leukocyte subsets to sites of inflammation or immune response. The potential role of these chemoattractants in selective trafficking of leukocyte subsets into lymph nodes was assessed by determining the expression of chemokines on a range of pathological and normal human lymph nodes and by evaluating the cellular composition of each lymph node. In situ hybridization using chemokine riboprobes and immunohistochemistry using specific antibodies were performed in order to correlate the mRNA and protein expression of the chemokines. The cellular source(s) of each chemokine was assessed by immunohistochemical staining of adjacent sections using antibodies directed against distinctive cellular markers. Substantial, but varied, expression of macrophage inflammatory protein (MIP)-1alpha, MIP-1beta, RANTES, macrophage chemotactic protein (MCP)-1, eotaxin, and interleukin 8 (IL-8) were detected in the pathological lymph nodes by diverse cell types. Control lymph nodes showed expression only of RANTES, mainly by high endothelial venules. In all lymph nodes, except the nodes infiltrated with breast cancer, chemokine mRNA expression was highly concordant with the corresponding protein. In contrast with in vitro studies that have suggested discrete target cell specificity of chemokines, this study showed that with the possible exception of the neutrophil chemoattractant, IL-8, no chemokine appeared to be uniquely associated with the accumulation of a specific leukocyte subset. These data implicate chemokines in the recruitment of leukocytes to lymph nodes affected by diverse disease states.     

Indirect enhancement of neutrophil activity and adhesion to cultured human umbilical vein endothelial cells by isoprostanes (iPF2alpha-III and iPE2-III).

Isoprostanes are metabolites of arachidonic acid found in blood under various conditions of oxidative stress. Because arachidonic acid derivatives are major mediators of inflammation, we investigated the potential inflammatory effects of iPF2alpha-III (previously 8-isoPGF2alpha) and iPE2-III (8-isoPGE2) on human polymorphonuclear granulocytes (PMN), as well as on human umbilical vein endothelial cells (HUVECs). The early activation marker CD11b on PMN and the adhesion molecules ICAM-1, E-selectin, and P-selectin on HUVECs were quantified by flow cytometry. Levels of the cytokines interleukin (IL)-6 and IL-8 were measured in the culture supernatant by enzyme-linked immunosorbent assay. Furthermore, adhesion of PMN to HUVECs was assessed. Neither isoprostane showed any direct stimulatory effects on PMN or HUVECs at concentrations of 0.1 or 1 microM: there was no acute elevation in expression of CD11b or P-selectin and no change of ICAM-1 or E-selectin after 4 or 24 h of incubation, respectively. The levels of interleukin IL-6 and IL-8 were also unaltered. However, PMN adhesion was significantly enhanced both after 4 and 24 h of incubation of HUVECs with iPF2alpha-III, and CD11b expression on PMN was elevated by contact of these cells with the supernatant of pre-exposed HUVECs. Neither of these actions were inhibited by an endothelin receptor antagonist (bosentan) or a combined thromboxane A2/isoprostane-receptor antagonist (SQ29548). Thus, although not having a direct pro-inflammatory potential, isoprostanes might indirectly accentuate PMN stimulation. This seems to occur via a receptor-independent mechanism, perhaps the production of an active metabolite of isoprostanes by endothelial cells.