Methods for analysis of matrix metalloproteinase regulation of neutrophil-endothelial cell adhesion

Recent evidence indicates novel role for matrix metalloproteinases (MMPs), in particular gelatinase A (MMP-2), in the regulation of vascular biology that are unrelated to their well-known proteolytic breakdown of matrix proteins. We have previously reported that MMP-2 can modulate vascular reactivity by cleavage of the Gly32-Leu33 bound in big endothelin-1 (ET-1) yielding a novel vasoactive peptide ET-1[1–32]. These studies were conducted to investigate whether gelatinolytic MMPs could affect neutrophil-endothelial cell attachment. ET-1[1–32] produced by MMP-2 up-regulated CD11b/CD18 expression on human neutrophils, thereby promoted their adhesion to cultured endothelial cells. ET-1[1–32] evoked release of gelatinase B (MMP-9), which in turn cleaved big ET-1 to yield ET-1[1–32], thus revealing a self-amplifying loop for ET-1[1–32] generation. ET-1[1–32] was rather resistant to cleavage by neutrophil proteases and further metabolism of ET-1[1–32] was not a prerequisite for its biological actions on neutrophils. The neutrophil responses to ET-1[1–32] were mediated via activation of ETA receptors through activation of the Ras/Raf-1/MEK/ERK signaling pathway. These results suggest a novel role for gelatinase A and B in the regulation of neutrophil functions and their interactions with endothelial cells. Here we describe the methods in detail as they relate to our previously published work. Published: October 28, 2002     

Leukocyte inhibitory factor (LIF) potentiates neutrophil responses to formyl-methionyl-leucyl-phenylalanine

The ability of purified (80,000-fold) human leukocyte inhibitory factor (LIF) to modulate several formyl-methionyl-leucyl-phenylalanine (f-met-leu-phe)-induced neutrophil functions was evaluated. Although not affecting directed migration itself, at low concentrations (1/2 to 2 U/ml), LIF was demonstrated to potentiate chemotaxis induced by f-met-leu-phe (40.3% +/- 8.1) and to reduce the concentration of f-met-leu-phe necessary for maximal chemotaxis (10(-8) to 10(-9) M). Similarly, LIF did not directly induce the respiratory burst, but potentiated both superoxide generation (151.6% +/- 77) and hydrogen peroxide production (54.9% +/- 15.5) in the presence of f-met-leu-phe (10(-7) M). LIF was also shown to induce degranulation of neutrophil-specific granules in a dose-dependent manner. Neutrophil-specific granules have been shown to contain an intracellular pool of receptors for f-met-leu-phe, and on degranulation provide the surface membrane with a fresh source of receptors. Our data suggested that LIF potentiation of neutrophil stimulation by f-met-leu-phe might be mediated, at least in part, by increasing the number of available membrane receptors as a result of its ability to induce degranulation. Radioligand receptor analysis using f-met-leu-[3H] phe was performed, and LIF was shown to mediate an increase in receptors for f-met-leu-phe from an average of 18,600 on untreated cells to 27,000 after pretreatment with LIF. This increase in receptors could "sensitize" the neutrophils for f-met-leu-phe and possibly explain the potentiation of neutrophil stimulation observed in the presence of the ligand. LIF was also found to have a more generalized effect on the transduction of neutrophil activation stimuli, mediating a 35.8% increase in superoxide production after exposure to calcium ionophore. The data do not permit a determination as to whether the increase in receptor number is responsible for the potentiation of f-met-leu-phe-mediated function, or whether this occurs secondary to the more generalized effect on neutrophil stimulation transduction.     

Leukocyte adhesion deficiency type II

Leukocyte adhesion deficiency type II (LAD II) is a rare disorder characterized by recurrent infections, persistent leukocytosis, and severe mental and growth retardation. LAD II neutrophils are deficient in expression of selectin ligand activity, and exhibit a correspondingly diminished ability to roll on endothelium and to traffic to inflammatory sites in vivo. LAD II patients exhibit a deficiency in the expression of cell surface fucosylated glycan structures that include the H and Lewis blood group determinants and the sialyl Lewis x epitope, yet the corresponding fucosyltransferase activities responsible for synthesis of these structures are expressed at normal levels. The molecular defect in LAD II has been localized to the pathway that synthesizes GDP-fucose from GDP-mannose. However, the two known component enzymes in this GDP-fucose biosynthetic pathway are normal in sequence and in expression levels in LAD II cells. The genetic lesion in LAD II that accounts for the generalized fucosylation defect in LAD II patients remains to be determined.     

Loss of pentameric symmetry of C-reactive protein is associated with promotion of neutrophil-endothelial cell adhesion.

The classic acute-phase reactant C-reactive protein (CRP) is a cyclic pentameric protein that diminishes neutrophil accumulation in inflamed tissues. When the pentamer is dissociated, CRP subunits undergo conformational rearrangement that results in expression of a distinctive isomer with unique antigenic and physicochemical characteristics (termed modified CRP (mCRP)). Recently, mCRP was detected in the wall of normal human blood vessels. We studied the impact and mechanisms of action of mCRP on expression of adhesion molecules on human neutrophils and their adhesion to human coronary artery endothelial cells. Both CRP and mCRP (0.1-200 microg/ml) down-regulated neutrophil L-selectin expression in a concentration-dependent fashion. Furthermore, mCRP, but not CRP, up-regulated CD11b/CD18 expression and stimulated neutrophil extracellular signal-regulated kinase activity, which was accompanied by activation of p21(ras) oncoprotein, Raf-1, and mitogen-activated protein kinase kinase. These actions of mCRP were sensitive to the mitogen-activated protein kinase kinase inhibitor PD98059. mCRP markedly enhanced attachment of neutrophils to LPS-activated human coronary artery endothelial when added together with neutrophils. This effect of mCRP was attenuated by an anti-CD18 mAb. Thus, loss of pentameric symmetry in CRP is associated with appearance of novel bioactivities in mCRP that enhance neutrophil localization and activation at inflamed or injured vascular sites.     

Leukocyte adhesion to endothelial cells

The adhesion of leukocytes to endothelium is a physiological phenomenon which is the first step for leukocyte emigration. The adhesion can be dramatically increased in pathological situations such as inflammation and vascular diseases. The molecular basis of leukocyte-endothelium interaction has been largely investigated in the last ten years. Using monoclonal antibodies it is possible to characterize the leukocyte adhesion molecule (LeuCAM) also named CD11/CD18 complex. These molecules responsible for leukocyte adhesion are heterodimers consisting of a common beta subunit and different subunit CD11a/CD18 corresponding to LFA-1; CD11b/CD18 to Mac1/Mol; CD11c/CD18 to GP150-95. Beside these receptors, other leukocyte structures such as the fibronectin receptors are involved in the adhesive process. On the endothelial cell side specialized structures implicated in leukocyte adhesion have been identified. Structures like Intercellular Adhesion Molecule (ICAM) are expressed on endothelial cells in the absence of stimulation, while other receptors Endothelial Leukocyte Adhesion Molecule (ELAM) are only detectable on activated endothelial cells. Cytokines such as IL-1 induced the expression of ELAM, increased the number of ICAM and Human Leukocyte Antigens (HLA) DR, DP, DQ. In various pathological circumstances, namely extracorporeal circulation, Acute Respiratory Distress Syndrome (ARDS), hypercholesterolemia and diabetes mellitus increased leukocyte adhesion has been reported and is potentially responsible for vascular damage. Therefore, the modulation of leukocyte-endothelial cell interactions is a possible target for antithrombotic and antiatherosclerotic therapy.     

Leukocyte adhesion and microvessel permeability

To investigate the direct effect of leukocyte adherence to microvessel walls on microvessel permeability, we developed a method to measure changes in hydraulic conductivity (L(p)) before and after leukocyte adhesion in individually perfused venular microvessels in frog mesentery. In 19 microvessels that were initially free of leukocyte sticking or rolling along the vessel wall, control L(p) was measured first with Ringer-albumin perfusate. Blood flow was then restored in each vessel with a reduced flow rate in the range of 30-116 microm/s to facilitate leukocyte adhesion. Each vessel was recannulated in 45 min. The mean number of leukocytes adhering to the vessel wall was 237 +/- 22 leukocytes/mm(2). At the same time, L(p) increased to 4.7 +/- 0.5 times the control value. Superfusion of isoproterenol (10 microM) after leukocyte adhesion brought the increased L(p) back to 1.1 +/- 0.2 times the control in 5-10 min (n = 9). Superfusing isoproterenol before leukocyte adhesion prevented the increase in L(p) (n = 6). However, the number of leukocytes adhering to the vessel wall was not significantly affected. These results demonstrated that leukocyte adhesion caused an increase in microvessel permeability that could be prevented or restored by increasing cAMP levels in endothelial cells using isoproterenol. Thus cAMP-dependent mechanisms that regulate inflammatory agent-induced increases in permeability also modulate leukocyte adhesion-induced increases in permeability but act independently of mechanisms that regulate leukocyte adhesion to the microvessel wall. Application of ketotifen, a mast cell stabilizer, and desferrioxamine mesylate, an iron-chelating reagent, attenuated the increase in L(p) induced by leukocyte adhesion, suggesting the involvement of oxidants and the activation of mast cells in leukocyte adhesion-induced permeability increase. Furthermore, with the use of an in vivo silver stain technique, the locations of the adherent leukocytes on the microvessel wall were identified quantitatively in intact microvessels.     

Hepatocyte growth factor decreases sensitivity to chemotherapeutic agents and stimulates cell adhesion, invasion, and migration

Hepatocyte growth factor (HGF), also known as scatter factor (SF), plays an important role in cell:cell adhesion, cell proliferation, motility, and invasiveness of epithelial cells and tumor cells. In this study, we examined the effects of HGF on these types of biological activities and chemosensitivity in Chinese hamster ovary (CHO) cells by stable transfection of the HGF gene. HGF-transfected clones produced very high titers of HGF protein, whereas control vector-transfected clones did not produce detectable HGF protein. HGF-transfected clones showed modestly increased proliferation rates and became more resistant to cell death and apoptosis caused by two anticancer drugs, adriamycin (ADR) and camptothecin (CPT), compared to controlvector-transfected clones. Furthermore, HGF-transfected clones also exhibited increased activities of cell adhesion, migration, and invasion. The current study is the first demonstration that overexpression of the HGF gene affects chemosensitivity and cell metastasis behaviors, suggesting that HGF signaling pathway is a promising new target of therapeutic intervention of tumors.     

Leukocyte migration inhibitory factor (LMIF) production in unidirectional mixed lymphocyte cultures.

Puromycin treatment of lymphocytes was used to develop a one-way test for leukocyte migration inhibitory factor (LMIF) production in the mixed lymphocyte culture (MLC) reaction. Lymphocytes incubated with this protein synthesis inhibitor induced a vigorous mediator production by nontreated allogeneic cells, being themselves unable to respond to stimulator cells. When puromycin-treated cells were stimulated with the mitogens PHA, ConA, or PWM, overall protein and DNA synthesis were significantly decreased with concomitant abolishment of LMIF production. Viability of stimulator lymphocytes was found to be essential for generation of the mediator in MLC reaction.     

Leukocyte migration in guinea pigs. II. Partial characterization of a leukocyte migration inhibitory factor distinct from macrophage migration inhibitory factor.

In this study we present data on the partial biological and biochemical characterization of guinea pig leukocyte migration inhibition factor (LIF) and migration inhibition factor (MIF). The results indicate that guinea pig LIF and MIF are distinct mediators of cellular immunity, in terms of indicator cells affected and molecular weight. This is in agreement with previous reports showing distinctions between human LIF and MIF. Partial characterization of guinea pig LIF suggested that it is a heat-stable protein of molecular weight 68,000–158,000 and does not contain terminal sialic acid groups.     

Leukocyte subpopulations in the uteri of leukemia inhibitory factor knockout mice during early pregnancy

Leukemia inhibitory factor (LIF) is transiently expressed on Day (D) 1 of pregnancy by the uterine epithelium and on D4 specifically by the glandular epithelium. The Lif knockout female mice are infertile because of uterine defects that affect embryo implantation, but pregnancy can be rescued in these mice by injections of LIF on D4 of pregnancy. Many of the specific actions of LIF in the uterus are unknown, especially with regard to uterine cell biology. Leukocytes, such as macrophages, natural killer (NK) cells, and eosinophils, are present in the pregnant uterus and are thought to be beneficial, because alterations in their proportions can adversely affect pregnancy. Immunocytochemistry and cell counting were used to compare the distributions and dynamics of leukocyte subpopulations in wild-type and Lif knockout mice. The percentage of macrophages was reduced by more than half in the Lif knockout mice on D3 of pregnancy, and their distribution was disrupted, suggesting that LIF is a chemokine for these cells. The NK cells were detected as early as D3 of pregnancy, but the Lif knockout mice had double the percentage of NK cells compared to wild-type mice at this time, indicating that LIF restricts the migration of NK cells to the uterus. The Lif knockout mice also had significantly higher percentages of eosinophils in the outer stroma on D3, and in the midstroma on D4, of pregnancy, suggesting that LIF also may restrict eosinophil migration to the uterus. These alterations of the uterine leukocyte subpopulations in Lif knockout mice may disrupt pregnancy and contribute to failure of implantation.     

Leukocyte inhibitory factor activates human neutrophils and macrophages to release leukotriene B4 and thromboxanes

Recent evidence has proved that cytokines can stimulate the production of 5-lipoxygenase products. Leukotriene B4 (LTB4) is a major mediator of leukocyte activation in acute inflammatory reactions, which produce chemotaxis, lysosomal enzyme release, and cell aggregation. Leukocyte inhibitory factor (LIF) also causes biological responses related to inflammation, i.e., LIF directly induces specific granule secretion by polymorphonuclears (PMNs) and potentiates many formyl-methionyl-leucyl-phenylalanine (FMLPs) mediated responses. Since arachidonic acid products are important mediators of inflammation, we have studied the effects of LIF on the arachidonic acid cascade products LTB4 and thromboxane A2 (TxA2). Resuspended at a final concentration of greater than 95% polymorphonuclear PMNs were isolated and tested with some cytokines on the release of LTB4 and TxA2. Peripheral blood mononuclear cells were isolated and seeded in Petri dishes and incubated for 60 min. Adherent macrophages were used for the cytokine stimulation study. Both types of leukocytes were treated with LIF, interleukin 6 (IL 6), and granulocyte-monocyte colony stimulating factor (GM-CSF) at different concentrations, and test agents A23187 and FMLP. Radioimmunoassay for LTB4 and TxB2 was determined by the resulting supernatants. Treatment of PMNs and macrophages with LIF at different concentrations proved to generate significant increases in LTB4 and TxA2 production. This was compared with IL 6 and GM-CSF, which had no effects. In these experiments, TxA2 generations could not be attributed to platelet contamination of PMN suspensions. The quantity of platelet contamination was not sufficient to influence how much TxB2 was produced. The similarities of LIF to other arachidonate stimulating cytokines suggest a similar mode of action in producing hematologic changes typical of tissue injury.(ABSTRACT TRUNCATED AT 250 WORDS)     

Leukocyte migration inhibitory factor (LIF) to sperm from autoimmune men in infertile couples

Leukocyte migration inhibitory factor (LIF) is produced by lymphocytes with receptors specific to sensitizing antigens. This principle was used to detect possible antigenic differences between sperm of autoimmune and nonautoimmune men. Sixteen fertile and 91 infertile couples were screened for cytotoxic and hemagglutinating antibodies to sperm from their husbands and controls. Their lymphocytes were tested for the production of LIF to sperm extracts and seminal plasma from the husbands and controls by a direct leukocyte migration inhibition assay. Twenty-nine of 35 men producing LIF to sperm and/or seminal plasma were positive for sperm antibodies (p = 0.0004, vs sperm antibody-negative controls). Twenty-three of 29 wives with LIF production had sperm-autoimmune husbands (p = 0.04). Leukocyte migration was significantly inhibited in sperm-autoimmune men by autologous sperm extracts and seminal plasma in contrast to control sperm extracts and seminal plasma (p = 0.0006 and 0.001, respectively). The wives of autoimmune men had significantly higher LIF responses to their husbands' sperm extracts than to other antigens (p = 0.02). Men with cytotoxic antibodies in their seminal plasma produced LIF to autologous sperm (p = 0.001). It is suggested that certain sperm and seminal plasma antigens of autoimmune men may lead to specific humoral and cell-mediated immune responses in both partners.     

Interferon-tau stimulates secretion of macrophage migration inhibitory factor from bovine endometrial epithelial cells

During early pregnancy in ruminants, the embryo not only prevents prostaglandin F2alpha release, but it also modifies protein synthesis in the endometrium. This is accomplished by the secretion of interferon-tau (IFN-tau) from the embryo. The objective of this study was to identify and characterize specific proteins secreted from endometrial epithelial cells in response to IFN-tau that could be important for endometrial function and/or embryo development. The epithelial cells were prepared and cultured to confluence and then incubated with or without 100 ng/ml IFN-tau. At the end of the incubation, the proteins in the medium were analyzed by two-dimensional PAGE. The result showed that two major protein spots were induced by IFN-tau. One has a molecular mass of approximately 12 kDa and an isoelectric point (pI) of 6.7; the other has a molecular mass of 76 kDa and pI of 4.8. Protein sequence analysis showed that the 12-kDa protein contained a partial amino acid sequence that corresponded to macrophage migration inhibitory factor (MIF). To determine whether MIF is expressed in endometrial cells, isolated stromal or epithelial cells were incubated with or without 100 ng/ml IFN-tau for 0, 3, 6, 12, 24, and 48 h. After incubation, the MIF protein in cells was examined by Western blotting analysis, and the steady-state mRNA for MIF was examined by Northern analysis. Results showed that MIF protein and mRNA were present in the epithelial cells but not the stromal cells. The presence of MIF in the luminal epithelium of endometrial tissue was confirmed by immunohistochemistry. However, there was no effect of IFN-tau on MIF expression in the epithelial cells. The concentration of MIF in the medium was quantified by Western blotting analysis to determine if IFN-tau altered MIF protein secretion from the epithelial cells. The results showed that IFN-tau significantly stimulated the secretion of MIF protein from the cells. These data show that MIF is expressed in the epithelial, but not the stromal, cells of the endometrium and that MIF secretion from the epithelial cells is stimulated by IFN-tau. It is therefore likely that MIF plays a role in early embryo development, and further characterization of MIF expression and its regulation in the endometrium will add significantly to our understanding of early embryo-uterine interactions.     

The multicomponent nature of teratoma cell adhesion factor

The multicomponent nature of teratoma cell adhesion factor has been demonstrated. Fractionation of crude ascites fluid on a DEAE cellulose ion exchange column shows that two or more components are involved in teratoma adhesion factor (TAF) activity. Glycoproteins (or proteoglycans) in fractionated ascites fluid were localized in polyacrylamide gels. The possible role of these sugar-containing molecules in teratoma cell adhesion and current hypotheses on the mechanism of carbohydrate involvement in intercellular adhesion are discussed.     

Leukocyte adhesion: High-speed cells with ABS.

In order to decide where to exit blood vessels and enter tissues, leukocytes roll along endothelial surfaces. Recent studies suggest that an 'automatic braking system' (ABS), involving selectin cell-adhesion molecules, enables leukocytes to roll at a fairly constant velocity despite large variations in blood flow rate.