Impaired transendothelial migration by neonatal neutrophils: abnormalities of Mac-1 (CD11b/CD18)-dependent adherence reactions

In order to evaluate the functions of lymphocyte function antigen-1 (LFA-1) (CD11a/CD18) and Mac-1 (CD11b/CD18) on neonatal neutrophils, we examined neutrophil adhesion to and migration through human umbilical vein endothelial cell (HUVEC) monolayers in vitro. Transendothelial migration of adult neutrophils was greatly enhanced by preincubation of HUVEC with interleukin-1 (IL-1). This migration was significantly inhibited by monoclonal antibodies (MoAbs) against LFA-1 (CD11a) and Mac-1 (CD11b) subunits. Migration of neonatal neutrophils was markedly diminished compared to adult neutrophils, and MoAbs against LFA-1 further reduced migration. In contrast, anti-Mac-1 MoAb was not inhibitory. Adhesion of adult neutrophils was significantly enhanced by prestimulation of HUVEC with IL-1, and was significantly inhibited by MoAbs against LFA-1. Adhesion of neonatal neutrophils was near adult levels and comparably inhibited by anti-LFA-1 MoAb. In addition, adhesion of neonatal and adult neutrophils to purified ICAM-1 in artificial planar membranes was comparable and almost completely inhibited by anti-LFA-1 MoAb. Chemotactic stimulation induced Mac-1-dependent adhesion of adult neutrophils to endothelial cells, purified intercellular adherence molecule-1 (ICAM-1) and protein-coated glass. In marked contrast, adhesion of neonatal neutrophils to these substrates was not significantly increased by chemotactic stimulation. These findings indicate that diminished transendothelial migration by neonatal neutrophils is related to abnormal interactions of Mac-1 with ICAM-1 and possibly other endothelial ligands. These functional deficits may contribute to impaired inflammation and infectious susceptibility in human neonates.     

Increased production of superoxide anion by neonatal polymorphonuclear leukocytes stimulated with a chemotactic peptide

In order to assess the functional property of neonatal polymorphonuclear leukocytes (PMNs), we studied their chemoattractant-stimulated superoxide anion (O2-) production using a soluble chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (FMLP) as a PMN stimulus. O2- production by neonatal PMNs was increased: when stimulated with 10(-7)M FMLP, O2- values (nanomoles; mean +/- SE) produced by 10(6) PMNs were 16.5 +/- 1.3 for neonatal PMNs and 12.6 +/- 0.6 for adult PMNs (p less than .02). When studied under various concentrations (10(-9) to 10(-6)M) of FMLP, neonatal PMNs produced more O2- than adult cells under the stimulation of a lower concentration (10(-8)M) as well as a higher concentration (10(-7)M) of FMLP. The increased O2- production by neonatal PMNs was also observed under their stimulation by phorbol myristate acetate. Neonatal and adult PMNs produced equal amounts of O2- when stimulated with a particulate stimulus of opsonized zymosan. To analyze further the mechanism for the increase in FMLP-induced O2- production by neonatal PMNs, we next studied whether there was some abnormality in their FMLP receptors. Neonatal PMNs had normal numbers of FMLP receptors; the receptor numbers (mean) per cell were 54,000 in neonatal PMNs and 56,000 in adult cells. Additionally, the FMLP receptors had normal affinity for the peptide. These results demonstrate that the increased production of O2- by FMLP-stimulated neonatal PMNs is not due to the abnormality of its binding to the receptors but is due to that of the subsequent events.     

Roles of Mac-1 and glycoprotein IIb/IIIa integrins in leukocyte-platelet aggregate formation: stabilization by Mac-1 and inhibition by GpIIb/IIIa blockers

Circulating platelet-leukocyte hetero-aggregates play an important role in acute cardiovascular events and hypersensitivity reactions. The association involves the receptor families of selectins and integrin. The objective of this study was to investigate the role of CD11b/CD18 integrin (Mac-1) in hetero-aggregate formation and search for a counter-receptor on platelets ready to interact with Mac-1. As a model of leukocytes, Mac-1 presenting Chinese hamster ovary (CHO) cells were used to evaluate the role of Mac-1 in hetero-aggregate formation. The amount of CHO cell-bound active and inactive platelets was measured by flow cytometry, while the counter-receptors on platelets were identified via using blocking antibodies. We observed significant platelet adhesion on Mac-1-bearing cells when platelet-rich plasma or activated platelets were present. Inactive platelets did not adhere to Mac-1-bearing cells. Addition of fibrinogen, a ligand of Mac-1 significantly increased platelet binding. CD40L was demonstrated to act similarly on Mac-1. Inhibition of platelet GpIIb/IIIa completely abolished CHO cell-platelet aggregation. In our study, we have shown for the first time that Mac-1 mediates the formation of hetero-aggregates without selectin tethering when Mac-1 ligands such as fibrinogen or CD40L are present and blockers of platelet GpIIb/IIIa are able to diminish this interaction.     

Psychosocial factors are independent risk factors for the development of Type 2 diabetes in Japanese workers with impaired fasting glucose and/or impaired glucose tolerance1

Aims We prospectively studied Japanese workers with impaired fasting glucose (IFG) and/or impaired glucose tolerance (IGT) and analysed possible risk factors for diabetes, including psychosocial factors such as stress. Methods The participants were 128 male Japanese company employees (mean age, 49.3 ± 5.9 years) with IFG and/or IGT diagnosed by oral glucose tolerance test (OGTT). Participants were prospectively studied for 5 years with annual OGTTs. The Kaplan–Meier method and Cox's proportional hazard model were used to analyse the incidence of diabetes and the factors affecting glucose tolerance, including anthropometric, biochemical and social–psychological factors. Results Of 128 participants, 36 (28.1%) developed diabetes and 39 (30.5%) returned to normal glucose tolerance (NGT) during a mean follow‐up of 3.2 years. Independent risk factors for diabetes were night duty [hazard ratio (HR) = 5.48, P = 0.002], higher fasting plasma glucose (FPG) levels within 6.1–6.9 mmol/l (HR = 1.05, P = 0.031), stress (HR = 3.81, P = 0.037) and administrative position (HR = 12.70, P = 0.045), while independent factors associated with recovery were lower FPG levels (HR = 0.94, P = 0.017), being a white‐collar worker (HR = 0.34, P = 0.033), non‐smoking (HR = 0.31, P = 0.040) and lower serum alanine aminotransferase (ALT) levels (HR = 0.97, P = 0.042). Conclusions In addition to FPG levels at baseline, psychosocial factors (night duty, stress and administrative position) are risk factors for Type 2 diabetes, while being a white‐collar worker, a non‐smoker and lower serum ALT levels are factors associated with return to NGT in Japanese workers with IFG and/or IGT.     

Abnormal B-cell function in rats with non-insulin-dependent diabetes induced by neonatal streptozotocin: effect of in vivo insulin, phlorizin, or vanadate treatments

Neonatal rats treated with streptozotocin on day 5 after birth (n5-STZ model) exhibited, when fully grown, a frank basal hyperglycemia (17.4 +/- 0.7 vs. 6.6 +/- 0.2 mmol/L in nondiabetic rats), a specific failure of glucose-induced insulin release, and hyperresponse to arginine. To investigate whether or not chronic correction of the hyperglycemia can improve the defects on insulin secretion, we tested diverse maneuvers, all of which aimed to lower chronically the hyperglycemia. Insulin secretion was studied with the isolated perfused pancreas preparation. A 16-day subcutaneous insulin therapy (approximately 10 U/kg/day) unevenly correcting the plasma glucose levels (10.8 +/- 1.6 mmol/L) did not improve the lack of insulin response to glucose while the arginine-induced insulin release returned to values close to normal. A 28-day intraperitoneal phlorizin infusion (50 mg/kg/day0 or a 20-day oral vanadate administration (40 mg/kg/day) caused near normalization of the basal plasma glucose level in the treated n5-STZ rats (7.8 +/- 0.5 and 8.2 +/- 0.5 mmol/L, respectively). Nevertheless, these treatments did not correct the insulin secretion in response to glucose nor the hyperresponsiveness to arginine. Furthermore, we investigated whether or not glucopenia in vitro could restore the glucose-induced insulin release in this diabetic model. After a 50 min glucose-free period, the insulin response to a subsequent glucose stimulation still did not materialize. These observations suggest that in the present n5-STZ diabetic model, (a) hyperresponsiveness to arginine cannot be solely regarded as a residual effect of hyperglycemia; (b) the return to normal values of insulin release in response to arginine after insulin therapy, despite a still prevailing mild hyperglycemia, suggests that exogenous insulin per se may regulate to some extent the diabetic B cells; and (c) near normalization of the basal glucose levels is not a sufficient condition to obtain improvement of the B-cell response to glucose, such a finding being consistent with the concept that stringent normalization of glycemia is a prerequisite.     

Adhesive receptor Mac-1 coordinates the activation of factor X on stimulated cells of monocytic and myeloid differentiation: an alternative initiation of the coagulation protease cascade.

Monocytes initiate coagulation through regulated surface expression of tissue factor and local assembly of a proteolytic enzymatic complex formed by tissue factor and factor VII/activated factor VII. We now show that, in the absence of these initiating molecules, monocytes and cell lines of monocytic/myeloid differentiation can alternatively initiate coagulation after exposure to ADP. The molecular basis for this procoagulant response consists of two distinct events. First, cell stimulation with ADP induces high-affinity binding of coagulation factor X to the surface-adhesive receptor Mac-1. Locally, Mac-1-concentrated factor X is then rapidly proteolytically cleaved to an active protease with size and activity characteristics of activated factor X, which supports the cell-associated formation of thrombin and the procoagulant response. We conclude that the monocytic/myeloid adhesive receptor Mac-1 has the unexpected, specifically inducible property to organize a molecular assembly culminating in rapid fibrin formation that is independently regulated from tissue factor and factor VII/activated factor VII.     

The growth of cultured rabbit articular chondrocytes is stimulated by pituitary growth factors but not by purified human growth hormone or ovine prolactin

Recent reports describing a direct stimulation by GH of bone and cartilage growth led us to compare the in vitro mitogenic effects of highly purified human GH and PRL and two pituitary-derived growth factors in rabbit articular chondrocytes. These preparations were tested for their ability to promote [3H]thymidine incorporation into growth-arrested monolayer chondrocyte cultures and were also assayed in cell growth experiments. The factors tested included 22,000-dalton and 20,000-dalton human GH ovine PRL, glycosylated ovine PRL, bovine pituitary fibroblast growth factor (bpFGF), and a partially purified pituitary growth factor distinct from bpFGF. We found that no significant mitogenic effect was produced by either of the human GH or PRL preparations. Both of the pituitary-derived growth factors were potent mitogens, with bpFGF active at a final medium concentration of 10 pg/ml. These studies support the large body of evidence that GH has no significant direct in vitro effect on chondrocyte growth. The very potent effects of the pituitary-derived growth factors raise the possibility that their presence in GH preparations may be responsible for the in vitro mitogenic effects attributed to these preparations.     

Adherence of human polymorphonuclear leukocytes to endothelial monolayers: effects of temperature, divalent cations, and chemotactic factors on the strength of adherence measured with a new centrifugation assay

Polymorphonuclear leukocytes (PMNs) adhere to endothelial cells at sites of acute inflammation. To examine this phenomenon in vitro, we have developed a new assay to measure adherence of PMNs to cultured endothelial cells. Human PMNs were labeled with 111indium-oxine and incubated in microtiter wells with monolayers of either human umbilical vein or bovine aortic endothelial cells. Following incubation, the wells were sealed, inverted, and centrifuged at varying speeds. Results are expressed as the percentage of PMNs added initially that remained attached to the monolayers after being subjected to dislodgment forces (ie, relative centrifugal forces) ranging from 1 to 1,200 g. Adherence of PMNs to endothelial monolayers was temperature dependent, dependent on the concentration of extracellular Mg2+ (but not Ca2+), and enhanced significantly by the chemotactic peptides, N-formyl-methionyl-leucyl- phenylalanine (fMLP) and human C5a. It was found that fMLP and C5a not only increased the number of PMNs that adhered to endothelial cells, but also increased the strength of adherence.     

The effect of anti-inflammatory agents and inflammation on granulocyte adherence: Evidence for regulation by plasma factors

Significant inhibition of granulocyte adherence to nylon fiber columns followed the administration of alcohol, aspirin, sodium salicylate, acetaminophen, indomethacin, phenylbutazone, colchicine or prednisone to normal subjects. The addition of salicylates and glucocorticoids to blood in vitro had no effect on adherence, but plasma from volunteer subjects treated with either drug contained a factor which inhibited the adherence of normal granulocytes. The factor is heat stable, nondialyzable and not present in serum; it produces a linear dose response in normal cells. When mixed with the adherence-increasing factor found in inflammatory diseases, it neutralizes the augmenting effect and normal granulocyte adherence results.The effect of anti-inflammatory therapy on inflammatory disease was studied in aspirin-treated patients with rheumatoid arthritis. Their granulocyte adherence fell into two categories based on the clinical control of their disease: patients in good control had only slightly increased granulocyte adherence, but those in poor control had an average adherence more than twice normal. Mean blood aspirin levels were equivalent for the two groups ($\text{11.0}\text{mg}\text{100}\text{ml}$ for the well controlled and $\text{13.4}\text{mg}\text{100}\text{ml}$ for those poorly controlled). Thus, clinical response to anti-inflammatory therapy correlates well with granulocyte adherence, not with aspirin levels. The potential pathogenetic role of adherence-modifying factors in inflammatory diseases remains to be determined.     

Metabolism of endogenous arachidonic acid by isolated lungs of neonatal and adult rabbits stimulated with calcium ionophore: effect of hypoxia.

We have determined the effect of hypoxia on arachidonic acid metabolism by rabbit lungs stimulated with calcium ionophore A23187. Isolated lungs of neonatal and adult rabbits were perfused during normoxia (pO2 greater than 100 torr) or hypoxia (pO2 less than 40 torr) and arachidonic acid metabolism stimulated by the addition of A23187 (5 microM) to the perfusate. Cyclooxygenase metabolites PGE2, TxA2, and PGI2 were measured by radioimmunoassay and lipoxygenase metabolites LTB4, LTC4, LTD4, and LTE4 by HPLC. During normoxia, neonatal lungs synthesized the three cyclooxygenase metabolites that we measured in similar amounts. LTC4 constituted 56% of the leukotrienes produced. Hypoxia caused a 100% increase in the amount of PGI2 synthesized by neonatal lungs; however, total TxB2 and PGE2 production was not altered significantly. LTC4 production decreased significantly during hypoxia, whereas LTE4 production increased. Adult lungs synthesized significantly lower amounts of both cyclooxygenase and 5-lipoxygenase products than neonatal lungs. During normoxia, PGE2 was measured in highest amount in adult lungs. Similar to the neonatal lungs, LTC4 was the predominant leukotriene (56%) measured. During hypoxia, there was a 100% increase in PGI2 production by adult lungs, as was observed in neonatal lungs. There was also a small but significant increase in PGE2 production, with no change in TxA2 production. LTC4 production also decreased, but there was a marked increase in synthesis of LTB4 by adult lungs. Our data demonstrate that hypoxia alters the profile of arachidonic acid metabolites produced by rabbit lungs stimulated with A23187. Also, the age of the rabbit significantly affects both the amount and profile of metabolites synthesized by stimulated lungs during normoxia and hypoxia.     

Age-dependent decline of in vitro migration (basal and stimulated by IGF-1 or insulin) of human vascular smooth muscle cells

Since biological aging causes a decrease in functions such as cell proliferation, we have studied the possible effect of age on the migration capacity of human vascular smooth muscle cells (SMCs). To this aim, the migration activity of cultured SMCs from arteries of male human donors ranging in age from 43-77 years was determined in a Boyden chamber, under basal conditions and after insulin-like growth factor-1 (IGF-1) or insulin stimulation. Migration activity decreased with donor age (r2 = 87%, 85%, and 78%, respectively). IGF-1 and insulin significantly reduced the age-dependent relationship observed in basal conditions, so that, comparing young with old, both IGF-1 and insulin stimulated SMC migration similarly, although the effect of age remained in absolute terms. In this article, we conclude that the age-dependent decline of migration activity--similar to what has already been shown for SMC proliferation--may be part of the biological ageing phenotype, which is not overcome by hormone stimulation.     

Effects of cross-linking ICAM-1 on the surface of human vascular smooth muscle cells: induction of VCAM-1 but no proliferation

OBJECTIVE: Intercellular adhesion molecule (ICAM)-1 is an immunoglobulin-like cell adhesion molecule expressed by several cell types, including proliferating vascular smooth muscle cells (VSMC). Cross-linking ICAM-1 on the surface of different cell types has previously been shown to cause an increase in cellular activation within the cytoplasm. Here, our objective was to examine events following ligation of ICAM-1 on the surface of human VSMC. METHODS: VSMC were isolated by explant from human pulmonary arteries or aortic tissue from cardiac transplant donors. ICAM-1 was ligated with monoclonal antibodies, followed by cross-linking with a secondary antibody. Activation of signalling pathways, proliferation and expression of a second adhesion molecule, vascular cell adhesion molecule (VCAM)-1 were investigated. RESULTS: ICAM-1 cross-linking caused an increase in activation of extracellular regulated kinase (Erk)-1/-2 and Jun N-terminal kinase (JNK)-1/-2. mRNA and protein for VCAM-1 was observed after ICAM-1 cross-linking, and this was abrogated by addition of an upstream inhibitor of Erk-1/-2, PD98059. No increase in cell proliferation was observed. CONCLUSIONS: Ligation of ICAM-1 on the surface of vascular smooth muscle cells in vitro, leads to the expression of adhesion molecules associated with monocyte infiltration, but does not contribute to smooth muscle cell proliferation. In vivo, this might lead to prolongation of the inflammatory response within diseased blood vessels, by arresting monocytes within atherosclerotic plaques.     

Chemokine expression by systemic sclerosis fibroblasts: Abnormal regulation of monocyte chemoattractant protein 1 expression

Objective

Chemokines are important mediators in the chemoattraction of leukocytes to sites of inflammation. This study investigated the potential contribution of systemic sclerosis (SSc) fibroblasts to chemokine production and its potential relevance to the pathogenesis of SSc.

Methods

The expression of messenger RNA (mRNA) for different C‐C and C‐X‐C chemokines by SSc and normal fibroblasts was studied by RNase protection assay. Monocyte chemoattractant protein 1 (MCP‐1) protein production was analyzed by enzyme‐linked immunosorbent assay. The chemotactic effect of fibroblast‐derived MCP‐1 on monocytic cells was analyzed in a transmigration assay. Nuclear factor κB (NF‐κB) and activator protein 1 (AP‐1) activation in fibroblasts was studied by electromobility shift analysis. MCP‐1 expression in SSc skin sections was studied by immunohistochemistry.

Results

Among all chemokine genes studied, only MCP‐1 and interleukin‐8 mRNA were expressed by nonstimulated normal and SSc fibroblasts. SSc fibroblasts displayed increased constitutive expression of MCP‐1 mRNA and protein and showed a blunted response to oxidative stress. Increased MCP‐1 production was associated with higher chemotactic activity for monocytic cells. Increased NF‐κB or AP‐1 activation was not responsible for the constitutive overexpression of MCP‐1 by SSc fibroblasts. In SSc skin sections, MCP‐1 expression was detected in fibroblasts, keratinocytes, and mononuclear cells, whereas it was undetectable in normal skin.

Conclusion

SSc fibroblasts display a specific pattern of chemokine gene expression that is characterized by constitutively increased and abnormally regulated expression of MCP‐1 in vitro. MCP‐1 is also expressed in lesional skin and can participate in the pathogenesis of SSc.

     

Recombinant human granulocyte colony-stimulating factor as an activator of human granulocytes: potentiation of responses triggered by receptor- mediated agonists and stimulation of C3bi receptor expression and adherence

Recombinant human granulocyte colony-stimulating factor (rhG-CSF) enhanced superoxide release and membrane depolarization in parallel in human granulocytes stimulated by the receptor-mediated agonists, N- formyl-methionyl-leucyl-phenylalanine and wheat germ agglutinin, but not by the Ca2+ ionophore ionomycin and phorbol myristate acetate, which bypass the receptors to stimulate the cells. The optimal effect was obtained by pretreatment of cells with 25 to 50 ng/mL (1.3 to 2.6 nmol/L) rhG-CSF for 10 minutes at 37 degrees C. rhG-CSF produced by bacteria and mammalian cells had identical biological effects on a molar basis. rhG-CSF neither affected stimulus-induced increase in cytoplasmic free Ca2+ nor changed the number and affinity of N-formyl- methionyl-leucyl-phenylalanine receptors. The priming effect of rhG-CSF was temperature dependent and did not require new protein synthesis. rhG-CSF increased the expression of C3bi receptors on human granulocytes and enhanced granulocyte adherence to nylon fiber. The optimal effect was obtained by pretreatment of cells with 25 to 50 ng/mL rhG-CSF for 30 minutes at 37 degrees C. rhG-CSF had no effect on human monocytes. These findings demonstrate that rhG-CSF can selectively stimulate mature granulocyte functions.     

Endothelial nuclear factor-kappaB translocation and vascular cell adhesion molecule-1 induction by complement: inhibition with anti-human C5 therapy or cGMP analogues

We have previously shown that reoxygenation of hypoxic human umbilical vein endothelial cells (HUVECs) leads to the activation and deposition of complement. In the present study, we investigated whether the terminal complement complex (C5b-9) influences HUVEC nuclear factor-kappaB (NF-kappaB) translocation and vascular cell adhesion molecule-1 (VCAM-1) protein expression after hypoxia/reoxygenation by decreasing endothelial cGMP. Additionally, we investigated the action of anti-human C5 therapy on endothelial cGMP, NF-kappaB translocation, and VCAM-1 protein expression. Reoxygenation (0.5 to 3 hours, 21% O(2)) of hypoxic (12 hours, 1% O(2)) HUVECs in human serum (HS) significantly increased C5b-9 deposition, VCAM-1 expression, and NF-kappaB translocation compared with hypoxic/reoxygenated HUVECs treated with the recombinant human C5 inhibitor h5G1.1-scFv. Acetylcholine (ACh)-induced cGMP synthesis was significantly higher in normoxic HUVECs compared with hypoxic HUVECs reoxygenated in HS but did not differ from hypoxic HUVECs reoxygenated in buffer or HS treated with h5G1.1-scFv. Treatment of hypoxic/reoxygenated HUVECs with h5G1.1-scFv or cGMP analogues significantly attenuated NF-kappaB translocation and VCAM-1 protein expression. Treatment with NO analogues, but not a cAMP analogue, cGMP antagonists, or an NO antagonist, also significantly attenuated VCAM-1 expression. We conclude that (1) C5b-9 deposition, NF-kappaB translocation, and VCAM-1 protein expression are increased in hypoxic HUVECs reoxygenated in HS; (2) reoxygenation of hypoxic HUVECs in HS, but not buffer alone, attenuates ACh-induced cGMP synthesis; and (3) treatment of hypoxic/reoxygenated HUVECs with h5G1.1-scFv attenuates C5b-9 deposition, NF-kappaB translocation, and VCAM-1 expression while preserving ACh-induced cGMP synthesis. C5b-9-induced VCAM-1 expression may thus involve an NO/cGMP-regulated NF-kappaB translocation mechanism.     

Cell surface adhesiveness of mouse sarcoma lines evaluated by latex particle adherence assay: correlation with growth behavior and electrophoretic mobility

Using the latex particle adherence assay and five mouse sarcoma cell lines of the identical origin, etiology and genotype but differing in malignancy we attempted to correlate the degree of cell surface adhesiveness with growth behavior and electrophoretic mobility of cells. Higher tumorigenicity of four of the cell lines (Mc11--Mc14) was associated with lower cell surface adhesiveness and, conversely, lower malignancy of the fifth line (Mc15) with higher cell surface adhesiveness. No simple correlation or causal relationship was found among the electrophoretic mobility of the lines and other cellular characteristics.