Propofol inhibits human neutrophil functions

Neutrophils play important roles in the antibacterial host defense mechanism and in the pathogenesis of tissue injury. Propofol has been reported to impair the production of reactive oxygen species from neutrophils. We examined the effect of propofol (2,6-diisopropylphenol), at clinically relevant concentrations and at 10 and 100 times this concentration, on several aspects of human neutrophil functions using an in vitro system. Propofol significantly inhibited chemotaxis, phagocytosis, and reactive oxygen species (ROS) (O2-, H2O2, OH) production of neutrophils in a dose-dependent manner. At clinically relevant concentrations, propofol suppressed these neutrophil functions, but it did not decrease ROS generation by the cell-free (xanthine-xanthine oxidase) system. Increase in intracellular calcium concentrations in neutrophils stimulated by N-formyl-L-methionyl-L-leucyl-L-phenylalanine was dose-dependently attenuated by propofol. This decreasing effect on [Ca2+]i in neutrophils may represent one of the mechanisms responsible for the inhibition of neutrophil functions by propofol. IMPLICATIONS: Neutrophils play a pivotal role in the antibacterial host defense system and tissue injury. We found that at clinically relevant concentrations, propofol impaired neutrophil functions. Further studies may determine whether this impairment, observed in vitro, leads to clinical immunological suppression.     

The intravenous administration of tumor necrosis factor alpha, interleukin 8 and macrophage-derived neutrophil chemotactic factor inhibits neutrophil migration by stimulating nitric oxide production

1. The i.v. administration of tumor necrosis factor-alpha (TNF-alpha), interleukin-8 (IL-8) and the recently described macrophage-derived neutrophil chemotactic factor (MNCF) inhibits the recruitment of neutrophils to the inflammatory site. 2. Pretreatment of mice with the NO synthase antagonist, NG-monomethyl-L-arginine (L-NMMA, 15-60 mg kg(-1)), but not the inactive enantiomer D-NMMA (30 mg kg(-1)), prevented in a dose-dependent manner the TNF-alpha, IL-8 and MNCF-mediated inhibition of neutrophil migration into thioglycollate-challenged peritoneal cavities. 3. Treatment of the neutrophils with TNFalpha (10(-7) M), IL-8 (10(-7) M) or MNCF blocked their migration towards FMLP in the chemotaxis assay. The pretreatment of the neutrophils with L-NMMA (50-200 microM) prevented in a dose-dependent manner the inhibition of FMLP-induced chemotaxis by IL-8, but did not alter the inhibition caused by TNF-alpha or MNCF. Different concentrations of the NO donors, S-nitroso-N-acetylpenicillamine (SNAP) or 3-morpholino-sydnonimine (SIN-1), did not alter this chemotaxis. 4. Preincubating the neutrophils with L-NMMA (200 microM) significantly increased the TNF-alpha (10(-7) M) and MNCF-mediated neutrophil adhesion to unstimulated endothelial cells, but had no effect on IL-8 (10(-7) M)-mediated adhesion. 5. Although NO donors did not directly affect the mechanisms of neutrophil motility, NO is involved in the in vitro inhibitory action of IL-8 on chemotaxis. The TNF-alpha and MNCF-mediated inhibition of neutrophil migration seems to be indirect, by affecting the mechanisms of adhesion. It was concluded that TNF-alpha-, IL-8- and MNCF-mediated inhibition of neutrophil migration is associated with the stimulation of NO production.     

Cellular memory: neutrophil orientation reverses during temporally decreasing chemoattractant concentrations

Cell directional orientation or shape polarization is the first cellular step in neutrophil locomotion. To better understand how chemoattractants interact with cells, we studied neutrophil polarization (or shape changes) during exposure to a temporally decreasing chemoattractant signal of N-formyl-methionyl-leucyl-phenylalanine (FMLP) in the absence of a spatial concentration gradient. To accomplish this objective, we used a manifold of differing FMLP concentrations attached to a stopped-flow microscope chamber. Spatial gradients of a fluorescent chemotactic peptide could not be detected in the chamber by using microfluorometry. When FMLP was injected at continually increasing concentrations at 10-s intervals, the shape and relative direction of the neutrophil persisted. However, when temporally decreasing FMLP concentrations were injected, approximately 80% of the cells changed their direction with 44% of the total cells swinging about to 180 degrees +/- 15 degrees. Most of these directional changes involved dissolution of both the lamellipodium and uropod and reformation of these structures 180 degrees from their original positions. This research suggests that neutrophils reverse their morphological polarity when exposed to temporally decreasing ligand concentrations by "remembering" their ligand exposure history and relative direction.     

Cytokine-induced neutrophil chemoattractant gene expression in the rat hypothalamus by osmotic stimulation

Cytokine-induced neutrophil chemoattractant (CINC) is one of the chemokines and has chemotaxity for neutrophils. Recently, we found the presence of stress-sensitive CINC expression in the hypothalamic nuclei such as the paraventricular nucleus. Since CINC was predominantly co-localized with vasopressin in the supraoptic nucleus (SON), we investigated the effect of hyperosmotic challenge on CINC mRNA in the hypothalamus. We found that CINC mRNA expression in the hypothalamus was augmented within 30 min following osmotic stimulation and immediately returned to the basal level. The suckling, which is a stimulation to oxytocin neurons in the SON, has no effect on CINC mRNA expression in the hypothalamus. This is the first evidence that the chemokine in the brain is activated by osmotic stimulation.     

Neutrophil elastase inhibitor reduces neutrophil chemoattractant production after ischemia-reperfusion in rat liver

BACKGROUND & AIMS: Neutrophils are important in the development of tissue injury induced by ischemia-reperfusion. The ability of an inhibitor of neutrophil elastase (ONO-5046) to protect against ischemia-reperfusion injury in rat liver was investigated by measuring serum concentrations of cytokine-induced neutrophil chemoattractant. METHODS: Liver ischemia was induced in rats by occluding the portal vein for 30 minutes, and ONO-5046 or anticoagulants were injected intravenously 5 minutes before vascular clamping. RESULTS: Serum concentration of cytokine-induced neutrophil chemoattractant increased after reperfusion, reached a maximum at 6 hours, and then gradually decreased. However, pretreatment of animals with heparin (50 U/kg), antithrombin III (250 U/kg), or ONO-5046 (10 mg/kg) resulted in significantly smaller increases in the serum concentration of cytokine-induced neutrophil chemoattractant after reperfusion. Pretreatment with both ONO-5046 and heparin, or both ONO-5046 and antithrombin III, produced additive effects. Pretreatment of rats with both ONO-5046 and heparin or both ONO-5046 and antithrombin III also inhibited the increase in cytokine-induced neutrophil chemoattractant mRNA in liver. These combined treatments significantly reduced the increases in both the number of neutrophils accumulated in the liver and the hepatic activity of myeloperoxidase. CONCLUSIONS: Cytokine-induced neutrophil chemoattractant production after ischemia-reperfusion in the liver is mediated by neutrophil elastase and activation of coagulation within the hepatic microcirculation.     

Regulation of cytokine-induced neutrophil chemoattractant in rat bone marrow-derived macrophages by inflammatory mediators

During the course of inflammation, macrophages are highly influenced by their local environment and changes in the cytokine milieu. Exposure of macrophages to various factors during different phases of the inflammatory response may have a strong influence on the pattern of gene expression, which a macrophage exhibits. We examined how these mediators affect the regulation of the expression and production of cytokine-induced neutrophil chemoattractant (CINC). Our study demonstrates that CINC can be induced in bone marrow-derived macrophages by lipopolysaccharide, interleukin-1 beta, tumor necrosis factor-alpha (TNFalpha), and interferon-gamma/TNF alpha. These mediators are factors which a macrophage would be expected to encounter early in an inflammatory process. In contrast, transforming growth factor-beta (TGFbeta), which is expressed late in the inflammatory process during mesenchymal cell proliferation and tissue repair, did not induce detectable amounts of CINC and functioned to suppress CINC production stimulated by early inflammatory mediators. Suppression of CINC production occurred whether TGF beta was added simultaneously, 12 or 24 h prior to the stimulus.     

Interleukin-10 inhibits neutrophil phagocytic and bactericidal activity

Effective host defense against bacterial invasion is characterized by the vigorous recruitment and activation of inflammatory cells, which is dependent upon the coordinated expression of both pro- and anti-inflammatory cytokines. Interleukin-10 (IL-10) is a recently described cytokine with potent anti-inflammatory properties in vivo and in vitro. In this study we investigated whether IL-10 could directly regulate the ability of neutrophils (PMN) to phagocytose and kill bacteria. Initial studies demonstrated that human recombinant IL-10 (hrIL-10) inhibited the ability of PMN to phagocytose Escherichia coli in vitro. Inhibition of phagocytosis occurred in the absence of changes in CR1 (C3b) or Fc receptor expression, as treatment of PMN with IL-10 failed to induce significant changes in Fc gamma IIR, Fc gamma IIIR or CR1 cell surface expression. However, incubation of PMN with IL-10 resulted in a dose-dependent decrease in CDIIb (Mac-1) expression. In addition to effects on PMN phagocytosis, hrIL-10 significantly attenuated PMN microbicidal activity, as bactericidal assays revealed that co-incubation of PMN with hrIL-10 resulted in a marked decrease in killing of phagocytosed bacteria. Furthermore, IL-10 inhibited the production of superoxide from PMA-stimulated PMN, suggesting that the detrimental effects of IL-10 on PMN microbicidal activity were due, in part, to suppression of respiratory burst. In summary, our studies indicate that IL-10 inhibits PMN-dependent phagocytosis and killing of E. coli in vitro, and suggest that this cytokine may impair effective antibacterial host defense in vivo.     

Role of cytokine-induced neutrophil chemoattractant (CINC) in ozone-induced airway inflammation and hyperresponsiveness

Cytokine-induced neutrophil chemoattractant (CINC) is a rat chemokine with potent chemoattractant effects on neutrophils. We determined the involvement of CINC in ozone-induced airway neutrophilia and bronchial hyperresponsiveness (BHR) in the rat. We found a marked increase in lung CINC messenger RNA (mRNA) within 2 h after cessation of ozone exposure (1 ppm for 3 h), as measured by Northern blot analysis, whereas rats exposed to room air had no detectable CINC mRNA. Ozone exposure induced a significant neutrophilia in bronchoalveolar lavage fluid (BALF) at 24 h after exposure (air-exposed rats: 4.2 +/- 2.0 x 10(4), versus ozone-exposed rats: 16.1 +/- 3.7 x 10(4)); prior treatment with a goat anti-CINC antibody (1 mg, intravenously) suppressed the neutrophilia (3.1 +/- 0.9 x 10(4)). When administered intratracheally, the antibody (230 micrograms) partially inhibited the influx of neutrophils. The increase in bronchial responsiveness to acetylcholine observed after ozone exposure was not inhibited by the anti-CINC antibody. The anti-CINC antibody (1 mg, intravenously) also inhibited BALF neutrophilia induced by exposure to a higher concentration of ozone (3 ppm, 3 h), without an effect on BHR. CINC is an important chemokine causing ozone-induced neutrophil chemoattraction, but is not involved in the induction of ozone-induced BHR. The neutrophil is unlikely to contribute to BHR in this model.     

Immunosuppressants decrease neutrophil chemoattractant and attenuate ischemia/reperfusion injury of the liver in rats

BACKGROUND: Neutrophils may play an important role in the development of liver ischemia/reperfusion injury. We investigated the effects of the immunosuppressants azathioprine (AZA), cyclosporine A (CsA), tacrolimus (FK506), and rapamycin (RPM) on the expression of cytokine-induced neutrophil chemoattractant (CINC) after ischemia/reperfusion of the liver. METHODS: Liver ischemia was induced in male Wistar rats by occluding the portal vein with a microvascular clip for 30 minutes. Rats received two intramuscular injections of AZA (4 mg/kg), CsA (5 mg/kg), FK506 (0.5 mg/kg), or RPM (0.5 mg/kg) 3 and 24 hours before ischemia/reperfusion of the liver. RESULTS: Serum CINC concentrations in untreated animals increased, peaked 6 hours after reperfusion, and thereafter decreased gradually. Pretreatment with AZA, CsA, FK506, and RPM, however, inhibited the increase in serum CINC concentrations after reperfusion. CINC mRNA in liver tissue increased and peaked 3 hours after reperfusion, but was significantly lower in animals treated with AZA, CsA, FK506, and RPM. In vitro CINC production by Kupffer cells harvested from animals treated with AZA, CsA, FK506, or RPM 3 hours after reperfusion was also significantly lower than that observed in untreated animals. Both myeloperoxidase activity and the number of neutrophils accumulating in the liver 24 hours after reperfusion in animals treated with AZA, CsA, FK506, and RPM were significantly lower than in untreated animals. This correlated with lower serum aspartate transaminase, alanine transaminase, and lactate dehydrogenase levels in animals treated with AZA, CsA, FK506, and RPM 24 hours after reperfusion. CONCLUSION: The immunosuppressants AZA, CsA, FK506, and RPM reduce neutrophil accumulation and attenuate ischemia/reperfusion injury of the liver.     

Downregulation of cytokine-induced neutrophil chemoattractant and prolongation of rat liver allograft survival by interleukin-10

Deficiency of the complement protein C2 (C2D), one of the most common genetic deficiencies of the complement system, is associated with rheumatological disorders and increased susceptibility to infection. Two types of C2D have been recognized, each in the context of specific major histocompatibility complex (MHC) haplotypes; type I, a deletion, frameshift and premature stop codon resulting in absence of detectable C2 protein synthesis, and type II, missense mutations resulting in a block in secretion of C2 proteins. Analysis of C2 expression in a child with C2 deficiency, a MHC haplotype different from those associated with type I or II C2D, and recurrent infections revealed additional molecular heterogeneity among C2 deficient patients. No detectable C2 protein was synthesized in the child's fibroblasts under conditions supporting C2 synthesis and secretion in normals and the child's C2 mRNA was reduced to 42% of normal. Nucleotide sequencing of RT-PCR fibroblast mRNA and genomic DNA revealed a type I C2 deficiency (28 base-pair deletion) on one allele and a previously unrecognized two base-pair deletion in exon 2 on the other. Expression of the closely linked factor B gene was markedly decreased (Bf mRNA 25% of normal), though Bf was up-regulated appropriately by interferon-gamma and the flanking sequence containing the Bf promoter was normal in this C2-deficient patient. Moreover, the concentration of Bf protein was normal in the patient's plasma.     

Vicia villosa B4 lectin inhibits nucleotide pyrophosphatase activity toward UDP-GalNAc specifically

A new integrative vector (pCJR24) was constructed for use in the erythromycin producer Saccharopolyspora erythraea and in other actinomycetes. It includes the pathway-specific activator gene actII-ORF4 from the actinorhodin biosynthetic gene cluster of Streptomyces coelicolor. The actI promoter and the associated ribosome binding site are located upstream of an NdeI site (5'-CATATG-3') which encompasses the actI start codon allowing protein(s) to be produced at high levels in response to nutritional signals if these signals are faithfully mediated by the ActII-ORF4 activator. Several polyketide synthase genes were cloned in pCJR24 and overexpressed in S. erythraea after integration of the vector into the chromosome by homologous recombination, indicating the possibility that the S. coelicolor promoter/activator functions appropriately in S. erythraea. pCJR24-mediated recombination was also used to place the entire gene set for the erythromycin-producing polyketide synthase under the control of the actI promoter. The resulting strain produced copious quantities of erythromycins and precursor macrolides when compared with wild-type S. erythraea. The use of this system provides the means for rational strain improvement of antibiotic-producing actinomycetes.     

The role of calcium ions in DEAE-dextran-induced stimulation of neutrophil migration

The polycation DEAE-dextran caused a strong enhancement of non-directed migration of rabbit peritoneal neutrophils. The activating effect on migration was completely annulled in the presence of the polyanion poly-D-glutamic acid, indicating that the effect depended on the positive charge of the macromolecule. Chemotaxis activated by the chemotactic peptide fMLP was only slightly affected by the polycation. In contrast with fMLP-activation, stimulation of migration by DEAE-dextran was dependent on the presence of extracellular Ca2+. DEAE-dextran also stimulated migration of electroporated neutrophils. The stimulation was absent when calcium was not present; the increase of migration was strongest at Ca(2+)-concentrations between 100 nM and 1 microM Ca2+. This indicates that the requirement for extracellular Ca2+ in intact cells is a reflection of the intracellular requirement. Several types of calcium blockers gave a moderate inhibition of DEAE-dextran activated migration. Activation of migration by DEAE-dextran of electroporated neutrophils was completely inhibited by calcium channel blockers, at very low concentrations. The results suggest that both Ca(2+)-fluxes across the plasma membrane and Ca2+ from intracellular stores are required for DEAE-activated migration, and that the calcium from the intracellular source is required on a place where the extracellular Ca2+ has no, or limited, admittance.     

Heparin inhibits human coronary artery smooth muscle cell migration

Heparin, an anticoagulant, has been shown to reduce neointimal proliferation and restenosis following vascular injury in experimental studies, but the clinical trials of heparin in coronary balloon angioplasty have been negative. The current study, therefore, examined the effect of heparin on basal or stimulated migration by serum and platelet-derived growth factor (PDGF)-BB in cultured human coronary artery smooth muscle cells (SMCs) by Boyden's chamber method. In addition, the reversibility of the heparin effect on human coronary artery SMC migration was examined. Fetal calf serum (FCS) and PDGF-BB stimulated SMC migration in a concentration-dependent manner. Heparin in moderate to high concentration (10 to 100 U/mL) exhibited concentration-related inhibition of FCS- and PDGF-BB-stimulated SMC migration; however, a low concentration (1 U/mL) of heparin had no inhibitory effects. Heparin also had weak inhibitory effects on nonstimulated SMC migration. The SMCs that were exposed to a high concentration (100 U/mL) of heparin for 6 hours were capable of migrating after a short lag period of removal of heparin from the culture medium. These SMCs also showed recovery of responses to FCS and PDGF-BB by migrating significantly greater than the nonstimulated level. Furthermore, heparin-containing medium did not contain detached cells. These results indicate that heparin inhibits human coronary artery SMC migration, especially when stimulated by FCS or PDGF-BB, and that this inhibitory effect of heparin is reversible and not simply a function of killing cells.     

The effects of photodynamic therapy on human neutrophil migration using bacteriochlorin a

Bacteriochlorin a photodynamic therapy (BCA-PDT) caused inhibition of interleukin (IL)-8-activated neutrophil migration, at concentrations that did not induce membrane damage. Random migration and migration induced by other chemoattractants were also inhibited, indicating that the effect of BCA-PDT was not at the level of chemoattractant-receptor interaction but down stream. The BCA-PDT completely blocked superoxide production of phorbol ester-stimulated neutrophils indicating that superoxide production by neutrophils present in the tumor before and during BCA-PDT is not the cause of inactivation of tumor cells. Both type I and type II quenchers prevented inhibition by BCA-PDT but only in electroporated cells. Confocal laser scanning microscopy showed that the fluorescence of BCA was located inside the cell. These results show that the effects of BCA-PDT are intracellular and of a mixed type I/type II character and that the neutrophils present in the tumor during illumination probably do not contribute to tumor eradication by releasing reactive oxygen species.     

Effect of anti-macrophage migration inhibitory factor antibody on lipopolysaccharide-induced pulmonary neutrophil accumulation

Macrophage migration inhibitory factor (MIF) is a recently rediscovered pro-inflammatory cytokine that has the unique potential to override the anti-inflammatory action of glucocorticoids. Since recent reports suggest the pivotal role of MIF in acute lung injury, we examined the protective effect of anti-MIF antibody on lipopolysaccharide (LPS)-induced acute lung injury in rats. Rats were injected with LPS (7 mg/kg) intraperitoneally with or without pretreatment with anti-MIF antibody. The anti-MIF antibody significantly attenuated LPS-induced migration of neutrophils to the lungs at 4 and 24 h as demonstrated by observation of the number of neutrophils per alveolus, the activity of myeloperoxidase of the lung tissue, and cell differentiation of neutrophils in bronchoalveolar lavage (BAL) fluid. The increased level of macrophage inflammatory protein-2, a powerful neutrophil chemokine, in BAL fluid was also significantly attenuated by pretreatment with the anti-MIF antibody as compared with the control group. Additionally, positive immunostaining for MIF was observed in bronchial epithelial cells and alveolar macrophages, and Northern blot analysis of lung tissues demonstrated increased MIF mRNA 24 h after LPS injection. These data suggest that the anti-MIF antibody has therapeutic potential for the treatment of acute lung injury by suppressing the level of neutrophil chemokine in the lungs.     

Antibody to Mac-1 or monocyte chemoattractant protein-1 inhibits monocyte recruitment and promotes tumor growth

Human tumors are frequently infiltrated by numerous monocytes/macrophages, which can be found within the tumor mass (intratumoral) or surrounding the tumor (peritumoral). The functional role that these monocytes/macrophages play in tumor growth is controversial. To address this issue we inhibited intratumoral monocyte/macrophage recruitment with mAbs that either blocked integrin function or neutralized a tumor-produced chemotactic protein. Both treatments significantly increased tumor formation and accelerated tumor growth. Surprisingly, the same results were obtained when recruitment of peritumoral or intratumoral monocytes/macrophages was blocked. Our findings are contrary to one of the purported roles of monocytes/macrophages, particularly in the peritumoral area, since we found no evidence for monocyte/macrophage-supported tumor growth. These results provide direct evidence that intratumoral as well as peritumoral monocytes/macrophages act to limit tumor size in the early stages following tumor inoculation and provide a mechanism that accounts for monocyte/macrophage recruitment to human tumors.     

Cyclosporine A inhibits lymphocyte migration into ovine peripheral nerve allografts

Lymphocyte migration into nerve allografts was measured to estimate the cyclosporine A (CsA) dose required to suppress rejection. Twelve outbred sheep received daily subcutaneous CsA at 0, 5, 10, or 15 mg/kg/day for 2 weeks prior to implantation of multiple heterotopic subcutaneous nerve grafts. Lymphocyte migration was determined after 7 days by an intravenous pulse of autologous 111indium-labeled lymphocytes and subsequent quantitation of gamma radioactivity in nerve tissue (CPM/g, mean +/- SEM). Measurement by radioimmunoassay revealed a dose-dependent increase in blood cyclosporine levels. Lymphocyte migration into autografts (404+/-44) was significantly less than migration into allografts (16,554+/-2,049), in control animals (P < 0.01). A dose-dependent inhibition of lymphocyte migration into nerve allografts was observed with counts of 7,662+/-1,692, 4,083+/-1,112, and 1,561+/-232 in sheep receiving 5, 10, or 15 mg/kg/day of CsA, respectively. Daily CsA administration produced effective blood levels and immunosuppression sufficient to inhibit lymphocyte migration into nerve allografts.     

Effects of propentofylline on tumor necrosis factor-alpha and cytokine-induced neutrophil chemoattractant production in rats with cerulein-induced pancreatitis and endotoxemia

Using rabbit polyclonal antiurinary protein 1 antibody to study the female prostate (Skene's gland) and the male prostate, characteristic localizations patterns appeared in single cells and groups of cells. The majority correspond to cells positive for neuroendocrine markers. In the cytoplasm, cells positive for protein 1 were most frequently found in the epithelial lining of the female urethra, in the pars prostatica of the male urethra, and in the ducts of the female and male prostate where the lining consisted of pseudostratified columnar epithelium. Their occurrence rate was far lower among secretory and basal cells of the male and female prostate glands. The cells with protein 1 corresponded to those displaying positivity for chromogranin A, silver staining by the Grimelius and less by the Sevier-Munger method, and by neuron specific enolase. Using the Masson-Hamperl argentaffin method, positive cells were only exceptionally found. The cells positive for protein 1, and particularly chromogranin A, and characterized by Grimelius positivity, contained different amounts of neuroendocrine granules and varied in size and shape. The majority of these cells had contact with the lumen of male and female prostatic ducts (open type of neuroendocrine cells). In some cases of the male and female urethra and of the great paraurethral ducts, a remarkably high number of cells containing protein 1 corresponded to cells only containing neuron-specific enolase but not chromogranin A and other neuroendocrine markers. These cells can be considered stem cells responsible for the renewal of the uroepithelium of the urethra and prostatic ducts. Protein 1 may thus be a further, though presumably not specific marker for the identification of cells of the neuroendocrine system in the prostate of the male and female. This marker could well be used to study uroepithelium maturation. The corresponding immunohistochemical distribution of human protein 1 in neuroendocrine and other cells of the male and the female prostate provides another analogous functional and morphological parameter of prostatic tissue in both sexes and further evidence supporting the non-vestigial concept of the prostate in the female.     

Endothelial cell cytosolic free calcium regulates neutrophil migration across monolayers of endothelial cells

Polymorphonuclear leukocytes (PMN) traverse an endothelial cell (EC) barrier by crawling between neighboring EC. Whether EC regulate the integrity of their intercellular adhesive and junctional contacts in response to chemotaxing PMN is unresolved. EC respond to the binding of soluble mediators such as histamine by increasing their cytosolic free calcium concentration ([Ca++]i) (Rotrosen, D., and J.I. Gallin. 1986. J. Cell Biol. 103:2379-2387) and undergoing shape changes (Majno, G., S. M. Shea, and M. Leventhal. 1969. J. Cell Biol. 42:617-672). Substances such as leukotriene C4 (LTC4) and thrombin, which increased the permeability of EC monolayers to ions, as measured by the electrical resistance of the monolayers, transiently increased EC [Ca++]i. To determine whether chemotaxing PMN cause similar changes in EC [Ca++]i, human umbilical vein endothelial cells (HUVEC) maintained as monolayers were loaded with fura-2. [Ca++]i was measured in single EC during PMN adhesion to and migration across these monolayers. PMN-EC adhesion and transendothelial PMN migration in response to formyl-methionyl-leucyl-phenylalanine (fMLP) as well as to interleukin 1 (IL-1) treated EC induced a transient increase in EC [Ca++]i which temporally corresponded with the time course of PMN-EC interactions. When EC [Ca++]i was clamped at resting levels with a cell permeant calcium buffer, PMN migration across EC monolayers and PMN induced changes in EC monolayer permeability were inhibited. However, clamping of EC [Ca++]i did not inhibit PMN-EC adhesion. These studies provide evidence that EC respond to stimulated PMN by increasing their [Ca++]i and that this increase in [Ca++]i causes an increase in EC monolayer permeability. Such [Ca++]i increases are required for PMN transit across an EC barrier. We suggest EC [Ca++]i regulates transendothelial migration of PMN by participating in a signal cascade which stimulates EC to open their intercellular junctions to allow transendothelial passage of leukocytes.