Chemotaxis Chamber Research Articles

Differential Chemotactic Responses Mediated by Platelet-Derived Growth-Factor α- and β-Receptors

We have performed a modified Boyden chamber chemotaxis assay using NRK-49F cells under serum-free condition, in order to characterize the chemotactic activity of recombinant platelet derived growth factor (PDGF)-AA, -AB, and -BB. All three PDGF isoforms showed similar chemotactic activity on NRK cells expressing both PDGF α- and β-receptors. A checkerboard analysis revealed that PDGF-AA was a true chemoattractant for NRK cells, indicating that α-receptors could transduce signals for the chemotactic response. We then examined the inhibitory effects of PDGF isoforms in the upper chamber on NRK cell migration to PDGF in the lower chamber. When higher concentrations of PDGF-AB or -BB were present in the upper chamber, cell migration to any of the PDGF isoforms was completely blocked, whereas PDGF-AA in the upper chamber could not induce complete inhibition of the chemotactic migration to PDGF-AB or -BB. Even when 100 ng/ml of PDGF-AA was present in the upper chamber, NRK cell migration to 5 ng/ml of PDGF-AB in the lower chamber was not completely blocked. These findings suggest that the chemotactic response induced by ligand binding to PDGF β-receptor may be different from that induced by ligand binding to PDGF α-receptors.

Enhanced migration of fibroblasts derived from lungs with fibrotic lesions.

The migration and proliferation of fibroblasts may be important in the pathogenesis of pulmonary fibrosis. Considerable data are available on the proliferation of fibroblasts, but very few on their migration. The migratory activity of fibroblasts obtained from lung biopsy specimens from 11 patients with idiopathic pulmonary fibrosis (IPF) was studied using a 96-well chemotaxis chamber. Fibroblasts from eight normal controls, seven patients with interstitial fibrosis associated with a collagen vascular disease (IP-CVD), and 13 patients with sarcoidosis were also examined. Migratory activity was tested in a serum-free medium in the presence and absence of platelet derived growth factor (PDGF), 30 ng/ml, as a chemoattractant. Migration of fibroblasts from patients with IPF was enhanced in serum-free maintenance medium alone (mean (SD) controls v IPF: 183 (86) v 689 (491) cells/field), and was also enhanced when cells were stimulated by PDGF (controls v IPF: 829 (222) v 1928 (600) cells/field). Fibroblasts from tissues with dense fibrosis had a greater capacity for migration than those from an earlier stage of fibrosis. No correlation was found between migratory activity and proliferative capacity of the individual cells. The fact that fibroblasts from fibrotic lungs migrate faster than those from controls suggests that migration is related to the initiation of the pulmonary fibrotic process. These in vitro studies suggest that fibroblasts derived from the lungs of patients with pulmonary fibrosis have a migratory phenotype. Such a change in fibroblast phenotype, if it occurred in vivo, may be important in the context of the pathogenesis of pulmonary fibrosis.

Experimental gene therapy of cancer using tumor cells engineered to secrete interleukin-13.

Cytokines locally delivered to the site of a tumor boost both specific and nonspecific host anti-tumor defenses. Interleukin (IL)-13 is a recently described cytokine produced by mouse type 2 helper T lymphocytes. The aim of this study was to evaluate the inhibition of tumor growth induced by IL-13 delivered locally within or around transplanted tumor cells in mice. We observed that local administration of IL-13 at the site of transplanted tumor cells in vivo had potent inhibitory effects on growth of both immunogenic (P815 mastocytoma, H-2d) or nonimmunogenic (3LL lung carcinoma, H-2b) tumor cells. Mice injected with transfected P815 cells secreting large amounts of IL-13 rejected the P815 tumor and developed systemic specific anti-tumor immunity leading to long-lasting specific anti-tumor protection. Less efficient anti-tumoral effects were obtained with the nonimmunogenic 3LL tumor model when local administration of IL-13 was achieved by co-inoculating xenogeneic chinese hamster ovary (CHO) IL-13 cells. Several local injections of CHO IL-13 cells were needed to obtain rejection of 3LL tumors and no induction of long-lasting anti-3LL memory was obtained. Several studies were performed to elucidate the IL-13 anti-tumoral effects. Experiments with nude mice indicated that Il-13 can also stimulate nonspecific anti-tumor defenses. The histological examination of P815 IL-13 cells undergoing rejection showed monocytic cells and neutrophils infiltrating the tumor. Studies indicated that IL-13 administered in vitro did not directly stimulate the cytotoxicity of peritoneal macrophages and natural killer cells. However, experiments with Boyden chemotaxis chambers indicated that IL-13 was chemotactic for macrophages. Finally, preliminary experiments in vitro suggest that IL-13 improved antigenic presentation of P815 membranes. Thus, anti-tumor effects of IL-13 in vivo most probably result from pleiotropic effects including recruitment of nonspecific cells and improved stimulation of immune-specific anti-tumor effectors.

Migration and proliferation of guinea pig and human airway epithelial cells in response to tachykinins.

Restoration of the epithelial lining of a damaged airway is a necessary component of airway repair. Tachykinins, including substance P (SP) and neurokinin A (NKA), are localized to sensory nerves within the airway mucosa. These tachykinins regulate several airway functions, but their role in the repair of the epithelium has not been explored. To determine whether tachykinins stimulate migration and proliferation of airway epithelial cells, guinea pig tracheal epithelial (GPTE) and human bronchial epithelial (HBE) cells were grown in primary culture for 4-5 days. Epithelial cell migration was assessed in a blindwell chemotaxis chamber, and proliferation was determined by immunohistochemistry after incorporation of the thymidine analogue 5-bromo-2'-deoxyuridine (BrdU). Both GPTE and HBE cells migrated after stimulation with 10(-11) M NKA [23.0 +/- 3.6 vs. 5.4 +/- 1.2 cells per 10 high-power fields (hpf), P < 0.001, n = 8 for GPTE cells; 18.4 +/- 2.3 vs. 3.8 +/- 0.5 cells per 10 hpf for control, P < 0.001, n = 4 for HBE cells]. Migration was stimulated within 2 h, was maximal after 6 h, and was attenuated substantially by the neurokinin 2 (NKA)-receptor antagonist SR-48968. NKA-stimulated migration was both chemokinetic and chemotactic, and it could be blocked by inhibition of protein synthesis with cyclohexamide, inhibition of microtubular function with colchicine, or inhibition of actin microfilament elongation with cytochalasin D.(ABSTRACT TRUNCATED AT 250 WORDS)

Peptides from the Amino Terminus of Rantes Cause Chemotaxis of Human T-Lymphocytes

RANTES is a potent chemotactic and activating agent for a variety of leukocytes including T-lymphocytes. To identify the region of the molecule responsible for this activity, we have made overlapping 10 amino acid peptides scanning the protein. A micro-Boyden chamber chemotaxis assay showed the most efficacious peptides came from at the N-terminus. EC50 values of 8 nM (± 2.2 nM), 3.7 nM (± 2.0 nM) and 3.1 nM (± 2.0 nM) were calculated from dose response curves for the peptides (1-10), (3-12), and (5-14). Control peptides from other regions are not active. In THP-1 cells, none of the peptides give a Ca2+ response. The active peptides are shown to be principally chemotactic rather than chemokinetic by a checker board analysis. The results imply that the principal region of RANTES responsible for chemotaxis is located in the amino terminus.

Follicular dendritic cells and B cell chemotaxis.

B cells isolated from germinal centers (GC) of immune mice 2-5 days after antigen (Ag) challenge migrate in response to chemotactic signals, whereas GC B cells isolated at other times and resting B cells do not. Since B cells are in direct contact with follicular dendritic cells (FDC) in GC we reasoned that FDC might play a role in enabling B cells to become chemotactically active. Resting B cells were co-cultured with FDC either with or without anti-mu-dextran (anti-mu-dex) as an Ag surrogate and/or recombinant interleukin (rIL)-4 as a T cell surrogate. After 3 days, the B cells were isolated and their migration to chemotactic factors contained in zymosan-activated serum assessed in microchemotaxis chambers. B cells incubated alone or with anti-mu-dex or rIL-4 showed minimal migration, which could be increased if both anti-mu-dex or rIL-4 were present. However, maximal migration was obtained when B cells were cultured with FDC, and this was not increased by addition of anti-mu-dex and/or rIL-4, indicating that the FDC signal was a primary signal and did not require pre-activation of the B cells. Checkerboard analysis using variation in concentration and location of the chemoattractant in chemotaxis chambers indicated that both chemotaxis and chemokinesis occurred. B cell migration began within 6 h of culture, peaked by 48 h and decreased thereafter. Removal of FDC or interference with FDC-B cell contact ablated or significantly decreased induction of B cell migration. Furthermore, induction did not require functional T cells. These data indicate that FDC can induce resting B cells to become responsive to chemotactic signals.

Endothelial cell differentiation into capillary-like structures in response to tumour cell conditioned medium: a modified chemotaxis chamber assay

We have developed a modified chemotaxis chamber assay in which bovine aortic endothelial (BAE) cells degrade Matrigel basement membrane and migrate and form capillary-like structures on type I collagen. This capillary formation occurs in the presence of conditioned media from highly metastatic tumour cell lines, such as B16F10 murine melanoma or MDA-MD-231 human breast adenocarcinoma, but not in the presence of conditioned medium (CM) from the less invasive B16F0 cell line. Replacement of tumour cell CM by 10 ng ml-1 basic fibroblast growth factor (bFGF) also results in capillary-like structure formation by BAE cells. An anti-bFGF antibody blocks this effect, showing that bFGF is one of the factors responsible for the angiogenic response induced by B16F10 CM in our assay. Addition of an anti-laminin antibody reduces significantly the formation of capillary-like structures, probably by blocking the attachment of BAE cells to laminin present in Matrigel. The anti-angiogenic compound suramin inhibits in a dose-dependent manner (complete inhibition with 100 microM suramin) the migration and differentiation of BAE cells on type I collagen in response to B16F10 CM. This assay represents a new model system to study tumour-induced angiogenesis in vitro.

Soluble factors from the olfactory bulb attract olfactory Schwann cells

Olfactory Schwann cells (OSCs) extend processes that ensheathe bundles of olfactory axons as they course from the olfactory epithelium to the olfactory bulb (OB). Results of morphological and immunohistochemical studies have led to speculation that OSCs may be involved in guiding the olfactory axons to their target tissue. In this study we have explored this possibility by investigating the relationship between OSCs and the OB. Olfactory Schwann cells labelled with 1,1'-dioctadecyl 3,3,3',3'-tetramethylindocarbocyanine perchlorate (Dil) were injected into the nasal region of E14 rat embryos and entire embryos were cultured for 24 hr. It was found in some embryos, that the OSCs had migrated toward the presumptive OB. Cocultures of neonatal OB explants on OSC monolayers showed that the OSCs were attracted to the OB and formed a ring-like aggregate around the explant after 48 hr culture. This attraction was absent when a piece of cerebrum was used in place of the OB. When medium conditioned by neonatal OBs was placed in the lower compartment of the chemotaxis chamber, OSCs seeded in the upper compartment migrated through the pores of the nucleopore filter to reach the underside which was in contact with the conditioned medium. After 6 hr of incubation, scanning electron microscopy was performed on the underside of the nucleopore filters. Cell counts of OSCs showed that the cell density was significantly higher when medium conditioned by OBs was used instead of unconditioned medium or medium conditioned by cerebrum. The results of these experiments show that the OSCs migrate toward the OB under the influence of soluble factor(s) secreted by the target tissue.

Induction of natural killer cell migration by monocyte chemotactic protein−1, −2 and −3

Under certain physiological and pathological conditions, natural killer (NK) cells rapidly accumulate in tissues. Chemokines are an essential component of the current paradigm of leukocyte recruitment. The present study was designed to investigate the responsiveness of NK cells to the prototypic C-C chemokine, monocyte chemotactic protein-1 (MCP-1). MCP-1 induced migration across filters of interleukin (IL)-2-activated NK cells, whereas it was a weak attractant for unstimulated cells. Maximal induction of migration required a positive concentration gradient between the lower and the upper compartment of the chemotaxis chamber. Preliminary characterization of the MCP-1 receptor on NK cells indicated that the chemotactic response to MCP-1 was blocked by pre-treatment of cells with Bordetella pertussis toxin, and MCP-1 but not IL-8 displaced 125I-labeled MCP-1 from IL-2-activated NK cells. The related chemokines MCP-2 and MCP-3 were also active--though less potent--attractants for activated NK cells. Thus the spectrum of action of MCP-1, -2 and -3 encompasses NK cells and chemokines are likely to play a role in regulating extravasation of these cells.

319 THE ROLE OF IL-8 IN PULMONARY INFLAMMATION DURING NEONATAL CHRONIC LUNG DISEASE

The development of chronic lung disease (CLD) following neonatal respiratory distress syndrome (RDS) is associated with pulmonary inflammation. We investigated the role of interleukin 8 (IL-8) in polymorphonuclear leucocyte (PMN) mediated inflammation during RDS and the early stages of CLD. Of the 14 pre-term infants (gestational age <31 weeks) recruited, 7 developed RDS only, and 6 developed CLD following RDS. Broncho-alveolar lavage fluid (BALF) was obtained from ventilated infants on days 1,5 and 14. The dilution of lung secretions was calculated using urea concentration. IL-8 and myeloperoxidase (MPO) concentrations and PMN counts of BALF were analysed. PMN chemotactic responses to IL-8 and BALF were assessed ex vivo using micro chemotaxis chambers (Neuro Probe). The median IL-8 concentrations of BALF from infants in the CLD group and the RDS only group were 218pg/ml and 1891pg/ml on day 1, 3585pg/ml and 2434pg/ml on day 5 and 6780pg/ml and 144pg/ml on day 14 respectively. The BALF IL-8 concentration correlated (Spearmans Rank) with the BALF PMN count (p=0.02) and with MPO (p=0.01). This study indicates that IL-8 may be associated with PMN activation and margination during neonatal RDS and CLD.

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The urokinase receptor is required for human monocyte chemotaxis in vitro.

Mononuclear phagocytes (Mphi) produce urokinase-type plasminogen activator (uPA) and also express a specific cell-surface receptor for urokinase, uPAR. The concomitant expression of these proteins provides a mechanism by which Mphi can degrade extracellular matrix proteins during directed cell migration. In this study, we sought to determine if uPAR plays a role in Mphi chemotaxis that is distinct from its role in matrix proteolysis. Exposing adherent monocytes to a chemotactic gradient causes plasma membrane uPAR to localize strongly to the leading edge of cell migration. Adherence alone or exposure to FMLP had no effect on uPAR expression. Using Boyden chamber chemotaxis assays, we demonstrate that treating mononuclear cells with an anti-uPAR mAb (either as an intact mAb or F[ab']2) ablates chemotaxis induced by FMLP and monocyte chemotactic peptide-1 (P < 0.001). Inactivating the catalytic activity of uPAR-bound uPA had no effect on chemotaxis. Similarly, blocking uPAR expression with an antisense oligonucleotide to uPAR completely ablates chemotaxis, but blocking uPA expression with an antisense oligonucleotide to uPA has a minimal effect. We therefore demonstrate that expression and unimpeded function of uPAR plays an obligate role in M phi chemotaxis by mechanisms that are largely independent of its ligand, uPA. Combined with its known role in mediating pericellular proteolysis, these observations demonstrate that uPAR is essential for both locomotion and traversing tissue barriers during M phi migration.

A rapid, multiwell colorimetric assay for chemotaxis

This paper describes a colorimetric assay for the rapid quantification of chemotaxis in multiple samples. In this assay, cells that have migrated through polycarbonate membrane filters are collected onto the bottom wells of a chemotaxis chamber after centrifugation then the number of viable cells collected in the bottom well is quantified by measurement of the reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenol tetrazolium bromide (MTT). The degree of MTT reduction, which corresponds to the relative cell number, is measured automatically with an ELISA reader. The MTT method of quantitation is as sensitive as the standard manual method, is especially useful for large numbers of samples and requires no specialized laboratory equipment.

Effect of antibiotics on sputum inflammatory contents in acute exacerbations of bronchiectasis

We studied the changes in sputum neutrophil chemotactic activity (NCA) and elastolytic activity (EA) in acute exacerbations of bronchiectasis before and after treatment with oral antibiotics. Twelve patients who chronically produced sputum were assessed in the stable state, and when they subsequently developed symptoms of acute exacerbations, prior to initiation of antibiotics, during 2 weeks of antibiotics, and at 2 and 6 weeks after stopping antibiotics. NCA was measured using modified Boyden's technique with multiwell chemotaxis chamber, and EA with N-succiny-trialanine- p-nitroanilide as elastase substrate. All 12 patients had NCA (49·3 ± 8·69% FMLP response) and EA (50·5 ± 17·1 mU per 100 μl) in their sputum in the stable state. At acute exacerbation, there was significant increase in NCA ( P < 0·001) and EA ( P < 0·05). All responded clinically after 1 week of antibiotics, and this was associated with a decrease in NCA and EA back to the levels in stable state. A further week of antibiotics did not result in further decline of NCA or EA. Three patients had another acute exacerbation clinically between 2–6 weeks after stopping antibiotics and their NCA and EA rose again. In the other nine patients, both NCA and EA at 2 and 6 weeks post-treatment were similar to pre-exacerbation levels. Our findings suggest that short course antibiotics effectively control the upsurge in inflammatory activity in acute axacerbations, but has little effect on chronic airway inflammation. This would challenge the validity of such a treatment strategy which is still the most common in clinical practice, when persistent airway inflammation is believed to lead to progressive lung destruction.

Serotonin and cardiac morphogenesis in the mouse embryo.

The possible involvement of the neurotransmitter serotonin (5-HT) and its binding protein (SBP) in cardiac morphogenesis was studied using mouse whole embryo culture (together with immunocytochemistry or 3H-thymidine autoradiography) and a cell migration assay. Embryos were cultured before and during the period of endocardial cushion formation, embryonic (E) days 9-12, in the presence of 5-HT, the monoamine oxidase (MAO) inhibitor nialamide, or an uptake inhibitor (fluoxetine or sertraline). For the migration assay, cells from the outflow tracts of E12 embryos were dissociated and placed in a chemotaxis chamber together with different concentrations of 5-HT. E9 embryos cultured in the presence of 10 microM 5-HT and nialamide exhibited intense 5-HT immunoreactivity (5-HT IR) throughout the myocardium. This staining was greatly diminished by fluoxetine, sertraline, or the absence of nialamide. As morphogenesis proceeded, myocardial staining in embryos exposed to 5-HT became restricted to developing endocardial cushion forming regions and was more completely blocked by uptake inhibitors. No evidence for 5-HT synthesis by myocardium was found at any age studied using the precursor L-tryptophan. SBP was present in endocardial cushions in cultured and uncultured embryos. 3H-thymidine autoradiography demonstrated that both fluoxetine and sertraline inhibited proliferation of cardiac mesenchyme, endocardium, and myocardium. These effects were most pronounced when exposure began at E9 (prior to cushion formation). Dose-dependent effects of 5-HT on migration of outflow tract cells were also observed. Taken together, these results suggest that 5-HT may play a role in cardiac morphogenesis during endocardial cushion formation.

Regulation of human mononuclear phagocyte migration by cell surface-binding proteins for advanced glycation end products.

Nonenzymatic glycation of proteins occurs at an accelerated rate in diabetes and can lead to the formation of advanced glycation end products of proteins (AGEs), which bind to mononuclear phagocytes (MPs) and induce chemotaxis. We have isolated two cell surface-associated binding proteins that mediate the interaction of AGEs with bovine endothelial cells. One of these proteins is a new member of the immunoglobulin superfamily of receptors (termed receptor for AGEs or RAGE); and the second is a lactoferrin-like polypeptide (LF-L). Using monospecific antibodies to these two AGE-binding proteins, we detected immunoreactive material on Western blots of detergent extracts from human MPs. Radioligand-binding studies demonstrated that antibody to the binding proteins blocked 125I-AGE-albumin binding and endocytosis by MPs. Chemotaxis of human MPs induced by soluble AGE-albumin was prevented in a dose-dependent manner by intact antibodies raised to the AGE-binding proteins, F(ab')2 fragments of these antibodies and by soluble RAGE. When MP migration in response to N-formyl-Met-Leu-Phe was studied in a chemotaxis chamber with AGE-albumin adsorbed to the upper surface of the chamber membrane, movement of MPs to the lower compartment was decreased because of interaction of the glycated proteins with RAGE and LF-L on the cell surface. The capacity of AGEs to attract and retain MPs was shown by implanting polytetrafluoroethylene (PTFE) mesh impregnated with AGE-albumin into rats: within 4 d a florid mononuclear cell infiltrate was evident in contrast to the lack of a significant cellular response to PTFE with adsorbed native albumin. These data indicate that RAGE and LF-L have a central role in the interaction of AGEs with human mononuclear cells and that AGEs can serve as a nidus to attract MPs in vivo.

Nitric oxide synthase inhibitors attenuate human monocyte chemotaxis in vitro.

Nitric oxide synthase (NOS) inhibitors have been shown to modulate neutrophil migration. We hypothesized that the NOS inhibitors NG-monomethyl-L-arginine (L-NMMA), NG-nitro-L-arginine methyl ester (L-NAME), and L-canavanine (L-CAN) also modulate human peripheral blood monocyte chemotaxis. To test this hypothesis, monocyte chemotaxis toward formylmethionylleucyl-phenylalanine (fMLP) was assessed using a modified blindwell chemotaxis chamber technique. L-NMMA and L-NAME, but not D-NMMA or L-CAN, significantly attenuated fMLP-induced monocyte chemotaxis (P < .05). L-Arginine and sodium nitroprusside, but not D-arginine, reversed NOS inhibitor-induced responses. Dibutryl cyclic guanyl monophosphate (cGMP) attenuated the inhibitory effects of L-NMMA on monocyte chemotaxis (P < .05). Finally, fMLP increased cGMP generation by monocytes, which was significantly attenuated by L-NMMA (P < .05). These data indicate that the L-arginine/NO biosynthetic pathway regulates human monocyte chemotaxis in vitro.

Neutrophil Chemotaxis to Leukotriene B4 In Vitro is Decreased for the Human Neonate

Leukotriene B4 (LTB4) is a product of arachidonic acid metabolism and a potent chemoattractant for adult polymorphonuclear leukocytes (PMN). LTB4 may be an important inflammatory mediator in neonatal lung disorders such as bronchopulmonary dysplasia, but neonatal PMN chemotaxis to LTB4 has not been studied. We compared total PMN migration and its components, chemotaxis and chemokinesis, to LTB4 in newborns and adults. PMN from healthy adults and umbilical blood of healthy, full-term newborns (n = 21 pairs) were incubated in a 48-well chemotaxis chamber using 10-microns thick polycarbonate membranes. Membranes with pore sizes of either 3 or 5 microns (diameter) were used to assess the influence of PMN deformability on chemotaxis. For both 3- and 5-microns filter pore sizes, total PMN migration increased in a dose-dependent manner from an LTB4 concentration of 10(-9) to 10(-6) M. The increase in total PMN migration was due entirely to chemotaxis (no chemokinesis) for newborns and adults. However, chemotaxis for the newborn was markedly attenuated, specifically, 14 and 24% of adult values at LTB4 concentrations of 10(-8) and 10(-7) M, respectively, with the 3-microns pore size. With the 5-microns filter pore size, newborn chemotaxis significantly increased to 40 and 49% of adult values at LTB4 concentrations of 10(-8) and 10(-7) M, respectively. We conclude that PMN chemotaxis to LTB4 in vitro is lower in newborns than in adults and part of this impairment may be caused by a decreased deformability of the newborn PMN.(ABSTRACT TRUNCATED AT 250 WORDS)

Vasoactive intestinal polypeptide (VIP) inhibits rat alveolar macrophage phagocytosis and chemotaxis in vitro

Vasoactive intestinal polypeptide (VIP) has been shown to inhibit lymphocyte function and is believed to modulate the immune response. We explored the possible immunomodulatory effects of VIP on alveolar macrophage (AM) function by examining its influence on AM phagocytosis and chemotaxis. Rat AMs were collected by bronchoalveolar lavage and incubated for 90 min with polystyrene beads in the presence or absence of VIP in concentrations from 10 −11 M to 10 −5 M. VIP significantly ( P < 0.0001) inhibited AM phagocytosis of polystyrene beads at concentrations of 10 −11 to 10 −6 M, with a maximal inhibition of 35% at 10 −6 M (but no inhibition at 10 −5 M). AMs were also incubated for 90 min in a chemotaxis chamber with endotoxin-activated rat serum (EARS) as a chemoattractant, with or without VIP in concentrations from 10 −9 to 10 −6 M. VIP significantly ( P < 0.0001) inhibited AM chemotaxis by at least 30% at concentrations of 10 −9 to 10 −6 M, with a maximal inhibition of 46% at 10 −7 M. These results indicate that VIP, in concentrations from 10 −11 to 10 −6 M, inhibits rat AM function as assessed by phagocytosis of polystyrene beads and chemotaxis to EARS. The inhibition of alveolar macrophage function is another mechanism by which VIP may modulate the immune response in the lung.

Interleukin-1 alpha and -beta augment pulmonary artery transendothelial albumin flux in vitro.

Human recombinant interleukin-1 alpha (rIL-1 alpha) and -beta were studied to determine whether either could alter the permeability of bovine pulmonary artery endothelial cell monolayers. Endothelial cells were grown to confluence on filters mounted in chemotaxis chambers placed in wells. Barrier function of the monolayers was assessed by placing 14C-labeled bovine serum albumin ([14C]BSA) in the upper chamber and sampling the lower well for [14C]BSA. rIL-1 alpha induced a significant (P less than 0.01) dose- and time-dependent increase in transendothelial [14C]BSA flux. rIL-1 alpha exposures as brief as 30 min increased permeability, but the increased albumin transfer could not be demonstrated before 4 h after exposure. Exposures up to 6 h were reversible at 24 h. rIL-1 alpha induced significantly (P less than 0.01) greater increments in [14C]BSA flux than did equivalent exposures to rIL-1 beta. No important differences between bovine and human rIL-1 beta were demonstrated. Increased transendothelial flux could not be ascribed to either endothelial cytotoxicity or growth inhibition. There was no additive or synergistic relationship between rIL-1 alpha and human recombinant tumor necrosis factor-alpha. Our studies suggest that IL-1 alpha and -beta may play a role in the pathogenesis of pulmonary vascular leak.