Changes in the motility, morphology, and F-actin architecture of human dendritic cells in an in vitro model of dendritic cell development.

An in vitro model has been developed for analyzing the two developmental phases of human dendritic cell (DC) migration. Employing the age of the culture and the addition of GM-CSF, IL-4, and serum to regulate cellular phenotype, and glass coated with acid-precipitated human plasma proteins to facilitate persistent DC translocation, the model produces three sequential in vitro phenotypes with the following suggested in vivo counterparts: (1) DCs recently isolated from blood, which are highly polar and motile, and reflect the behavior of "undifferentiated" DCs that must extravasate from the blood stream and migrate into peripheral tissue; (2) large, nonmotile, stellate DCs, which reflect the highly "differentiated" signature phenotype of DCs in peripheral tissue, whose function is to capture foreign antigens; and (3) the large, motile "dedifferentiated" DCs, which reflect the behavior of "veiled cells" that have captured an antigen, retracted dendritic processes, migrated out of peripheral tissue, and are in the process of transporting a captured antigen to a proximal draining lymph node for presentation to T cells. Computer-assisted motion analysis of the three sequential phenotypes and fluorescent staining of F-actin reveal three unique behavioral states and unique cellular architecture consistent with inferred in vivo function. This in vitro model should serve as a starting point for elucidating the cues and molecular mechanisms involved in the regulation of DC differentiation and motility.

Cytokine-induced sequential migration of neutrophils through endothelium and epithelium.

OBJECTIVE AND DESIGN: To better understand the mechanisms by which cytokines induced neutrophils to migrate into the airways, we constructed a novel in vitro model system. MATERIALS: Human umbilical vein endothelial cell (HUVE) monolayers were grown on top of permeable filters and human lung type II-like alveolar epithelial cell (A549) monolayers were grown on the undersurface of the filters. METHODS: The sequential migration of human neutrophils through the endothelium (apical to basal movement) and subsequently through the epithelium (basal to apical movement) in response to IL-1 beta or TNF alpha located basally to the epithelium was measured. RESULTS: We found that IL-1 beta and TNF alpha induced dose-responsive and time-dependent migration through the double monolayers-filter complex. The pattern of migration was similar, and the amount greater than or equal to that observed through either single monolayer/filter complex. Neutrophil migration through naked filters was generally less than that observed through the cellular barriers. The contribution of the monolayer orientation was also examined and found to favor the more physiologic directional migration of neutrophils through an endothelial and epithelial barrier, apical to basal and basal to apical, respectively. In contrast, FMLP-induced neutrophil migration was not dependent upon either the orientation or presence of the monolayer(s). CONCLUSIONS: Thus, we have established an in vitro model system to examine cytokine-induced sequential migration of neutrophils through endothelium and the respiratory epithelium in a manner analogous to that occurring with an in vivo airway stimulus causing neutrophil-rich airway inflammatory responses.

Combination of IL-8 plus TNF alpha induces additive neutrophil migration.

We have previously reported that TNF alpha's ability to induce neutrophil transendothelial and transepithelial migration was largely dependent on the generation of IL-8 by the endothelial or pulmonary epithelial cells. To further explore the interrelationship of IL-8 with TNF alpha, we examined the migration of human neutrophils through noncellular barriers in response to these cytokines alone or in combination. We directly compared neutrophil migration through 3 microns-pore size polycarbonate Transwell filters in response to 10 nM TNF alpha or either 10 nM TNF alpha or buffer plus 10(-8) to 10(-11) M IL-8. We found that the combination of TNF alpha and IL-8 induced neutrophil migration that was generally additive, and sometimes synergistic, to that of either agent alone. In conclusion, these data support the role of cytokine networking in inducing neutrophil-rich lung inflammatory responses.

The sequential migration of neutrophils through endothelium and epithelium: a new model system.

To better understand the mechanisms by which neutrophils migrate into the airways, we constructed a novel in vitro model system with human umbilical vein endothelial cell (HUVE) monolayers grown on top of permeable filters and human lung Type II-like alveolar epithelial cell (A549) monolayers grown on the undersurface of the filters. The sequential migration of human neutrophils through the endothelium (apical to basal movement) and subsequently through the epithelium (basal to apical movement) in response to a stimulus located basally to the epithelium was measured. We found that the neutrophil chemoattractants, formylmethionylleucylphenylalanine (FMLP), leukotriene B4 (LTB4), and interleukin-8 (IL-8), induced dose-responsive migration through the double monolayer-filter complex. The pattern of migration was similar to that observed through either a naked filter or single monolayer-filter complex. Maximal chemotaxis through the double monolayer-filter complex was observed by 3 hours. Thus, we have established an in vitro model system to examine the sequential migration of neutrophils through endothelium and the respiratory epithelium in a manner analogous to that occurring with an in vivo airway stimulus causing neutrophil-rich airway inflammatory responses.

T cell syncytia induced by HIV release. T cell chemoattractants: demonstration with a newly developed single cell chemotaxis chamber.

A chemotaxis chamber has been developed to analyze both the velocity and the directionality of individual T cells in gradients of high molecular mass molecules over long periods of time. Employing this chamber, it is demonstrated that syncytia induced by HIV in SUP-T1 cell cultures release two T cell chemoattractants with approximate molecular masses of 30 and 120 kDa. Neither uninfected single cells nor polyethylene glycol-induced syncytia release detectable chemoattractant, suggesting that these chemoattractants are linked to HIV infection. Soluble gp120 functions as a T cell chemoattractant and the addition of anti-gp120 antibody to syncytium-conditioned medium blocks the high molecular mass chemoattractant activity but not the low molecular mass activity. The addition of anti-CD4 antibody to syncytium-conditioned medium also blocks the high molecular mass chemoattractant activity but not the low molecular mass activity. These results demonstrate that HIV-induced T cell syncytia release a low and a high molecular mass T cell chemoattractant, and suggest that the high molecular mass factor is gp120 and that it functions through the CD4 receptor.

Cytokine-induced neutrophil transepithelial migration is dependent upon epithelial orientation.

The mechanisms by which mediators and cytokines stimulate neutrophils to migrate across the lung epithelium are still unclear. We hypothesized that neutrophil transepithelial migration depends upon polarity of the epithelium. We therefore compared neutrophil migration through human lung Type II-like alveolar epithelial cell line (A549) monolayers grown on the upper versus lower surface of permeable filters to simulate apical-to-basal and basal-to-apical movement of neutrophils, respectively. The classic chemoattractants formyl-methionylleucylphenylalanine (FMLP), leukotriene B4 (LTB4), and interleukin-8 (IL-8) induced equivalent neutrophil transepithelial migration in the apical-to-basal and basal-to-apical directions. However, the degree of neutrophil transepithelial migration was significantly greater in the basal-to-apical direction in response to either IL-1beta or tumor necrosis factor-alpha (TNF-alpha). Enhanced TNF-alpha-induced neutrophil migration through A549 monolayers in the basal-to-apical direction occurred regardless of whether the TNF-alpha was above or below the filter/monolayer complex. Actinomycin D pretreatment of A549 monolayers had no effect on FMLP-induced neutrophil transepithelial migration, but markedly (about 75%) inhibited both TNF-alpha- and IL-1beta-induced neutrophil transepithelial migration, regardless of monolayer orientation. Thus, in contrast to classic chemoattractants, IL-1beta and TNF-alpha induced greater neutrophil transepithelial migration in a basal-to-apical direction, and this occurred independently of the cytokine location, but depended upon intact metabolic capacity of the A549 cells. These data suggest that the mechanisms important for neutrophil transepithelial migration in response to classic chemoattractants differ from those important for migration in response to inflammatory cytokines.

Neutrophil transepithelial migration is dependent upon epithelial characteristics.

To better understand the mechanisms by which neutrophils migrate to the airway lumen during an inflammatory response, we constructed an in vitro model system to examine the interactions of human neutrophils, human lung epithelial cells, mediators, and proinflammatory cytokines. We directly compared neutrophil movement through three lung epithelial cell lines, A549, H441, and 16-HBE-14o, in response to three chemoattractants, FMLP, LTB4, and IL-8, and the proinflammatory cytokines IL-1 alpha and beta and TNF alpha. While there was variation in the responses to the chemotaxins, there was no correlation between the transmonolayer electrical resistance and the ability of the neutrophils to migrate across the epithelia in response to the agents used. FMLP, IL-8, and LTB4 induced dose- and time-dependent neutrophil migration across all three epithelia. However, TNF alpha- and IL-1-induced neutrophil migration occurred only through monolayers that produced soluble chemoattractants in response to these cytokines. Although all three epithelia produced low amounts of IL-8 constitutively, the capacity of IL-1 and TNF alpha to induce transepithelial migration was directly associated with the ability of the epithelia to produce large amounts of IL-8 in response to IL-1 and TNF alpha. We conclude that the phenotype of the epithelial cell (e.g., capacity to produce IL-8) affects stimulated neutrophil transepithelial migration.

Effects of neuropeptides on neutrophil migration through noncellular and endothelial barriers.

BACKGROUND: Neuropeptides and neutrophils are postulated to be important immune effector molecules and cells, respectively, in a variety of lung inflammatory conditions. METHODS: We examined the effects of three lung neuropeptides, substance P, vasoactive intestinal peptide (VIP), and somatostatin and two relatively protease-resistant peptides, helodermin and sandostatin, on human neutrophil migration across 3 microns pore filters and human endothelial monolayers cultured on these filters. RESULTS: At concentrations of 1 to 10 mumol/L, substance P induced significant neutrophil migration that was dose-responsive and equivalent through endothelium and filters. Neither VIP, helodermin, somatostatin, nor sandostatin induced significant neutrophil migration through either barrier at doses of 10(-14) to 10(-5) mol/L. Because VIP and somatostatin have been reported to inhibit some inflammatory cell functions, we also examined their effects on chemoattractant-induced neutrophil migration. Pretreatment of neutrophils and/or endothelium with VIP or somatostatin at either 10(-5) or 10(-10) mol/L did not significantly inhibit formyl-methionyl-leucyl-phenylalanine-, leukotriene B4-, or platelet activating factor-induced neutrophil migration through naked filters or endothelium. Thus only substance P had significant effects on neutrophil chemotaxis. CONCLUSIONS: Substance P, but not VIP or somatostatin, may be important in directly influencing neutrophil migration across endothelium towards lung inflammatory sites. However, all three neuropeptides might still modulate neutrophil functions and lung inflammatory responses through effects on other lung cells.

Effects of nedocromil sodium and WEB 2086 on chemoattractant-stimulated neutrophil migration through cellular and noncellular barriers.

Nedocromil sodium (Tilade) is an effective therapeutic agent against asthma and has been shown to exhibit antiinflammatory activity in vitro; however, its mode of action is yet to be described fully. Using an in vitro assay designed to mimic the extravasation of neutrophils from the peripheral circulation through cellular barriers to sites of inflammation, the effect of nedocromil sodium on chemoattractant-stimulated neutrophil migration was examined. We also examined the effects of WEB 2086, a platelet-activating factor (PAF) receptor antagonist, in parallel. Neutrophils and the cellular barrier were pretreated and/or co-incubated with nedocromil or WEB 2086 and the effects on neutrophil chemotaxis measured. In all treatments, nedocromil did not significantly affect chemotaxis through cellular or noncellular barriers to N-formyl-methionyl-leucyl-phenylalanine (FMLP), leukotriene B4 (LTB4), or PAF. In contrast, WEB 2086 inhibited PAF-induced neutrophil migration through both naked filters and endothelial and epithelial monolayers cultured on these filters. We conclude that while nedocromil has been shown to have inhibitory effects on neutrophils and is an effective therapeutic agent for asthma and inflammatory conditions, its activity is not primarily mediated by inhibition of neutrophil chemotaxis. Platelet-activating factor antagonists may partially be effective in asthma through inhibitory effects on neutrophil chemotaxis.

Degree of neutrophil chemotaxis is dependent upon the chemoattractant and barrier.

Stimulated neutrophil migration across lung endothelial and epithelial barriers is important in lung inflammatory processes. To better understand the interaction between chemoattractants, neutrophils, and endothelium and epithelium, we compared the ability of leukotriene B4 (LTB4), formylmethionylleucylphenylalanine (FMLP), and platelet-activating factor (PAF) to induce human neutrophil migration across 3-microns-pore filters alone and human umbilical vein endothelial (HUVE) cells and two different epithelial cell types, Madin-Darby canine kidney (MDCK) cells and human lung A549 cells, cultured in monolayers on these filters. LTB4, FMLP, and PAF induced neutrophil migration through naked filters, endothelial cells, and epithelial cells in a dose-related fashion. At optimal chemoattractant doses, LTB4, FMLP, and PAF induced relatively equivalent neutrophil migration through filters and endothelial and epithelial monolayers. However, the doses at which optimal neutrophil migration was observed to occur as well as the degree of neutrophil migration through the three barriers varied depending upon the chemoattractant. Based on dose-response experiments, the relative rank order of potency for the three chemoattractants was: LTB4 = FMLP greater than PAF for filter alone barrier; LTB4 greater than FMLP greater than PAF for HUVE cell barrier; and FMLP greater than LTB4 greater than PAF for MDCK and A549 epithelial cell barriers. Our data suggest that neutrophil chemotactic and subsequent lung inflammatory responses are interrelatedly influenced by both the quantity and type of chemoattractant present and the barrier through which the neutrophil must migrate.

Degree of platelet activating factor-induced neutrophil migration is dependent upon the molecular species.

Multiple molecular species of platelet activating factor (PAF) are produced as a result of inflammatory processes. PAF-induced neutrophil migration across endothelium is intrinsic to inflammatory responses. We therefore compared the ability of three naturally occurring PAF species (C16:0, C18:0, and C18:1), which only varied at carbon 1, to induce 51Cr-labeled human neutrophil migration across a naked 3-microns pore filter and human umbilical vein endothelial (HUVE) monolayers cultured on these filters. Time-course experiments indicated that all species of PAF tested induced significant neutrophil migration between 15 and 45 min. PAF-induced neutrophil migration through both filters alone and HUVE monolayers occurred at lower doses with C16:0 PAF. The rank order of chemotactic potency for the PAF species was C16:0 greater than C18:0 greater than C18:1 with both filters and endothelium coated filters as barriers. Intrinsic differences in the potency of these PAF molecular species to induce neutrophil chemotaxis were greater when HUVE cells were the barriers vs when filters alone were the barriers. Regardless of the molecular species used, at optimal PAF doses the degree of neutrophil migration through HUVE cells was often greater than that through filters alone. The specific PAF antagonist WEB 2086 inhibited neutrophil migration induced by all three PAF species equally. WEB 2086 pretreatment of the neutrophil or WEB 2086 coincubation with PAF, but not WEB 2086 pretreatment of the HUVE cell monolayers, significantly inhibited (65 to 80%) neutrophil migration. We conclude that the degree of PAF-induced neutrophil migration is dependent upon the molecular species of PAF. Moreover, the barrier through which the neutrophil must migrate appears to be important in influencing the overall chemotactic response to the various PAF species.

Some murine thymic lymphocytes can form gap junctions.

Analysis of thymic lymphocytes isolated from weanling mice has revealed a minority population able to form permeable, intercellular (gap) junctions. This population is largest in mice aged between 3 and 6 weeks, much smaller in fetal and new-born mice and undetectable in mice aged 12 weeks or more. Fractionation of the thymocytes on Percoll gradients or with peanut agglutinin (PNA) shows the cells able to form junctions are enriched in lower density fractions and agglutinated by PNA, suggesting they are among the most immature. Fractionation by complement mediated cytotoxicity (CMC) and by fluorescence activated cell sorting (FACS) using monoclonal antibodies to specific cell surface determinants shows the junction forming cells are Lyt-1+/Lyt-2- and that the phenotype is associated with both high and low Thy-1 and H-2K epitope densities.