Different pathways leading to cutaneous leukocytoclastic vasculitis in mice.

To investigate the pathomechanisms of leukocytoclastic vasculitis (LcV) we compared mouse models of LcV with non-vasculitic irritant contact dermatitis (ICD). Criteria for LcV as met by the immune complex-mediated Arthus reaction (Art-r) were also fulfilled by the localized Shwartzman reaction (Shw-r) and by cutaneous Loxoscelism (Lox) (injection of venom from Loxosceles reclusa containing sphingomyelinase D). After depletion of PMN (by gamma-irradiation) vessel damage could not be elicited in these models, distinguishing them from models of direct endothelial insult (necrotizing ICD). Depletion of complement could only delay, but not inhibit the Art-r, and did not change ICD, Lox or the Shw-r. The Shw-r exclusively revealed a sustained local expression of vascular adhesion molecules for 24 h in the preparatory phase (LPS s.c.), not observed in the Art-r, in Lox or ICD. Subsequent challenge with LPS i.p. was associated with upregulation of Mac-1 and ICAM-1 on PMN, but not of VLA-4 or LFA-1 (FACS analysis). Cytokines which were able to replace LPS in priming for LcV in the Shw-r (TNF-alpha and IL-1beta) also induced sustained expression of adhesion molecules, whereas IL-12 and IFN-gamma did neither. Neutralizing IL-12 or IFN-gamma also inhibited neither LcV nor sustained expression of adhesion molecules, whereas anti-TNF-alpha inhibited both. Anti-TNF-alpha had no marked inhibitory effects in the Art-r, in Lox or ICD. Combined (but not separate) neutralization of both E-selectin and VCAM-1 by antibodies suppressed LcV independent from reducing influx of PMN, proving that their sustained expression is decisive for the Shw-r and interferes with normal diapedesis. Since Loxosceles venom is known to dysregulate diapedesis and degranulation of PMN in vitro, since adherent immune complexes activate PMN at the vessel wall, and since adhesion molecules are dysregulated in the Shw-r, we suggest that LcV develops when activation of PMN coincides with vascular alterations which interfere with normal diapedesis.

Species-specific and conserved epitopes on mouse and human E-selectin important for leukocyte adhesion.

Selectins are C-type, cell surface lectins that are key players in leukocyte adhesion to the blood vessel wall endothelium. We describe here epitopes for a series of novel monoclonal antibodies (moAbs), UZ4-UZ7, directed against mouse E-selectin. All four antibodies specifically bind to mouse E-selectin, but not to P- or L-selectin, and all inhibit the adhesion of granulocytes, peripheral blood lymphocytes, and promyelocytic HL-60 cells to cytokine-activated mouse endothelium. Three moAbs, UZ5, UZ7, and UZ6, specifically inhibit mouse E-selectin-mediated adhesion by binding to epitopes in domains CR1 or CR2. moAb UZ4 inhibits leukocyte adhesion to both human and murine endothelium activated with IL-1 or other proinflammatory stimuli. UZ4 is the first described moAb that detects an epitope in the lectin domain which is conserved in both murine and human E-selectin (CXKKKL), but is not present in the other members of the selectin family, P- and L-selectin. Interestingly, UZ5, UZ6, and UZ7 more efficiently interfere with lymphocyte than with granulocyte adhesion to cytokine-activated endothelium, while UZ4 completely blocks adhesion of PMN, lymphocytes, and HL-60 and U937 cell lines. The data suggest that E-selectin-ligand engagement differs between lymphocytes and PMN, and that these differences may be accentuated by the CR1 and CR2 domains in the E-selectin cell adhesion molecule.

The EPS of Acidithiobacillus ferrooxidans--a model for structure-function relationships of attached bacteria and their physiology.

To dissolve pyrite or sulphur, leaching bacteria like Acidithiobacillus ferrooxidans attach to these substrata by extracellular polymeric substances (specifically, lipopolysaccharides). The primary attachment to pyrite at pH 2 is mediated by exopolymer-complexed iron(III) ions in an electrostatic interaction with the negatively charged pyrite surface. Cells grown on sulphur exhibit a different composition of the extracellular lipopolysaccharides, namely with increased hydrophobic properties, and do not attach to pyrite. Thus, the cells adapt the chemical composition of their exopolymers to the substrate/substratum. It is concluded that the mechanism of bacterial pyrite oxidation is basically indirect. The actual corrosive agents are iron(III) ions. Preliminary data indicate that active strains complex more iron(III) ions in their EPS than less active ones. Obviously, the exopolymeric layer comprises a reaction space for the regeneration of these ions by the activity of the iron oxidising bacteria.

Endothelial cell laminin isoforms, laminins 8 and 10, play decisive roles in T cell recruitment across the blood-brain barrier in experimental autoimmune encephalomyelitis.

An active involvement of blood-brain barrier endothelial cell basement membranes in development of inflammatory lesions in the central nervous system (CNS) has not been considered to date. Here we investigated the molecular composition and possible function of the extracellular matrix encountered by extravasating T lymphocytes during experimental autoimmune encephalomyelitis (EAE). Endothelial basement membranes contained laminin 8 (alpha4beta1gamma1) and/or 10 (alpha5beta1gamma1) and their expression was influenced by proinflammatory cytokines or angiostatic agents. T cells emigrating into the CNS during EAE encountered two biochemically distinct basement membranes, the endothelial (containing laminins 8 and 10) and the parenchymal (containing laminins 1 and 2) basement membranes. However, inflammatory cuffs occurred exclusively around endothelial basement membranes containing laminin 8, whereas in the presence of laminin 10 no infiltration was detectable. In vitro assays using encephalitogenic T cell lines revealed adhesion to laminins 8 and 10, whereas binding to laminins 1 and 2 could not be induced. Downregulation of integrin alpha6 on cerebral endothelium at sites of T cell infiltration, plus a high turnover of laminin 8 at these sites, suggested two possible roles for laminin 8 in the endothelial basement membrane: one at the level of the endothelial cells resulting in reduced adhesion and, thereby, increased penetrability of the monolayer; and secondly at the level of the T cells providing direct signals to the transmigrating cells.

Cell adhesion and migration properties of beta 2-integrin negative polymorphonuclear granulocytes on defined extracellular matrix molecules. Relevance for leukocyte extravasation.

Regulated adhesion of leukocytes to the extracellular matrix is essential for transmigration of blood vessels and subsequent migration into the stroma of inflamed tissues. Although beta(2)-integrins play an indisputable role in adhesion of polymorphonuclear granulocytes (PMN) to endothelium, we show here that beta(1)- and beta(3)-integrins but not beta(2)-integrin are essential for the adhesion to and migration on extracellular matrix molecules of the endothelial cell basement membrane and subjacent interstitial matrix. Mouse wild type and beta(2)-integrin null PMN and the progranulocytic cell line 32DC13 were employed in in vitro adhesion and migration assays using extracellular matrix molecules expressed at sites of extravasation in vivo, in particular the endothelial cell laminins 8 and 10. Wild type and beta(2)-integrin null PMN showed the same pattern of ECM binding, indicating that beta(2)-integrins do not mediate specific adhesion of PMN to the extracellular matrix molecules tested; binding was observed to the interstitial matrix molecules, fibronectin and vitronectin, via integrins alpha(5)beta(1) and alpha(v)beta(3), respectively; to laminin 10 via alpha(6)beta(1); but not to laminins 1, 2, and 8, collagen type I and IV, perlecan, or tenascin-C. PMN binding to laminins 1, 2, and 8 could not be induced despite surface expression of functionally active integrin alpha(6)beta(1), a major laminin receptor, demonstrating that expression of alpha(6)beta(1) alone is insufficient for ligand binding and suggesting the involvement of accessory factors. Nevertheless, laminins 1, 8, and 10 supported PMN migration, indicating that differential cellular signaling via laminins is independent of the extent of adhesion. The data demonstrate that adhesive and nonadhesive interactions with components of the endothelial cell basement membrane and subjacent interstitium play decisive roles in controlling PMN movement into sites of inflammation and illustrate that beta(2)-integrins are not essential for such interactions.

TNF-alpha-induced expression of adhesion molecules in the liver is under the control of TNFR1--relevance for concanavalin A-induced hepatitis.

TNF-alpha has been clearly identified as central mediator of T cell activation-induced acute hepatic injury in mice, e.g., Con A hepatitis. In this model, liver injury depends on both TNFRs, i.e., the 55-kDa TNFR1 as well as the 75-kDa TNFR2. We show in this report that the hepatic TNFRs are not transcriptionally regulated, but are regulated by receptor shedding. TNF directly mediates hepatocellular death by activation of TNFR1 but also induces the expression of inflammatory proteins, such as cytokines and adhesion molecules. Here we provide evidence that resistance of TNFR1(-/-) and TNFR2(-/-) mice against Con A hepatitis is not due to an impaired production of the central mediators TNF and IFN-gamma. Con A injection results in a massive induction of ICAM-1, VCAM-1, and E-selectin in the liver. Lack of either one of both TNFRs did not change adhesion molecule expression in the livers of Con A-treated mice, presumably reflecting the fact that other endothelial cell-activating cytokines up-regulated adhesion molecule expression. However, treatment of TNFR1(-/-) and TNFR2(-/-) mice with murine rTNF revealed a predominant role for TNFR1 for the induction of hepatic adhesion molecule expression. Pretreatment with blocking Abs against E- and P-selectin or of ICAM(-/-) mice with anti-VCAM-1 Abs failed to prevent Con A hepatitis, although accumulation of the critical cell population, i.e., CD4(+) T cells was significantly inhibited. Hence, up-regulation of adhesion molecules during acute hepatitis unlikely contributes to organ injury but rather represents a defense mechanism.

UVB-irradiated T-cells undergoing apoptosis lose L-selectin by metalloprotese-mediated shedding.

PURPOSE: L-selectin (CD62L) is a prerequisite for leucocyte adhesion to endothelial cells of blood vessels and consequently for transmigration. Its expression on the cell surface therefore regulates the ability of lymphocytes to enter lymph nodes, to re-enter blood vessels or to invade tissues at sites of inflammation. The aim of this study was to determine the expression of CD62L on apoptotic lymphocytes after UVB irradiation. MATERIALS AND METHODS: Peripheral blood mononuclear cells (PBMC) were isolated from peripheral blood of normal healthy volunteers. Cells were stimulated with phorbol myristate acetate (PMA) and ionomycin for activation. Apoptosis in peripheral T-cells and Jurkat cells was induced by irradiation with UVB (120 mJ/cm2). In addition, T-cells or Jurkat cells were cultured for the indicated time with anti-Fas antibody CH11. The CH11-induced apoptosis was inhibited by the pan-caspase inhibitor zVAD-fmk. For detection of apoptosis, cells were analysed by cytofluorometry for morphological changes typical for apoptosis. The reliability of the apoptotic cell gate was confirmed by staining with FITC-labelled annexin-V in the presence ofpropidium iodide (PI). For FACS analysis of CD62L expression on the cell-surface immunofluorescence was performed using FITC-conjugated anti-CD62L and PE-conjugated anti-CD3 antibodies. Soluble CD62L (sCD62L) in the cell supernatants was measured by standard ELISA technique. Assays were performed in the presence and absence of metalloprotease inhibitor KB8301. RESULTS: PBMC from healthy volunteers undergoing apoptosis following UVB irradiation selectively shed CD62L, whereas the expression of the lineage-specific marker CD3 showed only minor changes. Shedding was blocked by the hydroxamic acid-based metalloprotease inhibitor KB8301. When Jurkat cells were treated with the caspase inhibitor zVAD-fmk, anti-CD95 antibodies did not induce apoptosis, and the expression of CD62L remained unaltered. CONCLUSION: UVB or ionizing radiation induce apoptosis in lymphocytes. The loss of CD62L is associated with apoptosis and will influence lymphocyte trafficking and, by excluding them from CD62L-mediated adhesion and tissue invasion, might contribute to the regulation of inflammation.

Characterization of iron uptake from transferrin by murine endothelial cells.

Iron is required by the brain for normal function, however, the mechanisms by which it crosses the blood-brain barrier (BBB) are poorly understood. The uptake and efflux of transferrin (Tf) and Fe by murine brain-derived (bEND3) and lymph node-derived (m1END1) endothelial cell lines was compared. The effects of iron chelators, metabolic inhibitors and the cellular activators, lipopolysaccharide (LPS) and tumour necrosis factor-alpha (TNF-alpha), on Tf and Fe uptake were investigated. Cells were incubated with 59Fe-125I-Tf; Fe uptake was shown to increase linearly over time for both cell lines, while Tf uptake reached a plateau within 2 h. Both Tf and Fe uptake were saturable. bEND3 cells were shown to have half as many Tf receptors as m1END1 cells, but the mean cycling times of a Tf molecule were the same. Tf and Fe efflux from the cells were measured over time, revealing that after 2 h only 25% of the Tf but 80% of the Fe remained associated with the cells. Of 7 iron chelators, only deferriprone (L1) markedly decreased Tf uptake. However, Fe uptake was reduced by more than 50% by L1, pyridoxal isonicotinoyl hydrazone (PIH) and desferrithiocin (DFT). The cellular activators TNF-alpha or LPS had little effect on Tf turnover, but they accelerated Fe uptake in both endothelial cell types. Phenylarsenoxide (PhAsO) and N-ethyl maleimide (NEM), inhibitors of Tf endocytosis, reduced both Tf and Fe uptake in both cell lines, while bafilomycin A1, an inhibitor of endosomal acidification, reduced Fe uptake but did not affect Tf uptake. The results suggest that Tf and Fe uptake by both bEND3 and m1END1 is via receptor-mediated endocytosis with release of Fe from Tf within the cell and recycling of apo-Tf. On the basis of Tf- and Fe-metabolism both cell lines are similar and therefore well suited for use in in vitro models for Fe transport across the BBB.

Low-dose radiotherapy selectively reduces adhesion of peripheral blood mononuclear cells to endothelium in vitro.

BACKGROUND AND PURPOSE: The anti-inflammatory effect of low-dose radiotherapy (LD-RT) still is not understood. The adhesion of leukocytes to endothelial cells (EC) of the vessel wall is the initial event of tissue invasion, and thus, crucially contributes to the regulation of inflammation. We investigated the influence of LD-RT on the adhesion process in vitro. MATERIALS AND METHODS: Isolated peripheral-blood-mononuclear-cells (PBMC) were incubated with an activated murine endothelioma cell-line under shear conditions at 4 degrees C after irradiation with single doses between 0.1 and 10.0 Gy. Adherent cells were counted microscopically and compared to a non-irradiated control. In parallel, viability and expression of adhesion molecules, especially of L-selectin, and lineage-specific markers on the cell surface were determined by dye exclusion and cytofluorometry, respectively. Modulation of adhesion by soluble L-selectin was tested in the adhesion assay. RESULTS: Radiation doses of 0.1-0.5 Gy reduced the adhesion of viable PBMC to EC in vitro by 70% of the control level 4 h after irradiation. Leukocytes showed a marked reduction of L-selectin expression after LD-RT. Soluble L-selectin can inhibit the adhesion of PBMC to EC. CONCLUSION: The anti-inflammatory effect of LD-RT might, in part, be due to the reduction in the adhesion of PBMC to EC. This reduction in adhesion might be a consequence of the reduced expression of L-selectin on the surface of PBMC, and the inhibition of adherence by soluble L-selectin shed by PBMC in vitro.

Specialized patterns of vascular differentiation antigens in the pregnant mouse uterus and the placenta.

The success of pregnancy depends on the ability of trophoblast cells to infiltrate the maternal decidua and breach uterine vessels. To ask whether the antigenic phenotype of maternal endothelial cells (EC) in the vascular zone and central decidua basalis may reflect a specialized programming of these vessels for interaction with the trophoblast, we did a survey of several mouse EC differentiation antigens, including MECA-32, MECA-99, and endoglin. Our results revealed striking differences in the phenotype of endothelial lining of vessels in the distinct compartments of the pregnant uterus during Day 9 of pregnancy and at midgestation. Vessels in the central decidua basalis and the vascular zone showed strong expression of MECA-99 but only weak expression of MECA-32, contrasting with the MECA-99(lo), MECA-32(hi) vessels in the capsularis. The vascular zone in addition stained brightly with anti-endoglin. Importantly, invading trophoblast as well as trophoblast cells lining maternal blood spaces were MECA-99(+), MECA-32(-), and endoglin(-), suggesting that the expression of MECA-99 may reflect a specialized co-programming of these trophoblast and EC for future interaction, but also that trophoblast cells may mimic selected antigenic characteristics of endothelium in association with their role in lining maternal blood spaces. In the term pregnant uterus the expression of all differentiation antigens decreased dramatically, suggesting that trophoblast cells as well as maternal EC lose their selected antigenic characteristics when the process of placentation is complete.

Evidence of specialized leukocyte-vascular homing interactions at the maternal/fetal interface.

In normal pregnancy, the maternal immune system fails to reject the fetus or the placenta as an allogeneic graft. We hypothesize that specialized mechanisms of leukocyte recruitment might limit access of circulating maternal immune cells to the maternal/fetal interface. During the critical period of initial trophoblast invasion there is an elegantly orchestrated progression of leukocyte homing events in the decidua basalis, associated with highly regulated expression of vascular addressins and segregation of specialized leukocyte subsets into well-defined decidual microdomains. Neutrophils are limited to the region of necrosis associated with enzymatic digestion at the leading edge of the invading trophoblast, where an almost linear array of maternal blood vessels displays the neutrophil ligand E-selectin. Cells with the phenotype of monocytes but expressing alpha4beta7 integrin are localized in the blood vessels of the specialized "vascular zone", which display the unusual combination of P-selectin (partially associated with platelets) and the alpha4beta7 ligand mucosal vascular addressin-1 (MAdCAM-1). Granulated metrial gland cells (alpha4+beta7-, probably alpha4beta1+) constitute a well-defined cluster positioned in the central decidua basalis around venules prominently expressing the alpha4beta1 ligand VCAM-1 (but not MAdCAM-1). T and B lymphocytes are rare. Our results suggest that selective mechanisms for regulating leukocyte access, associated with microdomain specialization within the decidua basalis, may play a fundamental role in immune regulation during the invasive period of placental development.

Expression of laminin alpha1, alpha2, alpha4, and alpha5 chains, fibronectin, and tenascin-C in skeletal muscle of dystrophic 129ReJ dy/dy mice.

The dy/dy mouse is an animal model for human merosin-negative congenital muscular dystrophy (CMD), which has been reported to have reduced or no expression of the basement membrane protein laminin alpha2. We here investigate various myogenic and nonmyogenic tissues of mature dy/dy and control 129ReJ mice histologically and for laminin alpha2 expression. In addition, expression patterns of laminin alpha1, alpha2, alpha4, and alpha5 chains, the interstitial proteins fibronectin and tenascin-C, and the adhesion molecules VCAM-1, ICAM-1, and alpha4 integrin were characterized in skeletal muscle of 1- and 7-day and mature (>6 weeks old) dy/dy and control 129ReJ mice. The laminin alpha2 chain remained detectable in myogenic tissues of dy/dy mice by immunofluorescence using two different monoclonal antibodies and by Northern blot analysis. However, laminin alpha2 expression was significantly reduced or not detectable in nonmyogenic tissues of dy/dy mice, including skin, lung, kidney, brain, thymus, and eye. Focal lesions were observed in mature skeletal muscle only, characterized by necrotic tissue, isolated VCAM-1- and ICAM-1-positive cells indicative of inflammatory processes, and regenerating muscle fibers surrounded by intense tenascin-C and fibronectin expression. In contrast to studies on human CMD muscle, laminin alpha1 was not detectable in either dy/dy or control skeletal muscle using immunofluorescence or Northern blot analysis. Immunofluorescence localized laminin alpha4 to basement membranes of blood vessels, the endoneurium of the intramuscular nerves, and the neuromuscular junction in skeletal muscle of 1- and 7-day-old dy/dy and control mice. In mature muscle, laminin alpha4 expression shifted to the perineurium of intramuscular nerves in both dy/dy and control mice. Furthermore, strong upregulation of laminin alpha4 in the basement membranes of blood vessels, the perineurium of intramuscular nerves, and of isolated regenerating muscle fibers in the dy/dy mice was apparent. Investigation of 1-day-old animals revealed expression of laminin alpha5 in skeletal muscle fiber basement membranes of dy/dy but not control animals. This difference between dy/dy and control animals was no longer apparent at 7 days after birth, indicating a temporary shift in expression pattern of laminin alpha5 in dy/dy animals. Analysis of the extracellular matrix components of 1- and 7-day-old dy/dy and control skeletal muscle revealed an early onset of the dystrophy, even before histopathological features of the disease were evident. Our data confirm the absence of laminin alpha1 chain in myogenic tissues of both dy/dy and control mice and suggest compensation for reduced laminin alpha2 in dy/dy skeletal muscle by laminin alpha4 and, in early development, also laminin alpha5. These results have significant ramifications in the diagnosis of human merosin-negative CMD.

Control of neonatal tolerance to tissue antigens by peripheral T cell trafficking.

Self tolerance is acquired by the developing immune system. As reported here, particular properties of the neonatal tissue contribute to this process. Neonatal skin, but not adult skin, was accessible for naïve CD8 T cells. In mouse bone marrow chimeras generated at different ages, recent thymic emigrants were tolerized to a skin-expressed major histocompatibility complex class I antigen only during a neonatal period but not during adulthood. Blockade of T cell migration neonatally prevented tolerance induction. Thus, T cell trafficking through nonlymphoid tissues in the neonate is crucial for the establishment of self tolerance to sessile, skin-expressed antigens.

CD4+ T cells migrate into inflamed skin only if they express ligands for E- and P-selectin.

Previous data suggested a role of endothelial selectins in skin homing of lymphocytes. In the current study, we have analyzed the expression and functional role of E-and P-selectin ligands on CD4+ T cells induced in vivo upon skin sensitization, using soluble selectin-Ig chimera and blocking Abs. Only low numbers of CD4+ cells expressing significant levels of E- or P-selectin ligands were present in s.c. lymph nodes of untreated mice (0.5-1.5% and 2-4%, respectively). Induction of a delayed-type hypersensitivity reaction increased the percentage of E-selectin-binding CD4+ cells in the draining lymph nodes up to 6 to 9% and that of P-selectin-binding cells up to 14%. The majority of E- and P-selectin-binding cells displayed an activated phenotype as judged by the increase in IL-2R, CD71, or cell size. The populations of E- and P-selectin-binding cells were largely overlapping; all E-selectin-binding cells also bound to P-selectin, whereas only a subfraction of P-selectin-binding cells reacted with E-selectin. Both E- and P-selectin-binding CD4+ cells, isolated by FACS, efficiently migrated into inflamed, but not normal skin, whereas P- or E-selectin ligand-negative CD4+ T cells did not. Abs against one of the two endothelial selectins partially inhibited the entry of isolated, ligand-positive cells, whereas a combination of Abs against both selectins almost completely abrogated skin homing. These data indicate that the expression of functional ligands for E- and for P-selectin is essential for homing of CD4+ T cells into the inflamed skin.

E- and P-selectin are not involved in the recruitment of inflammatory cells across the blood-brain barrier in experimental autoimmune encephalomyelitis.

In experimental autoimmune encephalomyelitis (EAE) inflammatory cells cross the endothelial blood-brain barrier (BBB) and gain access to the central nervous system (CNS). Here we show that E- and P-selectin are not involved in the recruitment of inflammatory cells across the BBB. Neither expression of E- nor P-selectin is induced in BBB-forming endothelium at any time after initiation of EAE. Some of the inflammatory cells present in the CNS during EAE express ligands for E- or P-selectin. However, anti-E- and P-selectin antibodies influence neither immigration of inflammatory cells across the BBB nor the development of EAE. In general, suppression of E- and P-selectin expression on BBB endothelium is dependent on factors derived from the CNS microenvironment, eg, astrocytes. Our results suggest that during EAE suppression of E- and P-selectin expression on the BBB provides a CNS-specific mechanism to reduce leukocyte recruitment into the CNS.

Integrin-mediated intracellular Ca2+ signaling in Jurkat T lymphocytes.

T lymphocytes interact with components of the extracellular matrix after transendothelial migration on their way to sites of inflammation. To characterize the molecular basis of the interaction between T lymphocytes with different extracellular matrix proteins, we investigated the role of intracellular Ca2+ as a signal mediating such interactions and identified the cell surface integrins involved in this process. When Jurkat T lymphocytes loaded with the calcium-sensitive fluorescent dye fura-2 were placed on coverslips coated with human fibronectin, human collagen types I, IV, and VI, human tenascin, human laminin I, or mouse laminin I, an elevation in intracellular Ca2+ concentration was observed. In contrast, contact of the Jurkat T lymphocytes with vitronectin and thrombospondin did not induce Ca2+ signals in more cells as compared with control measurements in which cells were in contact with only BSA or polylysine. Furthermore, the percentage of Jurkat T lymphocytes responding with Ca2+ signals to collagen types I and IV, fibronectin, and laminin I was completely reduced to levels observed on BSA or polylysine when the cells were pretreated with specific anti-integrin Abs, suggesting a role for cell surface integrins as mediators of cell matrix-induced intracellular Ca2+ signaling. Similar results were obtained with peripheral human T lymphocytes activated by phytohemagglutinin.

P-selectin glycoprotein ligand-1 (PSGL-1) on T helper 1 but not on T helper 2 cells binds to P-selectin and supports migration into inflamed skin.

We have shown recently that mouse Th1 cells but not Th2 cells are selectively recruited into inflamed sites of a delayed-type hypersensitivity (DTH) reaction of the skin. This migration was blocked by monoclonal antibodies (mAb) against P- and E-selectin. Here we show that Th1 cells bind to P-selectin via the P-selectin glycoprotein ligand-1 (PSGL-1). This is the only glycoprotein ligand that was detectable by affinity isolation with a P-selectin-Ig fusion protein. Binding of Th1 cells to P-selectin, as analyzed by flow cytometry and in cell adhesion assays, was completely blocked by antibodies against PSGL-1. The same antibodies blocked partially the migration of Th1 cells into cutaneous DTH reactions. This blocking activity, in combination with that of a mAb against E-selectin, was additive. PSGL-1 on Th2 cells, although expressed at similar levels as on Th1 cells, did not support binding to P-selectin. Thus, the P-selectin-binding form of PSGL-1 distinguishes Th1 cells from Th2 cells. Furthermore, PSGL-1 is relevant for the entry of Th1 cells into inflamed areas of the skin. This is the first demonstration for the importance of PSGL-1 for mouse leukocyte recruitment in vivo.

P- and E-selectin mediate recruitment of T-helper-1 but not T-helper-2 cells into inflammed tissues.

When activated, T helper cells differentiate into one of two subsets, Th1 and Th2, characterized by distinct profiles of cytokine production. Th1 cells activate pro-inflammatory effector mechanisms involved in protection and autoimmunity, whereas Th2 cells induce humoral and allergic responses and downregulate local inflammation. Apart from differences in the repertoire of cytokines, no phenotypic attributes are established that distinguish the two subsets. Here we show that Th1 cells, but not Th2 cells, are able to bind to P-selectin and E-selectin. Moreover, only Th1 cells can efficiently enter inflamed sites in Th1-dominated models, such as sensitized skin or arthritic joints, but not in a Th2-dominated allergic response. Immigration of Th1 cells into inflamed skin can be blocked by antibodies against P- and E-selectin. These results provide evidence for adhesion mechanisms to distinguish between the two T helper subsets and mediate their differential trafficking. They indicate that selective recruitment is an additional level of regulation for both effector function profile and character of a local immune response.

Mouse polymorphonuclear granulocyte binding to extracellular matrix molecules involves beta 1 integrins.

The mechanism of adhesion of purified mouse polymorphonuclear granulocytes (PMN) to extracellular matrix proteins characteristic of basement membranes and the interstitium has been investigated and compared with the adhesion of a mouse progranulocytic cell line, 32DC13, and a mouse monocytic cell line, WEHI 78/24. All three cell types bound specifically to fibronectin and vitronectin to different degrees under different cellular activation states. 32DC13 bound to fibronectin and vitronectin strongly, and this binding increased upon cellular activation with phorbol 12-myristate-13-acetate (PMA) but not with formyl-Met-Leu-Phe. Only 32DC13 showed significant binding to laminin-1. By contrast, WEHI 78/24 and PMN bound only fibronectin and vitronectin; this binding was weak and was altered only marginally upon activation with PMA. In the case of WEHI 78/24, a slight increase in adhesion both to fibronectin and to vitronectin was observed after cellular activation with PMA, while PMN adhesion to both substrates was slightly reduced. The mechanism of binding to fibronectin and vitronectin was similar in the three cell types. The integrin alpha5 beta1 mediated fibronectin adhesion, demonstrating for the first time the existence of a functionally active beta1 integrin on mouse PMN. Vitronectin binding was mediated by alpha(v) beta3, as demonstrated by the ability of alpha(v)-specific cyclic L-Arg-L-Gly-L-Asp-D-Phe-L-Val (RGDfV) peptide (EMD66203), and anti-beta3 antibody to inhibit cell adhesion. 32DC13 adhesion to laminin-1 was via the alpha6 beta1 integrin. None of the three cell types tested bound to the basement membrane proteins collagen type IV and perlecan, or to the interstitial stromal constituents tenascin, collagen types I, V and VI. Interestingly, perlecan and collagen type IV were found to repel all three cell types. The relative inability of PMN, WEHI 78/24, and 32DC13 to bind to extracellular matrix proteins characteristic of basement membranes and their ability to bind inflammatory markers of the interstitium is discussed with respect to leukocyte extravasation processes.