Global expression analysis of the fibroblast transcriptional response to TGFbeta.

OBJECTIVES: Transforming Growth Factor-beta (TGFbeta) is the predominant cytokine in all forms of fibrotic reactions. As well as being secreted by immune modulators of fibrosis such as macrophages, it is involved in an autocrine feedback loop of fibroblast stimulation whose regulation is still poorly understood. We wished to gain some insight into the mechanisms of the fibroblast response to TGFbeta. METHODS: We undertook an exhaustive transcript profiling experiment using a widely validated restriction enzyme based method for identifying differentially expressed genes (GeneCalling). Transcriptional responses throughout a 24-hour time course were examined at multiple time points and classified. RESULTS: By 24 hours of TGF treatment over 1000 bands, representing a large number of transcripts, were down- or upregulated greater than 2-fold. All of the known genes responsive to TGFbeta, such as collagen and connective tissue growth factor, were upregulated. CONCLUSIONS: This encyclopedic method revealed many unknown transcriptional responses to TGFbeta including the upregulation of a variety of less expected cytoskeletal and matrix components, as well as interactions between the TGFbeta and tumor necrosis factor (TNF) pathways and alterations in cell death-related pathways. These may in part explain the idiosyncratic responses of mesenchymal cells to TGFbeta.

IgM and stromal cell-associated heparan sulfate/heparin as complement-independent ligands for CD19.

The more severe phenotype of mice lacking CD19 as compared to CD21 suggests that a complement-independent ligand for the CD19/CD21 complex exists. We sought ligands for CD19 by examining binding reactions with fusion proteins comprised of the extracellular region of CD19 and the Fc region of IgG1. A fusion protein containing the third extracellular domain (D3-Fc) bound to WEHI-231 cells, and this was competed by soluble IgM. This function of IgM was confirmed by the binding of D3-Fc to beads coated with IgM. A second ligand for D3-Fc was found on stromal cells, and was shown to be heparin/heparan sulfate. These two ligands would be considered to reside on follicular dendritic cells, and may account for the observed ability of D3-Fc to bind to sites in germinal centers containing these cells.

Reprogramming T cells: the role of extracellular matrix in coordination of T cell activation and migration.

The stable immunological synapse between a T cell and antigen-presenting cell coordinates migration and activation. Three-dimensional collagen gels transform this interaction into a series of transient hit-and-run encounters. Here we integrate these alternative modes of interaction in a model for primary T cell activation and effector function in vivo.

Regulation of inflammation by collagen-binding integrins alpha1beta1 and alpha2beta1 in models of hypersensitivity and arthritis.

Adhesive interactions play an important role in inflammation by promoting leukocyte attachment and extravasation from the vasculature into the peripheral tissues. However, the importance of adhesion molecules within the extracellular matrix-rich environment of peripheral tissues, in which cells must migrate and be activated, has not been well explored. We investigated the role of the major collagen-binding integrins, alpha1beta1 and alpha2beta1, in several in vivo models of inflammation. mAb's against murine alpha1 and alpha2 were found to significantly inhibit effector phase inflammatory responses in animal models of delayed-type hypersensitivity (DTH), contact hypersensitivity (CHS), and arthritis. Mice that were alpha1-deficient also showed decreased inflammatory responses in the CHS and arthritis models when compared with wild-type mice. Decreased leukocyte infiltration and edema formation accompanied inhibition of antigen-specific models of inflammation, as nonspecific inflammation induced by croton oil was not inhibited. This study demonstrates the importance in vivo of alpha1beta1 and alpha2beta1, the collagen-binding integrins, in inflammatory diseases. The study also extends the role of integrins in inflammation beyond leukocyte attachment and extravasation at the vascular endothelial interface, revealing the extracellular matrix environment of peripheral tissues as a new point of intervention for adhesion-based therapies.

Global expression analysis of extracellular matrix-integrin interactions in monocytes.

Central to immune and inflammatory responses are the integrin-mediated adhesive interactions of cells with their extracellular matrix (ECM)-rich environment. Using a comprehensive and quantitative mRNA profiling technique, we analyzed the effect of ECM-induced attachment on monocyte gene expression, its regulation by growth factors, and the integrin specificity of this event. Adhesion of cells to fibronectin resulted in increased expression of a large number of genes, which was strongly potentiated by the presence of growth factors. Adhesion activated both the NF-kappaB and Jak/STAT pathways of gene transcription and increased expression of genes involved in inflammatory and immune responses, revealing the importance of ECM-integrin interactions in these processes.

Immunological prevention of spontaneous early embryo resorption is mediated by non-specific immunosimulation.

PROBLEM: Spontaneous early embryo resorption following implantation occurs in many species, but little is known regarding the causes or the prevention of early pregnancy failure. Embryo and fetal loss have widely been assumed to be due to maternal allospecific immune rejection. Alloimmunization therapy with paternal tissues has been successfully used in human and murine pregnancies to prevent early embryo demise. The mechanisms of this treatment have been assumed to be the induction of antigen specific, fetal "graft" enhancing antibodies and suppressor cells. The purpose of this study was to investigate the validity of this assumption. METHOD: To investigate these general assumptions, female CBA/J mice were immunized with either specific or nonspecific antigens prior to mating with DBA/2 or Balb/c males. Further, a model system for the study of bacterial lipopolysaccharide (LPS) induced abortion was used to demonstrate the nature of antigen specific immune protection against abortion. RESULTS: Whereas the administration of 1 microgram of LPS to CFW female x CFW male pregnant mice on day 7 of gestation induced complete fetal resorption, prior immunization with 20 micrograms of LPS completely prevented LPS induced abortion as long as the anti-LPS antibody titers remained above a threshold value of about 1/500. Therefore, early embryo loss could be induced by a bacterial infection and could be prevented by appropriate immunity to abortogenic factors. However, due to the short half-life of IgM antibodies, immunity to LPS was short-lived and the protective effect of LPS immunization against LPS induced abortion waned after 5 wk. Through the use of the CBA/J female x DBA/2 male model system to study spontaneous early embryo loss, previous vaccination of CBA/J female mice with Balb/c spleen cells expressing paternal MHC antigens, complete Freund's adjuvant (CFA) or LPS, all decreased the incidence of spontaneous resorption in subsequent pregnancies. Similarly, a previous mating with a Balb/c male prevented spontaneous embryo loss for a period of up to 6 wk. However, none of the immunotherapeutic vaccinations or matings had a permanent effect on CBA/J female x DBA/2 male embryo survival, which one would have expected if specific immune mediators were involved. CONCLUSION: The results of this study indicated that the decrease in the incidence of spontaneous embryo resorption following alloimmunization was more likely to be due to nonspecific immunomodulatory effects on the immune system of the female mice, as opposed to specific antipaternal immunity. This may, in part, explain the placebo effects observed for alloimmunization therapy for human habitual pregnancy loss. The relevance of these results to the development of immunotherapy strategies for the prevention of habitual abortion is discussed.

Self-renewal of B-1 lymphocytes is dependent on CD19.

The B-1 subset of B lymphocytes is maintained by self-renewal of mature cells, and this process may involve signaling through membrane immunoglobulin (mIg). We determined whether CD19, a membrane protein that co-stimulates B cells by mIg, has a role in this process. Pre-natal treatment of mice with 1D3, a rat anti-mouse CD19 monoclonal antibody, down-regulated CD19 expression and reduced by sixfold the number of B-1a cells at birth; B-2 cells were relatively unaffected. Prolonged treatment of adult mice with 1D3 caused the loss of approximately 2% per day of peritoneal B-1a cells, without diminishing the recovery of splenic B-2 cells. The loss of B-1a cells was associated with inhibition of their replication rather than with accelerated turnover. Therefore, CD19 is involved in the development and self-renewal of B-1a cells, perhaps through its ability to amplify signaling through mIgM.

Ideas crystallized on immunoglobulin superfamily-integrin interactions.

Interactions between immunoglobulin superfamily (IgSF) members and integrins are central to lymphocyte homing, leukocyte emigration into tissues at inflammatory sites, and in cell-cell interactions that lead to immune responses. Recent X-ray crystal structures reveal that the interaction of a divalent cation found in the integrin structure with an acidic residue from the IgSF partner may be important in binding.

Heterogenous glycosylation of ICAM-3 and lack of interaction with Mac-1 and p150,95.

Intercellular adhesion molecule (ICAM)-1, ICAM-2, and ICAM-3 have been identified as counter-receptors for the leukocyte integrin lymphocyte function-associated antigen 1 (LFA-1). The other leukocyte integrins, Mac-1 and p150,95, also interact with ICAM-1. ICAM-1 and ICAM-3 are highly homologous, and an undefined ligand for Mac-1 is present on neutrophils where ICAM-3 is well expressed. In addition, glycosylation has been shown to affect the interaction of ICAM-1 with Mac-1. We therefore sought to characterize ICAM-3 heterogeneity and determine whether ICAM-3 was a ligand for either Mac-1 or p150,95. Despite extensive differences in N-linked glycosylation, ICAM-3 purified from lymphoid cells and from neutrophils supports adhesion of LFA-1-bearing cells equally well; however, neither supports adhesion of Mac-1 or p150,95-expressing chinese hamster ovary cell transfectants. Similarly, purified Mac-1 does not support adhesion of ICAM-2 or ICAM-3-expressing L cell transfectants. ICAM-3 on neutrophils does not participate in Mac-1-dependent homotypic aggregation. Thus, ICAM-3 is not a counter-receptor for either Mac-1 or p150,95.

Intercellular adhesion molecules (ICAM)-1 ICAM-2 and ICAM-3 function as counter-receptors for lymphocyte function-associated molecule 1 in human immunodeficiency virus-mediated syncytia formation.

It has been previously demonstrated that lymphocyte function-associated molecule 1 (LFA-1) plays a major role in human immunodeficiency virus (HIV)-mediated syncytia formation. In the present study we investigated the involvement of intercellular adhesion molecule-1 (ICAM-1), ICAM-2 and ICAM-3 in the process. The ability of monoclonal antibodies (mAb) directed against ICAM-1, ICAM-2 and ICAM-3 to block syncytia was analyzed either in phytohemagglutinin (PHA)-activated lymphocytes infected in vitro with primary or laboratory strains of HIV or by coculturing a T cell line stably expressing HIV envelope with PHA-activated lymphocytes. Complete inhibition of syncytia formation was observed only by the simultaneous addition to the cell cultures of all (i.e. anti-ICAM-1, anti-ICAM-2 and anti-ICAM-3) mAb. These results indicate that the interaction between LFA-1 and ICAM is a critical step in HIV-mediated syncytia formation, and that ICAM-1, ICAM-2 and ICAM-3 are the receptor molecules for the LFA-1-dependent syncytia formation.

Characterization of the function of intercellular adhesion molecule (ICAM)-3 and comparison with ICAM-1 and ICAM-2 in immune responses.

We have characterized the immunobiology of the interaction of intercellular adhesion molecule 3 (ICAM-3; CD50) with its counter-receptor, leukocyte function-associated antigen 1 (LFA-1; CD11a/CD18). Purified ICAM-3 supported LFA-1-dependent adhesion in a temperature- and cation-dependent manner. Activation of cells bearing LFA-1 increased adhesiveness for ICAM-3 in parallel to adhesiveness for ICAM-1. Although CBR-IC3/1 monoclonal antibody (mAb) blocked adhesion of cells to purified LFA-1, when tested alone, neither CBR-IC3/1 nor five novel ICAM-3 mAbs characterized here blocked adhesion of cells to purified ICAM-3 or homotypic adhesion. Two ICAM-3 mAbs, CBR-IC3/1 and CBR-IC3/2, were required to block LFA-1-dependent adhesion to purified ICAM-3- or LFA-1-dependent, ICAM-1-, ICAM-2-independent homotypic adhesion of lymphoid cell lines. Two ICAM-3 mAbs, CBR-IC3/1 and CBR-IC3/6, induced LFA-1-independent aggregation that was temperature and divalent cation dependent and was completely inhibited by ICAM-3 mAb, CBR-IC3/2, recognizing a distinct epitope. Purified ICAM-3 provided a costimulatory signal for proliferation of resting T lymphocytes. mAb to ICAM-3, together with mAbs to ICAM-1 and ICAM-2, inhibited peripheral blood lymphocyte proliferation in response to phytohemagglutinin, allogeneic stimulator cells, and specific antigen. Inhibition was almost complete and to the same level as with mAb to LFA-1, suggesting the most functionally important, and possibly all, of the ligands for LFA-1 have been defined.

Evaluation of the role of exogenous pathogens on the incidence of embryo loss during early pregnancy in mice.

The mating of CBA/j female mice (H2k) by DBA/2j male mice (H2d) typically results in an elevated incidence of spontaneous embryo loss thus providing an ideal genetically controlled laboratory model for the study of the factors causing early embryo loss during pregnancy. There is now considerable data on the cells and factors involved in fetal resorption but little is known about the events which activate this process. While the activation of the maternal response to the fetal implant could have endogenous or genetic origins, a role for exogenous factors including microbial pathogens could also be involved. In order to investigate these possibilities, the reproductive success of CBA/j female x DBA/2j male matings in a conventional animal care facility were compared with matings in a specific pathogen free (SPF) animal facility. All animals housed under these conditions were routinely screened by immunoassay and culture, for the presence of a number of viral and bacterial pathogens of mice. The incidence of spontaneous embryo loss in specific pathogen free CBA female mice mated by DBA and other male strains was found to be virtually identical to that of CBA female mice infected with multiple viral pathogens and housed under otherwise identical conditions (non-SPF). However, the numbers of implantation per pregnancy was significantly greater in an SPF facility. Therefore, exposure of mating mice to exogenous viral and bacterial pathogens did not appear to alter the overall incidence of spontaneous embryo resorption. It was concluded that the immunomodulatory effects of infection by common murine pathogens neither augmented nor reduced post-implantation embryo losses.

CDw50 and ICAM-3: two names for the same molecule.

CDw50 differentiation antigen is a molecule broadly expressed on hematopoetic cells but not on other cells. Previous experiments showed that CDw50 monoclonal antibodies (mAb) inhibited primary mixed lymphocyte culture (MLC). To understand the function of CDw50 better, we purified it and obtained peptide sequence. At the same time, intercellular adhesion molecule (ICAM)-3, the third ligand of lymphocyte function-associated molecule 1, was described by mAb and subsequent cDNA cloning. Immunochemical, functional, and protein sequencing studies show that ICAM-3 and CDw50 are the same glycoprotein, a 120-kDa surface molecule with presumably an important role in the immune responses.

Cloning and expression of intercellular adhesion molecule 3 reveals strong homology to other immunoglobulin family counter-receptors for lymphocyte function-associated antigen 1.

Based on protein sequence, we have isolated a cDNA for intercellular adhesion molecule 3 (ICAM-3), the most recently defined counter-receptor for lymphocyte function-associated antigen 1 (LFA-1). Expression of the cDNA yields a product that reacts with monoclonal antibody to ICAM-3 and functions as a ligand for LFA-1. The deduced 518-amino acid sequence of the predicted mature protein defines a highly glycosylated type I integral membrane protein with five immunoglobulin (Ig)-like domains. The five Ig-like domains of ICAM-3 are highly homologous with those of human ICAM-1 (52% identity) and human ICAM-2 (37% identity).

Isolation, characterization, and expression of mouse ICAM-2 complementary and genomic DNA.

Intercellular adhesion molecule-2 (ICAM-2), a cell surface glycoprotein, is a second counter-receptor for lymphocyte function-associated Ag-1 (LFA-1). We report here the isolation and characterization of the cDNA and the gene that encode murine ICAM-2 (Accession numbers X65493 and X65490, respectively). The deduced sequence of the cDNA has 60% amino acid identity with its human counterpart and has the same expression pattern in cells and tissues. Furthermore, COS cells transfected with mouse ICAM-2 complementary and genomic DNA bind to purified human LFA-1, demonstrating the conservation of the function of ICAM-2 as a ligand for LFA-1 and conservation across species of sequences that are critical for binding to human LFA-1. COS cells transfected with the ICAM-2 cDNA do not react with mAb PA3, previously suggested to define ICAM-2 in the mouse. The mouse ICAM-2 gene was isolated and its structural organization determined. The gene is present in a single copy in the mouse genome and contains four exons spanning about 5.0 kb of DNA. The exon/intron architecture correlates to the structural domains of the protein and resembles that of other Ig superfamily members. The gene for ICAM-2, which is constitutively expressed in endothelial cells, has several conserved sequence motifs in its promoter region, including a direct repeat, and lacks transcription factor-binding sites present in the ICAM-1 gene, which is inducible in endothelial cells.

Intercellular adhesion molecule 3, a third adhesion counter-receptor for lymphocyte function-associated molecule 1 on resting lymphocytes.

Recent studies suggest that some T and B lymphocyte cell lines bind to the integrin lymphocyte function-associated molecule 1 (LFA-1) chiefly through a pathway independent of its two known counter-receptors, intercellular adhesion molecules (ICAMs)-1 and -2. A monoclonal antibody (mAb) was raised that, in combination with blocking mAb to ICAM-1 and ICAM-2, can completely inhibit binding of these cell lines to purified LFA-1. This third ligand, designated ICAM-3 based on its functional relatedness to ICAM-1 and -2, is a highly glycosylated protein of 124,000 Mr. It is well expressed on all leukocytes and absent from endothelial cells. In assays of adhesion of resting lymphocytes to purified LFA-1, ICAM-3 is by far the most functionally important ICAM, implying an important role for ICAM-3 in the generation of immune responses.

Expression of intercellular adhesion molecule 1 (ICAM-1) in Alzheimer's disease.

In this study, 13 clinically and pathologically diagnosed cases of Alzheimer's disease were analyzed for the presence of intercellular adhesion molecule 1 (ICAM-1), ICAM-2, lymphocyte function associated antigen-1 (LFA-1), HLA-DR, LN-1, and LN-2. ICAM-1 was observed primarily on neuritic plaques and cerebrovascular endothelium. ICAM-1 was also shown to be present in brain tissue derived from 14 normal cases; however, the degree of immunoreactivity was quantitatively less compared to Alzheimer cases and was largely restricted to cerebrovascular endothelium. LFA-1 was shown to be present on microglial cells and leukocytes. Consistent with the findings of previous reports, HLA-DR was found to be expressed on microglial cells. In this study we failed to demonstrate dual immunolocalization for ICAM-1 and LFA-1, ICAM-1 and HLA-DR, or ICAM-1 and LN-2. As microglial cells express both HLA-DR and LFA-1, they may serve to mediate antigen presentation functions by interacting with lymphocyte ICAM-1. Alternately, the expression of these immune-associated glycoproteins on glial cells may be epiphenomenal occurring secondary to some aspect of the disease process. Finally, the presence of ICAM-1 within neuritic plaques raises the question as to whether adhesion may play some role in the process of neurite outgrowth and neurodegeneration.

Characterization of ICAM-2 and evidence for a third counter-receptor for LFA-1.

In an endeavor to further characterize human intercellular adhesion molecule-2 (ICAM-2), two murine monoclonal antibodies (mAb) were generated to ICAM-2 transfected COS cells, and designated CBR-IC2/1 and CBR-IC2/2. Immunoprecipitated, reduced ICAM-2 migrated as a broad band of Mr 60,000 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Treatment with N-glycanase revealed a peptide backbone of Mr 31,000, consistent with the size predicted from the cDNA. ICAM-2 had a broad distribution on hematopoietic cell lines and little expression on other cell lines, the sole exception being cultured endothelial cells which possess high levels of ICAM-2. Resting lymphocytes and monocytes expressed ICAM-2, while neutrophils did not. Staining of tissue sections with anti-ICAM-2 mAb confirmed their strong reactivity to vascular endothelium, but demonstrated a lack of ICAM-2 expression on other tissues. Small clusters of ICAM-2 positive cells were, however, seen in germinal centers. In contrast to ICAM-1 there was little or no induction of ICAM-2 expression on lymphocytes or cultured endothelium upon stimulation with inflammatory mediators. One of the two mAb, CBR-IC2/2, was found to totally inhibit binding of ICAM-2+ COS cells to purified lymphocyte function-associated antigen-1 (LFA-1). Using this mAb, LFA-1-dependent binding to both stimulated and unstimulated endothelium was found to be totally accounted for by ICAM-1 and ICAM-2. Homotypic aggregation of an Epstein-Barr virus-transformed B cell line, JY, was found to be solely ICAM-1 and ICAM-2-dependent, while in the case of the T cell lymphoma cell line, SKW3, anti- ICAM-2 mAb in conjunction with anti-ICAM-1 mAb could not inhibit the LFA-1-dependent aggregation. This suggests an additional LFA-1 ligand exists. Using a cell binding assay to purified LFA-1 in conjunction with anti-ICAM-1 and anti-ICAM-2 mAb, we have demonstrated that this putative third ligand for LFA-1 exists on SKW3 and other cell lines.

ICAM-1 (CD54): a counter-receptor for Mac-1 (CD11b/CD18).

While the leukocyte integrin lymphocyte function-associated antigen (LFA)-1 has been demonstrated to bind intercellular adhesion molecule (ICAM)-1, results with the related Mac-1 molecule have been controversial. We have used multiple cell binding assays, purified Mac-1 and ICAM-1, and cell lines transfected with Mac-1 and ICAM-1 cDNAs to examine the interaction of ICAM-1 with Mac-1. Stimulated human umbilical vein endothelial cells (HUVECs), which express a high surface density of ICAM-1, bind to immunoaffinity-purified Mac-1 adsorbed to artificial substrates in a manner that is inhibited by mAbs to Mac-1 and ICAM-1. Transfected murine L cells or monkey COS cells expressing human ICAM-1 bind to purified Mac-1 in a specific and dose-dependent manner; the attachment to Mac-1 is more temperature sensitive, lower in avidity, and blocked by a different series of ICAM-1 mAbs when compared to LFA-1. In a reciprocal assay, COS cells cotransfected with the alpha and beta chain cDNAs of Mac-1 or LFA-1 attach to immunoaffinity-purified ICAM-1 substrates; this adhesion is blocked by mAbs to ICAM-1 and Mac-1 or LFA-1. Two color fluorescence cell conjugate experiments show that neutrophils stimulated with fMLP bind to HUVEC stimulated with lipopolysaccharide for 24 h in an ICAM-1-, Mac-1-, and LFA-1-dependent fashion. Because cellular and purified Mac-1 interact with cellular and purified ICAM-1, we conclude that ICAM-1 is a counter receptor for Mac-1 and that this receptor pair is responsible, in part, for the adhesion between stimulated neutrophils and stimulated endothelial cells.

Modulation of the natural killer cell activity in pregnant mice alters the spontaneous abortion rate.

Effector cells associated with an aborting fetus appear to be both thymus derived (T) and natural killer (NK) cells. In order to test the hypothesis that NK cells are a major effector mediating early spontaneous abortion (less than day 8-10), CBA female mice mated by DBA/2 males were treated with either polyinosinic/cytidylic acid (poly I:C) to boost NK activity, or rabbit anti-asialo GM1 (RaASGM1) to decrease NK activity. The results of the NK assays of the spleens of treated mice confirmed that the reagents had the expected effect on NK activity and an inspection of the uteri indicated a significant increase in aborted embryos after poly I:C and a marked decrease in spontaneous abortions after RaASGM1 treatment. Therefore, spontaneous abortions may be mediated in part by the cytotoxic activity of unregulated NK cells.