Activated endothelium binds lymphocytes through a novel binding site in the alternately spliced domain of vascular cell adhesion molecule-1.

Vascular cell adhesion molecule-1 (VCAM-1) is induced on endothelial cells by inflammatory cytokines, and binds mononuclear leukocytes through the integrin very late antigen-4 (VLA-4) (alpha 4 beta 1). This adhesion pathway has been implicated in a diverse group of physiological and pathological processes, including B cell development, leukocyte activation and recruitment to sites of inflammation, atherosclerosis, and tumor cell metastasis. The major form of VCAM-1 (VCAM-7D) has seven extracellular immunoglobulin (Ig)-like domains, of which the three NH2-terminal domains (domains 1-3) are similar in amino acid sequence to domains 4-6. By functional analysis of VCAM-7D relative to VCAM-6D (a minor 6-domain form of VCAM-1 in which domain 4 is deleted because of alternative splicing), and chimeric constructs between VCAM-1 and its structural relative intercellular adhesion molecule-1 (ICAM-1), we show that either the first or the homologous fourth domain of VCAM-1 is required for VLA-4-dependent adhesion. Either of these binding sites can function in the absence of the other. When both are present, cell binding activity is increased relative to monovalent forms of the molecule. The homologous binding regions appear to have originated by internal duplication of a portion of a monovalent ancestral gene, before the mammalian radiation. Thus VCAM-1 exemplifies evolution of a functionally bivalent cell-cell adhesion molecule by intergenic duplication. We have also produced a new class of anti-VCAM-1 monoclonal antibodies that block domain 4-dependent adhesion, and demonstrate that both binding sites participate in the adhesion function of VCAM-1 on endothelial cells in vitro. Therefore both sites must be blocked in clinical, animal, or in vitro studies depending on the use of anti-VCAM-1 antibodies to inactivate the VCAM-1/VLA-4 adhesion pathway.

Signaling through the lymphotoxin beta receptor induces the death of some adenocarcinoma tumor lines.

Surface lymphotoxin (LT) is a heteromeric complex of LT-alpha and LT-beta chains that binds to the LT-beta receptor (LT-beta-R), a member of the tumor necrosis factor (TNF) family of receptors. The biological function of this receptor-ligand system is poorly characterized. Since signaling through other members of this receptor family can induce cell death, e.g., the TNF and Fas receptors, it is important to determine if similar signaling events can be communicated via the LT-beta-R. A soluble form of the surface complex was produced by coexpression of LT-alpha and a converted form of LT-beta wherein the normally type II LT-beta membrane protein was changed to a type I secreted form. Recombinant LT-alpha 1/beta 2 was cytotoxic to the human adenocarcinoma cell lines HT-29, WiDr, MDA-MB-468, and HT-3 when added with the synergizing agent interferon (IFN) gamma. When immobilized on a plastic surface, anti-LT-beta-R monoclonal antibodies (mAbs) induced the death of these cells, demonstrating direct signaling via the LT-beta-R. Anti-LT-beta-R mAbs were also identified that inhibited ligand-induced cell death, whereas others were found to potentiate the activity of the ligand when added in solution. The human WiDr adenocarcinoma line forms solid tumors in immunocompromised mice, and treatment with an anti-LT-beta-R antibody combined with human IFN-gamma arrested tumor growth. The delineation of a biological signaling event mediated by the LT-beta-R opens a window for further studies on its immunological role, and furthermore, activation of the LT-beta-R may have an application in tumor therapy.

BAFF binds to the tumor necrosis factor receptor-like molecule B cell maturation antigen and is important for maintaining the peripheral B cell population.

The tumor necrosis factor (TNF) family member B cell activating factor (BAFF) binds B cells and enhances B cell receptor-triggered proliferation. We find that B cell maturation antigen (BCMA), a predicted member of the TNF receptor family expressed primarily in mature B cells, is a receptor for BAFF. Although BCMA was previously localized to the Golgi apparatus, BCMA was found to be expressed on the surface of transfected cells and tonsillar B cells. A soluble form of BCMA, which inhibited the binding of BAFF to a B cell line, induced a dramatic decrease in the number of peripheral B cells when administered in vivo. Moreover, culturing splenic cells in the presence of BAFF increased survival of a percentage of the B cells. These results are consistent with a role for BAFF in maintaining homeostasis of the B cell population.

Arrangement of domains, and amino acid residues required for binding of vascular cell adhesion molecule-1 to its counter-receptor VLA-4 (alpha 4 beta 1).

Interaction of the vascular cell adhesion molecule (VCAM-1) with its counter-receptor very late antigen-4 (VLA-4) (integrin alpha 4 beta 1) is important for a number of developmental pathways and inflammatory functions. We are investigating the molecular mechanism of this binding, in the interest of developing new anti-inflammatory drugs that block it. In a previous report, we showed that the predominant form of VCAM-1 on stimulated endothelial cells, seven-domain VCAM (VCAM-7D), is a functionally bivalent molecule. One binding site requires the first and the other requires the homologous immunoglobulin-like domain. Rotary shadowing and electron microscopy of recombinant soluble VCAM-7D molecules suggests that the seven Ig-like domains are extended in a slightly bent linear array, rather than compactly folded together. We have systematically mutagenized the first domain of VCAM-6D (a monovalent, alternately spliced version mission domain 4) by replacing 3-4 amino acids of the VCAM sequence with corresponding portions of the related ICAM-1 molecule. Specific amino acids, important for binding VLA-4 include aspartate 40 (D40), which corresponds to the acidic ICAM-1 residue glutamate 34 (E34) previously reported to be essential for binding of ICAM-1 to its integrin counter-receptor LFA-1. A small region of VCAM including D40, QIDS, can be replaced by the similar ICAM-1 sequence, GIET, without affecting function or epitopes, indicating that this region is part of a general integrin-binding structure rather than a determinant of binding specificity for a particular integrin. The VCAM-1 sequence G65NEH also appears to be involved in binding VLA-4.

BAFF-R, a newly identified TNF receptor that specifically interacts with BAFF.

B cell homeostasis has been shown to critically depend on BAFF, the B cell activation factor from the tumor necrosis factor (TNF) family. Although BAFF is already known to bind two receptors, BCMA and TACI, we have identified a third receptor for BAFF that we have termed BAFF-R. BAFF-R binding appears to be highly specific for BAFF, suggesting a unique role for this ligand-receptor interaction. Consistent with this, the BAFF-R locus is disrupted in A/WySnJ mice, which display a B cell phenotype qualitatively similar to that of the BAFF-deficient mice. Thus, BAFF-R appears to be the principal receptor for BAFF-mediated mature B cell survival.

Attenuation of colitis in the cotton-top tamarin by anti-alpha 4 integrin monoclonal antibody.

Recent studies have demonstrated the induced expression of endothelial adhesion molecules including E-selectin (also called endothelial leukocyte adhesion molecule-1), vascular cell adhesion molecule and intercellular adhesion molecule in actively involved mucosa of patients with ulcerative colitis and Crohn's disease. Similar induction has been demonstrated in the colon of the Cotton-top tamarin (CTT), a New World primate that experiences a spontaneous acute and chronic colitis resembling ulcerative colitis. To assess the potential importance of leukocyte adhesion as a necessary step in acute colitis, the effect of parenteral mAb directed against adhesion molecules on CTT colitis was evaluated in placebo-controlled blinded trials. Serial administration of either of two anti-E-selectin mAb designated BB11 and EH8 effectively coated endothelial surfaces expressing this vascular adhesion molecule. Although colitis activity was slightly diminished after the 10-d treatment period in CTT receiving either BB11 or EH8, this reduction was not significantly different than that seen in animals given a placebo control when assessed by a previously validated standardized scale of inflammatory activity: mean histologic activity grade 2.2 +/- 0.2 pretreatment vs 1.5 +/- 0.5 posttreatment in group receiving mAb and 2.1 +/- 0.1 pretreatment vs 1.3 +/- 0.5 posttreatment in the placebo group (P > 0.2). In contrast, administration of an anti-alpha 4 integrin mAb designated HP1/2 that binds VLA4 (alpha 4 beta 1) and presumably alpha 4 beta 7 integrins resulted in significant attenuation of acute colitis when compared to both pretreatment activity index (P = 0.005) and the placebo control group (P < 0.01): mean histologic activity grade 1.6 +/- 0.3 pretreatment vs 0.2 +/- 0.1 posttreatment in the group receiving HP1/2 and 1.8 +/- 0.5 pretreatment and 1.2 +/- 0.2 posttreatment in the placebo control group. These studies using a model of spontaneous colitis in the CTT demonstrate the feasibility of modulation of leukocyte-vascular adhesion and/or other integrin-mediated events possibly including T cell aggregation and T cell-stromal interactions, as well as lymphocyte homing. These results suggest both that these processes are important and possibly essential elements in sustaining acute colitis and that their disruption may result in therapeutic benefit.

Alpha 4-integrins mediate antigen-induced late bronchial responses and prolonged airway hyperresponsiveness in sheep.

Eosinophils and T lymphocytes are thought to be involved in allergic airway inflammation. Both cells express the alpha 4 beta 1-integrin, very late antigen-4 (VLA-4, CD49d/CD29); alpha 4-integrins can promote cellular adhesion and activation. Therefore, we examined the in vivo effects of a blocking anti-alpha 4 monoclonal antibody, HP 1/2, on antigen-induced early and late bronchial responses, airway hyperresponsiveness, inflammatory cell influx, and peripheral leukocyte counts in allergic sheep. Sheep blood lymphocytes, monocytes, and eosinophils expressed alpha 4 and bound HP 1/2. In control sheep, Ascaris antigen challenge produced early and late increases in specific lung resistance of 380 +/- 42% and 175 +/- 16% over baseline immediately and 7 h after challenge, respectively, as well as airway hyperresponsiveness continuing for 14 d after antigen challenge. Treatment with HP 1/2 (1 mg/kg, i.v.) 30 min before antigen challenge did not affect the early increase in specific lung resistance but inhibited the late-phase increase at 5-8 h by 75% (P < 0.05) and inhibited the post-antigen-induced airway hyperresponsiveness at 1, 2, 7, and 14 d (P < 0.05, for each time). Intravenous HP 1/2 given 2 h after antigen challenge likewise blocked late-phase airway changes and postchallenge airway hyperresponsiveness. Airway administration of HP 1/2 (16-mg dose) was also effective in blocking these antigen-induced changes. Response to HP 1/2 was specific since an isotypic monoclonal antibody, 1E6, was ineffective by intravenous and aerosol administration. Inhibition of leukocyte recruitment did not totally account for the activity of anti-alpha 4 antibody since HP 1/2 neither diminished the eosinopenia or lymphopenia that followed antigen challenge nor consistently altered the composition of leukocytes recovered by bronchoalveolar lavage. Because airway administration of HP 1/2 was also active, HP 1/2 may have inhibited cell activation. Reduction of platelet-activating factor-induced eosinophil peroxidase release from HP 1/2-treated eosinophils supports such a mechanism. These findings indicate a role for alpha 4-integrins in processes that lead to airway late phase responses and persisting airway hyperresponsiveness after antigen challenge.

Vascular cell adhesion molecule-1 is expressed in human coronary atherosclerotic plaques. Implications for the mode of progression of advanced coronary atherosclerosis.

Endothelial attachment is the initial step in leukocyte recruitment into developing atherosclerotic lesions. To determine whether vascular cell adhesion molecule-1 (VCAM-1) expression may play a role in inflammatory cell recruitment into human atherosclerotic lesions, immunohistochemistry was performed with a polyclonal rabbit antisera, raised against recombinant human VCAM-1, on 24 atherosclerotic coronary plaques and 11 control coronary segments with nonatherosclerotic diffuse intimal thickening from 10 patients. Immunophenotyping was performed on adjacent sections to identify smooth muscle cells, macrophages, and endothelial cells. To confirm VCAM-1-expressing cell types, double immunostaining with VCAM-1 antisera and each of the cell-specific markers and in situ hybridization were performed. All atherosclerotic plaques contained some VCAM-1, compared to 45% of control segments. VCAM-1 was found infrequently on endothelial cells at the arterial lumen din both plaques (21%) and in control segments (27%), but was prevalent in areas of neovascularization and inflammatory infiltrate in the base of plaques. Double immunostaining and in situ hybridization confirmed that most VCAM-1 was expressed by subsets of plaque smooth muscle cells and macrophages. The results document the presence of VCAM-1 in human atherosclerosis, demonstrate VCAM-1 expression by human smooth muscle cells in vivo, and suggest that intimal neovasculature may be an important site of inflammatory cell recruitment into advanced coronary lesions.

Anti-CD154 (CD40L) prevents recurrence of diabetes in islet isografts in the DR-BB rat.

Islet transplantation for the treatment of autoimmune diabetes is more difficult because of the additional barrier presented by the autoimmunity. We tested the ability of hamster anti-rat CD154 to prevent recurrence of diabetes in renal subcapsular islet isografts in DR-BB (RT1uu) rats with established autoimmune diabetes. Experimental animals with established diabetes received intravenous injections of 15 mg/kg anti-CD154 on a specified schedule starting 2 days before renal subcapsular transplantation of an islet isograft. Control animals received either saline or hamster IgG. Plasma glucose levels >250 mg/dl over 3 days were used to indicate the recurrence of diabetes. Rats that received saline (n = 5) or control antibody (n = 3) had a recurrence of diabetes 6-11 days after transplantation. Histological examination of islet isografts from these rats showed complete destruction of the insulin-producing portion of the isograft with residual cells positive for glucagon. Recipient rats that received anti-CD154 at the 15-mg/kg dosage (n = 6) did not have a recurrence of diabetes for 308-461 days after transplantation. Islet isografts removed from the rats showed low levels of insulin immunoreactivity, high levels of insulin mRNA, and focal infiltration with lymphocytes but no evidence of islet destruction. Mean peak antibody concentration was 266 microg/ml and returned to undetectable levels by 67-88 days after transplantation. Rats that received anti-CD154 starting at 4-7 days after transplantation had a recurrence of diabetes within 11 days of the isotransplantation. Therefore, anti-CD154 as the sole immunomodulator prevented the recurrence of diabetes in islet isografts in rats with established autoimmune diabetes. This suggests that CD40/CD154 blockade is effective in preventing the insulitis or the effector phase of autoimmune diabetes.

Regular articles: conditional disruption of hedgehog signaling pathway defines its critical role in hair development and regeneration.

Members of the vertebrate hedgehog family (Sonic, Indian, and Desert) have been shown to be essential for the development of various organ systems, including neural, somite, limb, skeletal, and for male gonad morphogenesis. Sonic hedgehog and its cognate receptor Patched are expressed in the epithelial and/or mesenchymal cell components of the hair follicle. Recent studies have demonstrated an essential role for this pathway in hair development in the skin of Sonic hedgehog null embryos. We have further explored the role of the hedgehog pathway using anti-hedgehog blocking monoclonal antibodies to treat pregnant mice at different stages of gestation and have generated viable offspring that lack body coat hair. Histologic analysis revealed the presence of ectodermal placode and primodium of dermal papilla in these mice, yet the subsequent hair shaft formation was inhibited. In contrast, the vibrissae (whisker) development appears to be unaffected upon anti-hedgehog blocking monoclonal antibody treatment. Strikingly, inhibition of body coat hair morphogenesis also was observed in mice treated postnatally with anti-hedgehog monoclonal antibody during the growing (anagen) phase of the hair cycle. The hairless phenotype was reversible upon suspension of monoclonal antibody treatment. Taken together, our results underscore a direct role of the Sonic hedgehog signaling pathway in embryonic hair follicle development as well as in subsequent hair cycles in young and adult mice. Our system of generating an inducible and reversible hairless phenotype by anti-hedgehog monoclonal antibody treatment will be valuable for studying the regulation and mechanism of hair regeneration.

Human type I interferon receptor, IFNAR, is a heavily glycosylated 120-130 kD membrane protein.

Type I interferons (IFNs) bind and signal through cell surface receptors that share at least one common component. One candidate for such a component is the interferon-alpha receptor (IFNAR). Genetic studies have shown that the IFNAR gene product is required for response to many type I interferons. However, these studies also suggest that the IFNAR protein interacts with an additional receptor component(s) to form functionally complete type I IFN receptors. Although these genetic studies have contributed significantly to understanding the type I IFN receptors. Although these genetic studies have contributed significantly to understanding the type I IFN receptors, little biochemical characterization of IFNAR and its function has been reported. To facilitate biochemical studies of the IFNAR gene product, a monoclonal antibody, GB8, recognizing the extracellular domain of IFNAR was prepared. The epitope for GB8 maps to the second extracellular domain of IFNAR between amino acids 278 and 293. GB8 identifies IFNAR in western blots of cell membranes as a broad band with molecular mass ranging from 100 to 150 kD in membranes from CHO cells overexpressing the human IFNAR gene to 136-150 kD in Daudi cell membranes. Such variations in the mean value and the range of molecular mass between IFNAR in different cell lines suggest differences in glycosylation. The majority of glycosylation is N-linked, although there may also be a small amount O-linked oligosaccharide. Deglycosylation of IFNAR in Daudi cell membranes results in a 70 kD IFNAR species, indicating that nearly half of the apparent molecular mass of Daudi cell IFNAR is contributed by carbohydrate moieties.

Characterization of antihuman IFNAR-1 monoclonal antibodies: epitope localization and functional analysis.

The type I interferon receptor (IFNAR) is composed of two subunits, IFNAR-1 and IFNAR-2, encoding transmembrane polypeptides. IFNAR-2 has a dominant role in ligand binding, but IFNAR-1 contributes to binding affinity and to differential ligand recognition. A panel of five monoclonal antibodies (mAb) to human IFNAR-1 (HuIFNAR-1) was produced and characterized. The reactivity of each mAb toward HuIFNAR-1 on native and transfected cells and in Western blot and ELISA formats was determined. In functional assays, one mAb, EA12, blocked IFN-a2 binding to human cells and interfered with Stat activation and antiviral activity. Epitopes for the mAb were localized to subdomains of the HuIFNAR-1 extracellular domain by differential reactivity of the mAb to a series of human/bovine IFNAR-1 chimeras. The antibody EA12 seems to require native HuIFNAR-1 for reactivity and does not map to a single subdomain, perhaps recognizing an epitope containing noncontiguous sequences in at least two subdomains. In contrast, the epitopes of the non-neutralizing mAb FB2, AA3, and GB8 mapped, respectively, to the first, second, and third subdomains of HuIFNAR-1. The mAb DB2 primarily maps to the fourth subdomain, although its reactivity may be affected by other determinants.

Antibodies to lymphotoxin alpha (LT alpha) and LT beta recognize different glial cell types in the central nervous system.

The cytokine lymphotoxin (LT) is known to exist in two forms, secreted LT alpha and a membrane-bound LT alpha/beta complex. LT alpha shares the same receptor as tumor necrosis factor alpha and LT beta is recognized by its receptor, LT betaR. Since LT has been associated with oligodendrocyte pathology, the present study has examined the expression of these molecules by immunocytochemistry in diseased and normal CNS tissue, with a panel of monoclonal antibodies (mAb) to LT alpha, LT beta and LT betaR. Of three mAb to LT beta, two (B27 and C37) gave specific membrane staining on astrocytes, as well as lymphocytes. The third anti-LT beta mAb, B9, was selectively immunoreactive for oligodendrocytes, suggesting specific recognition sites. The reactivity was not specific for multiple sclerosis (MS) since oligodendrocytes in normal and non-MS CNS tissue also displayed positivity. MAb to LT betaR reacted with astrocytes only, giving a punctate membrane staining pattern suggestive of receptor sites. MAb to LT alpha gave strong reactivity on lymphocytes in active MS lesions and weak reactivity on microglia within lesion areas. These results show that mAb to LT alpha and LT beta recognize different cell types within the CNS. Furthermore, individual mAb against LT beta were capable of distinguishing between astrocytes and oligodendrocytes, perhaps indicative of different epitopes on LT beta. The presence of LT betaR on astrocytes suggests possible interactions between infiltrating lymphocytes and astrocytes via the LT pathway.

Expression of vascular cell adhesion molecule-1 by follicular dendritic cells.

Follicular dendritic cells are the major supporting cell of the germinal center microenvironment. The major function of follicular dendritic cells is to present antigen to B cells in secondary lymphoid tissues. Through cell-cell interactions, FDCs are hypothesized to be central to the regulation of normal B cell growth and differentiation. The major receptor-ligand pair which mediates B cell-FDC adhesion is the beta 1 integrin VLA-4, present on B cells and VCAM-1 expressed on FDCs. Follicular non-Hodgkin's lymphomas similarly employ this mechanism to bind to neoplastic germinal centers. The VCAM-1 molecule can exist as a 6 or 7 immunoglobulin domain form. The major form of VCAM-1 on activated endothelium is the 7 domain form. In this report we have determined by polymerase chain reaction of purified FDCs that they express predominantly mRNA for 7 domain VCAM-1. It is likely that the two forms of VCAM-1 are associated with distinct functions, therefore the expression of 7 domain VCAM-1 may be important in normal and neoplastic B cell-FDC interactions.

The concern for accurate, quality registry data.

The effective use of registries is critical to modern healthcare information management. But you've got to be careful in applying the data.

Costimulation and autoimmune diabetes in BB rats.

Costimulatory signals regulate T-cell activation. To investigate the role of costimulation in autoimmunity and transplantation, we studied the BB rat model of type 1 diabetes. Diabetes-prone BB (BBDP) rats spontaneously develop disease when 55-120 days of age. We observed that two anti-CD28 monoclonal antibodies (mAb) with different functional activities completely prevented diabetes in BBDP rats. Anti-CD154 mAb delayed diabetes, whereas treatment with CTLA4-Ig or anti-CD80 mAb accelerated disease. Anti-CD86 or anti-CD134L mAbs had no effect. Diabetes resistant BB (BBDR) rats are disease-free, but >95% of them develop diabetes after treatment with polyinosinic-polycytidylic acid and an mAb that depletes Treg cells. In the induced BBDR model, anti-CD154 mAb delayed onset of diabetes, whereas CTLA4-Ig, anti-CD134L or either of the anti-CD28 mAbs had little or no effect. In contrast, blockade of the CD134-CD134L pathway was highly effective for preventing autoimmune recurrence against syngeneic islet grafts in diabetic BBDR hosts. Blockade of the CD40-CD154 pathway was also effective, but less so. These data suggest that the effectiveness of costimulation blockade in the treatment of type 1 diabetes is dependent on both the costimulatory pathway targeted and the mechanism of induction, stage, intensity and duration of the pathogenic process.

A predominant idiotype on anti-hen egg white lysozyme antibodies from diverse mouses strains.

A predominant idiotype was detected on almost all antibody molecules directed against the multideterminant antigen, hen egg white lysozyme (HEL). This idiotypic set was defined by rabbit and quinea pig antiidiotypic antisera raised against a rare cross-reactive antibody population isolated and from a heterogeneous BALB/c anti-human lysozyme (HUL) humoral response. This isiotype was fond on the majority of all HEL-reactive antibody from all strain tested; its presence was independent of Ig-1 allotype of H-2 haplotype. The idiotype was also present on the majority of anti-HEL antibody from nonresponder mice immunized with keyhole limpet hemocyanin coupled to HEL. The idiotype was not found on normal mouse immunoglobulin or on antibodies specific to two other proteins, HUL and ribonuclease, which are similar to HEl in size and charge. Individual idiotype-positive anti-HEL antibody populations exhibited specificity differences when analyzed for reactivity toward other avian lysozymes, suggesting that distinct clonal products shared the idiotype specificities. Fractionation of heterogeneous anti-HEL antibodies by preparative isoelectric focusing yielded distinct antibody fractions that were demonstrated to share the relevant idiotype determinants. The data are discussed in terms of the HEL peptide specificites of idiotype-positive and -negative antibodies.