ICAM-2 peptides mediate lymphocyte adhesion by binding to CD11a/CD18 and CD49d/CD29 integrins.

Three fifteen-amino-acid polypeptides designated peptides 1, 2 and 3 were synthesised as likely candidates for mimicking the role of ICAM-2 as a ligand. The ability of each peptide to bind lymphoid cells was tested. Peptide 2 largely mediated cell attachment of unstimulated cells and this binding was only marginally increased by stimulating the cells with phorbol dibutyrate (P(Bu)2). Peptide 3 mediated minimal spontaneous cell attachment, but this binding was significantly enhanced following P(Bu)2 stimulation. Peptide 1 had no effect on cell attachment with or without stimulation. The cell attachment to peptide 2 was both temperature- and cation-dependent. Studies using specific monoclonal antibodies showed that with unstimulated cells, anti-VLA-4 alpha(CD49d) or beta chain (CD29) antibodies (KD4-13 and 4B4) and anti-CD18 (1B4) each partially inhibited the cell binding. Monoclonal antibodies against CD54 (ICAM-1; 84H10 or LB2), MHC class 1 (W6/32) and control mouse IgG had no effect. When anti-CD29 and anti-CD18 monoclonal antibodies were used concurrently, there was almost complete inhibition of the cell attachment. These observations indicated that cell adhesion via ICAM-2 is mediated: (i) predominantly by peptide 2 in unstimulated and P(Bu)2-stimulated cells, and also, to some extent, by peptide 3 in P(Bu)2-stimulated cells and (ii) by binding to both CD11/CD18 and CD49d/CD29 integrins.     

Characterization of ICAM-4 binding to the I domains of the CD11a/CD18 and CD11b/CD18 leukocyte integrins.

Intercellular adhesion molecule-4 (ICAM-4, LW blood group antigen), a member of the immunoglobulin superfamily expressed on red cells, has been reported to bind to CD11a/CD18 and CD11b/CD18 leukocyte integrins. The location of the ICAM-4 binding sites on CD11a/CD18 and CD11b/CD18 are not known. CD11/CD18 integrin I domains have been found to act as major binding sites for physiological ligands and a negatively charged glutamic acid in ICAMs is considered important for binding. ICAM-4 lacks such a residue, which is replaced by an arginine. However, we demonstrate here that ICAM-4 in red cells and transfected fibroblasts interacts specifically with the I domains of CD11a/CD18 and CD11b/CD18 integrins. The binding was inhibited by anti-I domain and anti-ICAM-4 antibodies and it was dependent on divalent cations. Interestingly, ICAM-4 negative red cells were still able to bind to the CD11b/CD18 I domain but the binding of these cells to the CD11a/CD18 I domain was clearly reduced. Using a solid phase assay, we were able to show that isolated I domains directly and specifically bind to purified recombinant ICAM-4 in a cation dependent manner. Competition experiments indicated that the binding sites in ICAM-4 for the CD11a and CD11b I domains are different. However, the ICAM-4 binding region in both I domains seems to overlap with the regions recognized by the ICAM-1 and ICAM-2. Thus we have established that the I domains contain an ICAM-4 binding region in CD11a/CD18 and CD11b/CD18 leukocyte integrins.     

Monoclonal antibodies to murine lipopolysaccharide (LPS)-binding protein (LBP) protect mice from lethal endotoxemia by blocking either the binding of LPS to LBP or the presentation of LPS/LBP complexes to CD14.

Cellular responses to LPS, the major lipid component of the outer membrane of Gram-negative bacteria, are enhanced markedly by the LPS-binding protein (LBP), a plasma protein that transfers LPS to the cell surface CD14 present on cells of the myeloid lineage. LBP has been shown previously to potentiate the host response to LPS. However, experiments performed in mice with a disruption of the LBP gene have yielded discordant results. Whereas one study showed that LBP knockout mice were resistant to endotoxemia, another study did not confirm an important role for LBP in the response of mice challenged in vivo with low doses of LPS. Consequently, we generated rat mAbs to murine LBP to investigate further the contribution of LBP in experimental endotoxemia. Three classes of mAbs were obtained. Class 1 mAbs blocked the binding of LPS to LBP; class 2 mAbs blocked the binding of LPS/LBP complexes to CD14; class 3 mAbs bound LBP but did not suppress LBP activity. In vivo, class 1 and class 2 mAbs suppressed LPS-induced TNF production and protected mice from lethal endotoxemia. These results show that the neutralization of LBP accomplished by blocking either the binding of LPS to LBP or the binding of LPS/LBP complexes to CD14 protects the host from LPS-induced toxicity, confirming that LBP is a critical component of innate immunity.     

Identification of TNF-alpha binding peptides from a D-amino acid hexapeptide library that specifically inhibit TNF-alpha binding to recombinant p55 receptor

Tumour necrosis factor alpha (TNF-alpha) is a pro-inflammatory cytokine with pleiotropic activity that binds to two transmembrane receptors. Its role in mediating the inflammatory response to injury or infection has been well documented and it has been shown to be a causative factor in rheumatoid arthritis, inflammatory bowel disease and septic shock. Using synthetic peptide libraries composed exclusively of D-amino acids, two distinct hexapeptide families that block the binding of TNF-alpha to its receptors were identified. In the deconvolution of the library, activity increased from submillimolar to the low micromolar range with the most active compound having an IC50 of 0.33 microM. With the aid of biotinylated constructs of these hexapeptides it was possible to demonstrate that their antagonistic effect is due to specific binding to TNF-alpha and not to its receptor.     

Two leukocyte receptors (CD11a/CD18 and CD11b/CD18) mediate transient adhesion to endothelium by binding to different ligands

Upon stimulation with C5a, TNF, or phorbol dibutyrate (PDB), polymorphonuclear leukocytes (PMN) exhibit first an increase then a decrease in adhesion to unstimulated endothelial cells (EC). Essentially all of this adhesion is mediated by the CD18 family of leukocyte integrins on PMN. To determine the individual roles of CD11a/CD18 (LFA-1), CD11b/CD18 (CR3, Mac-1) and CD11c/CD18 (p150,95) in adhesion of PDB-stimulated PMN to unstimulated EC, mAb against the CD11 chains were used. mAb against CD11a or CD11b each blocked adhesion of PMN to EC by approximately 50%, but mAb against CD11c had no effect. Inasmuch as a combination of anti-CD11a and CD11b mAb completely blocked adhesion, it appears that CD11a/CD18 and CD11b/CD18 make approximately equal contributions to binding, and CD11c does not participate. Anti-CD11a or CD11b each blocked adhesion by about 50% throughout the transient time course of PDB-stimulated adhesion, indicating that the capacity of each of these receptors to bind EC is transiently activated by PDB. We next examined the role of ICAM-1 on EC as a ligand for CD18. Two anti-ICAM-1 mAb (LB-2 and 84H10) each inhibited PMN adhesion in a dose-dependent fashion, reaching a maximal inhibition of approximately 50%. Anti-ICAM-1 mAb blocked the CD11a/CD18-dependent portion of adhesion because concomitant use of anti-CD11a and anti-ICAM-1 did not cause additive inhibition. In contrast, anti-CD11b plus anti-ICAM-1 resulted in complete blockade of adhesion. This result suggests that CD11a/CD18 recognizes ICAM-1 on EC, but CD11b/CD18 recognizes a different ligand(s). To determine if CD11b CD18 has the ability to recognize ICAM-1, human macrophages were plated on culture surfaces coated with purified ICAM-1. Interaction of CD11a/CD18 with the surface-bound ICAM-1 resulted in selective down-modulation of CD11a/CD18 from the apical portion of the macrophages. In contrast, ICAM-1-coated surfaces did not down-modulate CD11b/CD18. The data suggest that CD11b/CD18 does not recognize ICAM-1, and that this receptor functions in adhesion of PMN to EC by recognizing novel ligand(s) on EC.     

Human alpha-defensins inhibit BK virus infection by aggregating virions and blocking binding to host cells

BK virus (BKV) is a polyomavirus that establishes a lifelong persistence in most humans and is a major impediment to success of kidney grafts. The function of the innate immune system in BKV infection and pathology has not been investigated. Here we examine the role of antimicrobial defensins in BKV infection of Vero cells. Our data show that alpha-defensin human neutrophil protein 1 (HNP1) and human alpha-defensin 5 (HD5) inhibit BKV infection by targeting an early event in the viral lifecycle. HD5 treatment of BKV reduced viral attachment to cells, whereas cellular treatment with HD5 did not. Colocalization studies indicated that HD5 interacts directly with BKV. Ultrastructural analysis revealed HD5-induced aggregation of virions. HD5 also inhibited infection of cells by other related polyomaviruses. This is the first study to demonstrate polyomavirus sensitivity to defensins. We also show a novel mechanism whereby HD5 binds to BKV leading to aggregation of virion particles preventing normal virus binding to the cell surface and uptake into cells.     

Geldanamycin treatment ameliorates the response to LPS in murine macrophages by decreasing CD14 surface expression

Geldanamycin (GA) is an antibiotic produced by Actinomyces, which specifically inhibits the function of the heat shock protein 90 family. Treatment of a murine macrophage cell line (J774) with GA resulted in a reduced response to Escherichia coli lipopolysaccharide (LPS) as visualized by a decrease of NF-kappaB translocation into the nucleus and secretion of tumor necrosis factor alpha (TNF-alpha). To elucidate the mechanism of this effect, the expression of CD14, the formal LPS receptor, was analyzed. Cells treated with GA showed a reduced level of surface CD14 detected by immunostaining, whereas the expression of other surface receptors, such as FC-gamma receptor and tumor necrosis factor receptors (TNF-R1 and TNF-R2), was unaffected. The reduced surface level of CD14 was not due to a reduction in its expression because CD14 steady state mRNA levels or the total cellular pool of CD14 was not altered by GA treatment. Surface CD14 was more rapidly internalized after GA treatment (2-3 h) than after incubation with cycloheximide. Immunostaining of permeabilized cells after GA treatment revealed a higher intracellular content of CD14 colocalizing with calnexin, an endoplasmic reticulum (ER) protein. These results suggest that the decrease in CD14 surface expression after GA treatment is due to rapid internalization without new replacement. These effects may be due to the inhibition of Hsp90 and Grp94 by GA in macrophages.     

A novel family of hairpin peptides that inhibit IgE activity by binding to the high-affinity IgE receptor

A family of structured peptides that bind to FcepsilonRIalpha, the alpha-chain of the high-affinity receptor for IgE, has been identified. Binding selections using FcepsilonRIalpha and polyvalent peptide-phage libraries yielded a dominant 18-residue peptide-phage clone, as well as related sequences that did not resemble fragments of IgE. Synthetic peptides based on these sequences inhibited IgE binding to its receptor, and were found by NMR analysis to adopt a stable beta-hairpin structure in solution. Optimized peptides with micromolar receptor affinity exhibited high stability in biological fluids and inhibited cellular histamine release in an in vitro bioassay of IgE activity. The structure-activity relationships of these peptides, which are less than 1% of the size of IgE, suggest an overlap between their binding site and that of IgE on FcepsilonRI. Thus, the peptides demonstrate that blocking a small epitope on this receptor chain is sufficient to block IgE activity. Such structured peptides represent a possible starting point for the design of novel antagonists, and offer the potential for testing in vivo a new approach for treating allergic disease.     

The presence of activated CD4(+) T cells is essential for the formation of colony-forming unit-endothelial cells by CD14(+) cells

The number of colony forming unit-endothelial cells (CFU-EC) in human peripheral blood was found to be a biological marker for several vascular diseases. In this study, the heterogeneous composition of immune cells in the CFU-ECs was investigated. We confirmed that monocytes are essential for the formation of CFU-ECs. Also, however, CD4(+) T cells were found to be indispensable for the induction of CFU-EC colonies, mainly through cell-cell contact. By blocking or activating CD3 receptors on CD4(+) T cells or blocking MHC class II molecules on monocytes, it was shown that TCR-MHCII interactions are required for induction of CFU-EC colonies. Because the supernatant from preactivated T cells could also induce colony formation from purified monocytes, the T cell support turned out to be cytokine mediated. Gene expression analysis of the endothelial-like colonies formed by CD14(+) cells showed that colony formation is a proangiogenic differentiation and might reflect the ability of monocytes to facilitate vascularization. This in vitro study is the first to reveal the role of TCR-MHC class II interactions between T cells and monocytes and the subsequent inflammatory response as stimulus of monocytic properties that are associated with vascularization.     

Modulation of FcgammaRI (CD64) ligand binding by blocking peptides of periplakin

FcgammaRI requires both the intracellular domain of the alpha-chain and associated leukocyte Fc receptor (FcR) gamma-chains for its biological function. We recently found the C terminus of periplakin to selectively interact with the cytoplasmic domain of the FcgammaRI alpha-chain. It thereby enhances the capacity of FcgammaRI to bind, internalize, and present antigens on MHC class II. Here, we characterized the domains involved in FcgammaRI-periplakin interaction using truncated and alanine-substituted FcgammaRI mutants and randomly mutagenized periplakin. This allowed us to design TAT peptides that selectively interfered with endogenous FcgammaRI-periplakin interactions. The addition of these peptides to FcgammaRI-expressing cells modulated FcgammaRI ligand binding, as assessed by erythrocyte-antibody-rosetting. These data support a dominant-negative role of C-terminal periplakin for FcgammaRI biological activity and implicate periplakin as a novel regulator of FcgammaRI in immune cells.     

Paclitaxel may inhibit autoimmune diseases by sparing or increasing the number of CD4(+) CD25(+) regulatory T cells

Paclitaxel, a representative of taxanes, exhibits cytotoxic effects against a broad range of tumors. Strikingly, an emerging body of data suggests that paclitaxel also exerts effects on immune system by stimulating anti-tumor and anti-autoimmunity effects, supporting the idea that paclitaxel suppresses tumor through several mechanisms and not solely through inhibiting cell division. Based on the accumulating data, we hypothesized that paclitaxel may inhibit autoimmune diseases by sparing or actively increasing the number of CD4(+) CD25(+) Treg cells. The hypothesis, if proved to be correct, will significantly improve our understanding of the tumor immunity, autoimmunity and its related pathological effects. It will influence our choice on immunosuppressive drugs for cancer patients with autoimmune diseases. It will also impact the immunotherapy for tumors.     

CD36 peptides enhance or inhibit CD36-thrombospondin binding. A two-step process of ligand-receptor interaction.

CD36 (glycoprotein IV or IIIB) is an integral plasma membrane protein of wide cellular distribution and functions as a receptor site for thrombospondin (TSP), an adhesive protein important in cell-cell and cell-matrix interactions. OKM5, a monoclonal anti-CD36 antibody, has been reported to block CD36 cell adhesive functions suggesting that the OKM5 epitope on CD36 is functionally important. A panel of 10 synthetic CD36 peptides was made. One peptide, P139-155, specifically inhibited the immunoadsorption of CD36 by OKM5, and P139-155 was directly immunoadsorbed by OKM5, indicating that CD36 sequence 139-155 represents part of the OKM5 epitope. TSP bound to immobilized P139-155 in a dose-dependent and saturable manner. Surprisingly, P139-155 significantly augmented, instead of inhibited, binding of CD36 to TSP. This peptide did not induce platelet aggregation but augmented ADP- and collagen-induced aggregation in platelet-rich plasma. Another CD36 peptide, P93-110, which had no effect on OKM5 immunoadsorption, blocked binding of CD36 to immobilized TSP and partially inhibited collagen-induced platelet aggregation. P93-110 by itself did not bind to TSP; however, in the presence of P139-155, there was a marked enhancement of P93-110 binding to TSP, with a stoichiometry consistent with the trimeric nature of TSP. The data suggest that CD36-TSP interaction is a two-step process; the sequence 139-155 region of CD36 binds first to TSP, triggering a change in TSP to reveal a second site, which binds the 93-110 region of CD36 with high affinity. CD36 peptides can be used as stimulators or inhibitors in cellular adhesive events involving TSP-CD36 interaction. Conformational changes leading to the exposure or activation of high affinity binding sites may occur in both the receptor and the ligand upon cell-cell and cell-matrix adhesion.     

Differential regulation of CD44 expression by lipopolysaccharide (LPS) and TNF-alpha in human monocytic cells: distinct involvement of c-Jun N-terminal kinase in LPS-induced CD44 expression

Alterations in the regulation of CD44 expression play a critical role in modulating cell adhesion, migration, and inflammation. LPS, a bacterial cell wall component, regulates CD44 expression and may modulate CD44-mediated biological effects in monocytic cells during inflammation and immune responses. In this study, we show that in normal human monocytes, LPS and LPS-induced cytokines IL-10 and TNF-alpha enhance CD44 expression. To delineate the mechanism underlying LPS-induced CD44 expression, we investigated the role of the mitogen-activated protein kinases (MAPKs), p38, p42/44 extracellular signal-regulated kinase, and c-Jun N-terminal kinase (JNK) by using their specific inhibitors. We demonstrate the involvement, at least in part, of p38 MAPK in TNF-alpha-induced CD44 expression in both monocytes and promonocytic THP-1 cells. However, neither p38 nor p42/44 MAPKs were involved in IL-10-induced CD44 expression in monocytes. To further dissect the TNF-alpha and LPS-induced signaling pathways regulating CD44 expression independent of IL-10-mediated effects, we used IL-10 refractory THP-1 cells as a model system. Herein, we show that CD44 expression induced by the LPS-mediated pathway predominantly involved JNK activation. This conclusion was based on results derived by transfection of THP-1 cells with a dominant-negative mutant of stress-activated protein/extracellular signal-regulated kinase kinase 1, and by exposure of cells to JNK inhibitors dexamethasone and SP600125. All these treatments prevented CD44 induction in LPS-stimulated, but not in TNF-alpha-stimulated, THP-1 cells. Furthermore, we show that CD44 induction may involve JNK-dependent early growth response gene activation in LPS-stimulated monocytic cells. Taken together, these results suggest a predominant role of JNK in LPS-induced CD44 expression in monocytic cells.     

GPI-linked endothelial CD14 contributes to the detection of LPS

The inflammatory endothelial response to LPS is critical to the host's surviving a gram-negative bacterial infection. In this study we investigated whether human endothelial cells express the functional coreceptor for LPS, CD14, and most importantly whether it is glycosylphosphatidylinositol (GPI) linked. We also examined whether plasma proteins could reconstitute an LPS response in CD14-inhibited endothelium. RT-PCR- and CD14-specific MAbs demonstrated CD14 expression on primary human umbilical vein endothelial cells (HUVEC) but not passaged HUVEC. The amino acid sequence of endothelial CD14 was 99% homologous to CD14 on monocytes. Endothelium responded to relatively low levels of LPS in the absence of plasma, and this was entirely dependent on CD14. Removal of GPI-linked proteins with phosphatidylinositol-phospholipase C prevented LPS detection and subsequent protein synthesis (E-selectin expression). Endothelial CD14 was sufficient to initiate functional leukocyte recruitment, an event inhibited by blocking its LPS binding epitope and also by removing CD14 from the endothelial surface. Plasma proteins restored only approximately 30% of the LPS response in CD14-inhibited endothelium. In conclusion, our results strongly support an important role for endothelial membrane CD14 in the activation of endothelium for leukocyte recruitment.     

Evidence of a trimolecular complex involving LPS,LPS binding protein and soluble CD14 as an effector of LPS response

The kinetics of the interaction of lipopolysaccharide (LPS), lipopolysaccharide binding protein (LBP) and CD14 was studied using surface plasmon resonance. The association and dissociation rate constants for the binding of LPS and rsCD14 were 2.9 x 10(4) M(-1) s(-1) and 0.07 s(-1) respectively, yielding a binding constant of 4.2 x 10(5) M(-1). Significantly, the presence of LBP increased not only the association rate but also the association constant for the interaction between LPS and CD14 by three orders of magnitude. Our experimental results suggest that LBP interacts with LPS and CD14 to form a stable trimolecular complex that has significant functional implications as it allows monocytes to detect the presence of LPS at a concentration as low as 10 pg/ml or 2 pM, and to respond by secreting interleukin-6. Thus, LBP is not merely transferring LPS to CD14 but it forms an integral part of the LPS-rLBP-rsCD14 complex.     

Induction of CD4(+) and CD8(+) Bordetella pertussis toxin subunit S1 specific T cells by immunization with synthetic peptides.

In this study two synthetic peptides from the Bordetella pertussis toxin subunit S1 were conjugated to human anti-idiotypic antibodies and used as an immunogen in cancer patients to induce immunity. The aims of the present report are to explain why no carrier or adjuvant effect of the conjugated pertussis peptides could be established regarding induction of responses against the anti-idiotype and to explore the type and quality of induced anti-pertussis immune responses. The lack of carrier and adjuvant effect of the peptides might be related to the fact that the anti-idiotypic antibodies by themselves include helper epitopes and that none of the patients had a detectable T cell response against any of the selected peptides before immunization, which might be a requirement for an adjuvant effect. However, three of four immunized patients mounted a humoral as well as cellular response against the pertussis peptides used. The induced T cell immunity was restricted to one of the two peptides in responding patients. Established T cell lines and MHC blocking studies indicated that the T cell epitopes of the two peptides had a different MHC restriction. The type of T cell response induced seemed to govern the humoral response. The only durable antibody response was accompanied by the presence of a CD4(+) T cell response against the same peptide. Immunization with an anti-idiotype conjugated to synthetic peptides might thus induce both a B and a T cell response against the peptides and the type of induced T cells (CD4 or CD8) governs the quality of the humoral response. Moreover, the possibility of boosting or inducing a response against the antigen from which the peptide sequences were deduced also seemed feasible.     

Polymorphonuclear granulocyte expression of CD11a/CD18, CD11b/CD18 and L-selectin in normal individuals

Abstract In this study direct immunofluorescence and flow cytometry with calibration using quantitative bead standards were used to enumerate the cell surface receptors CD11a/CD18, CD11b/CD18 and L-selectin. Holding blood at room temperature and fixation of samples prior to staining induced changes in expression, while immediate staining of polymorphonuclear granulocytes (PMN) in whole blood followed by fixation produced accurate values. The ranges of PMN adhesion molecule expression in 10 normal individuals were CD11a/CD18: 14794–28725, CD11b/CD18: 5300–11939 and L-selectin: 35662–61654 receptors per cell. Differences within individuals over 4 h were also observed. Adhesion molecule expression is used as an index of the adhesive function and state of activation of the cell. The data presented here shows that there is inherent variability in the expression of the PMN adhesion molecules between and within individuals, thus direct comparisons of PMN adhesion molecule expression between patients and “normals” must be interpreted with caution.     

Cutting edge: cationic antimicrobial peptides block the binding of lipopolysaccharide (LPS) to LPS binding protein

We investigated the mechanism by which cationic antimicrobial peptides block the activation of macrophages by LPS. The initial step in LPS signaling is the transfer of LPS to CD14 by LPS binding protein (LBP). Because many cationic antimicrobial peptides bind LPS, we asked whether these peptides block the binding of LPS to LBP. Using an assay that measures the binding of LPS to immobilized LBP, we show for the first time that a variety of structurally diverse cationic antimicrobial peptides block the interaction of LPS with LBP. The relative ability of different cationic peptides to block the binding of LPS to LBP correlated with their ability to block LPS-induced TNF-alpha production by the RAW 264.7 macrophage cell line.