Involvement of CD14 and complement receptors CR3 and CR4 in nuclear factor-kappaB activation and TNF production induced by lipopolysaccharide and group B streptococcal cell walls

This study was undertaken to evaluate the role of CD14 and complement receptors type 3 (CR3) and 4 (CR4) in mediating TNF release and NF-kappaB activation induced by LPS and cell wall preparations from group B streptococci type III (GBS). LPS and GBS caused TNF secretion from human monocytes in a CD14-dependent manner, and soluble CD14, LPS binding protein, or their combination potentiated both LPS- and GBS-induced activities. Blocking of either CD14 or CD18, the common beta-subunit of CR3 and CR4, decreased GBS-induced TNF release, while LPS-mediated TNF production was inhibited by anti-CD14 mAb only. Chinese hamster ovary cell transfectants (CHO) that express human CD14 (CHO/CD14) responded to both LPS and GBS with NF-kappaB translocation, which was inhibited by anti-CD14 mAb and enhanced by LPS binding protein. While LPS showed fast kinetics of NF-kappaB activation in CHO/CD14 cells, a slower NF-kappaB response was induced by GBS. LPS also activated NF-kappaB in CHO cells transfected with either human CR3 or CR4 cDNA, although responses were delayed and weaker than those of CHO/CD14 cells. In contrast to LPS, GBS failed to induce NF-kappaB in CHO/CR3 or CHO/CR4 cells. Both C3H/OuJ (Lps[n]) and C3H/HeJ (Lps[d]) mouse peritoneal macrophages responded to GBS with TNF production and NF-kappaB translocation, whereas LPS was active only in C3H/OuJ macrophages. Thus, LPS and GBS differentially involve CD14 and CR3 or CR4 for signaling NF-kappaB activation in CHO cells and TNF release in human monocytes, and engage a different set of receptors and/or intracellular signaling pathways in mouse macrophages.     

Dual effects of LPS antibodies on cellular uptake of LPS and LPS-induced proinflammatory functions

Human phagocytes recognize bacterial LPS (endotoxin) through membrane CD14 (mCD14), a proinflammatory LPS receptor. This study tested the hypothesis that anti-LPS Abs neutralize endotoxin by blocking cellular uptake through mCD14. Ab-associated changes in the uptake and cellular distribution of FITC-LPS were assessed by flow cytometry and laser scanning confocal microscopy in human CD14-transfected Chinese hamster ovary fibroblasts (CHO-CD14 cells) and human peripheral blood monocytes. LPS core- and O-side chain-specific mAbs inhibited mCD14-mediated LPS uptake by both cell types in the presence of serum. O-side chain-specific mAb concurrently enhanced complement-dependent LPS uptake by monocytes through complement receptor-1 (CR1) and uptake by CHO-CD14 cells involving another heat-labile serum factor(s) and cell-associated recognition molecule(s). Core-specific mAb inhibited mCD14-mediated uptake of homologous and heterologous LPS, while producing less concurrent enhancement of non-mCD14-mediated LPS uptake. The modulation by anti-LPS mAbs of mCD14-mediated LPS uptake was associated with inhibition of LPS-induced nuclear factor-kappaB (NF-kappaB) translocation and TNF-alpha secretion in CHO-CD14 cells and monocytes, respectively, while mAb enhancement of non-mCD14-mediated LPS uptake stimulated these activities. LPS-specific Abs thus mediate anti-inflammatory and proinflammatory functions, respectively, by preventing target cell uptake of LPS through mCD14 and augmenting uptake through CR1 or other cell receptors.     

Physical association between CD155 and CD44 in human monocytes

Regulation of CD44-mediated binding to hyaluronan is critical in normal and diseased immune cell function. In earlier work by others (Shepley and Racaniello, J. Virol., 68, 1301 1309), anti-CD44 mAb blocked poliovirus binding to CD155 (the poliovirus receptor) in HeLa cells, suggesting that CD155 and CD44 may be physically associated. Here, we present evidence that CD155 and CD44 are physically associated in human monocytes. In co-modulation experiments in U937 monocytic cells, CD155 and CD44 reciprocally co-modulated. In primary human monocytes, CD 155 syn-capped with CD44. In immunofluorescence flow cytometric experiments, anti CD44 mAb inhibited up to 94% of binding by anti-CD155 mAb which blocks poliovirus binding to CD155. This inhibition was specific for CD155. Culturing monocytes increased the extent of inhibition. In addition, mAb against PRR2, a novel molecule that is related to CD 155, was inhibited by anti-CD44 in a dose-dependent manner, but not by anti-CD14. These data support the interpretation that CD155 (and related proteins) are physically associated with CD44 on monocyte cell surfaces. Although the current study does not address functional significance, we speculate that this interaction may have a role in regulating monocyte CD44 ligand binding which may be critical in pathological processes such as tumor metastasis and arthritis.     

Mutations of Gly to Ala in human glutathione transferase P1-1 affect helix 2 (G-site) and induce positive cooperativity in the binding of glutathione

Previous kinetic studies on human glutathione transferase P1-1 have indicated that the motions of an irregular alpha-helix (helix 2) lining the glutathione (GSH) binding site are viscosity dependent and may modulate the affinity of GSH binding. The effect of single amino acid residue substitutions (Gly to Ala) in this region is investigated here by site-directed mutagenesis. Three mutants (Gly41Ala, Gly50Ala and Gly41Ala/Gly50Ala) were overexpressed in Escherichia coli, purified, and characterized by kinetic, structural, and spectroscopic studies. All these mutant enzymes show kcat values similar to that of the wild-type enzyme, while the [S]0.5 for GSH increases about eight-fold in the Gly41Ala mutant and more than 100-fold in the Gly41Ala/Gly50Ala double mutant. This change in affinity towards GSH is accompanied by an induced positive cooperativity as reflected by Hill coefficients of 1.4 (Gly41Ala) and 1.7 (Gly41Ala/Gly50Ala) upon substrate binding. Taken together, these data suggest that the region around helix 2 is markedly altered leading to the observed intersubunit communication. Molecular modeling of the Gly41Ala/Gly50Ala mutant and of the inactive oxidized form of the native enzyme provides a structural explanation of our results.     

Utility of electron microscopy in the assessment of transthoracic needle lung biopsy specimens

The soluble form of the leukocyte membrane antigen CD14 is known to increase the sensitivity of endothelial and epithelial cell lines to bacterial lipopolysaccharide (LPS). This molecule also directly induces cytokine production in monocytes. Here, the effect of sCD14 and LPS on the release of IL-6 and IL-8 by human bronchial epithelial cells (HBECs) was studied. Soluble CD14 induced cytokine production both in the presence and absence of LPS. In addition, neither sCD14 nor anti-CD14 monoclonal antibody which blocks the interaction of LPS with CD14 had any effect on the binding of LPS to HBECs. These data suggest that sCD14 may induce the release of IL-6 and IL-8 from HBECs. However, the binding of LPS to bronchial epithelium appears to be mediated by CD14-independent mechanisms.     

A synthetic lipopolysaccharide-binding peptide based on amino acids 27-39 of serum amyloid P component inhibits lipopolysaccharide-induced responses in human blood

LPS-binding proteins in plasma play an important role in modifying LPS toxicity. Significant properties have already been attributed to the LPS-binding protein (LBP). It accelerates LPS toxicity as well as incorporation into high-density lipoproteins, leading to neutralization of LPS in serum. A search for other LPS-binding components in serum, using LPS-coated magnetic beads, revealed a new LPS-binding protein. N-terminal microsequencing identified this protein as serum amyloid P component (SAP). Purified SAP bound to smooth and rough types of LPS via the lipid A part. SAP inhibited the binding of FITC-labeled ReLPS (LPS from Salmonella minnesota strain R595) to human monocytes and the ReLPS-induced priming of the oxidative burst of human neutrophils only in the presence of low concentrations of LBP. In search for the LPS binding site of SAP, we found that pep27-39, a 13-mer peptide consisting of amino acids 27-39 of SAP, competitively inhibited the binding of LPS to SAP. In addition, pep27-39 significantly inhibited ReLPS-induced responses in phagocytes in the presence of serum, as well as in human whole blood. Carboxamidomethylated pep27-39 showed an even more pronounced reduction of the ReLPS-induced priming of phagocytes in human blood. Performing gel filtration of FITC-labeled ReLPS incubated with soluble CD14, we showed that SAP could not prevent binding of LPS to soluble CD14, in contrast to pep27-39. The ability of pep27-39 to antagonize specifically the effects of LPS in the complex environment of human blood suggests that pep27-39 may be a novel therapeutic agent in the treatment of gram-negative sepsis.     

Sensitization of rat glioblastoma multiforme to cisplatin in vivo following restoration of wild-type p53 function

A panel of seven monoclonal antibodies (mAbs) raised against cardiac troponin-I (CdTnI) isolated from canine and human hearts, which have been shown to be cardiac-specific but cross-species reactive [Cummins, B., Aukland, M. L. & Cummins, P. (1987) Amer. Heart J. 113, 1333-1344], were used in this study. These mAbs were tested against recombinant wild-type and mutant human CdTnI proteins to assess their value as probes for the phosphorylation status of CdTnI. Four mAbs were found to react positively with the recombinant wild-type protein and their epitopes were contained in residues 31-210 of the human cardiac protein. Two of these mAbs appeared to be directed against the same epitope site within this region. The remaining three mAbs only reacted against the recombinant wild-type protein when it was phosphorylated, showing that these three antibodies were directed against the phosphate group(s) on Ser23 and/or Ser24. In order to investigate this further, a series of single and double mutants of CdTnI were used in which either Ala (to direct the enzymatic phosphorylation) or Asp (to mimic the phosphate group) replaced the Ser23 and/or Ser24. It was found the all three mAbs were able to react with the mono-phosphorylated form of the [Ala23]CdTnI single mutant but not the mono-phosphorylated form of the [Ala24]CdTnI single mutant, showing that they specifically required phosphorylation at Ser24. Experiments with a synthetic peptide composed of residues 1-29 of human CdTnI confirmed these data. Two of the three phosphorylation-specific mAbs were able to react with mutants containing either two Asp residues replacing Ser23 and Ser24 or one Asp residue instead of Ser24, indicating that a negative charge at position Ser24 is sufficient to invoke a reaction. The other mAb was more specific in that it would only react with CdTnI species with a phosphate group on Ser24.     

Coexistence of CD14-dependent and independent pathways for stimulation of human monocytes by gram-positive bacteria

The cell wall is a key inflammatory agent of gram-positive bacteria. Possible receptors mediating cell wall-induced inflammation include CD14 and platelet-activating factor (PAF) receptor. To delineate the conditions under which these various receptors might be used, human monocytic THP-1 cells and heparinized whole human blood were stimulated with lipopolysaccharide (LPS), intact Streptococcus pneumoniae bacteria, or purified pneumococcal cell wall. THP-1 culture supernatant or cell-free plasma was analyzed for the presence of tumor necrosis factor, interleukin-1beta (IL-1beta), and IL-6. For the cultured monocytes, anti-CD14 inhibited induction of the inflammatory cytokines by the cell wall and LPS but not by intact pneumococcal bacteria. Despite the difference in CD-14 usage, the intracellular pathways induced by the three agents demonstrated similarities, as revealed in the presence of specific signal transduction inhibitors such as cholera toxin, pertussis toxin, and genistein. Cytokine production in whole human blood indicated that anti-CD14 failed to block responses to cell wall and intact pneumococci, whereas while LPS-induced responses were inhibited. PAF receptor antagonist had no effect under any conditions in both assays. These results indicate that although cell walls bind to both CD14 and PAF receptor, only CD14 appears to engender a cytokine response under restricted conditions. Furthermore, host cell responses to intact pneumococci are consistently independent of CD14 and PAF receptor.     

Bacterial lipopolysaccharide can enter monocytes via two CD14-dependent pathways

Host recognition and disposal of LPS, an important Gram-negative bacterial signal molecule, may involve intracellular processes. We have therefore analyzed the initial pathways by which LPS, a natural ligand of glycosylphosphatidylinositol (GPI)-anchored CD14 (CD14-GPI), enters CD14-expressing THP-1 cells and normal human monocytes. Exposure of the cells to hypertonic medium obliterated coated pits and blocked 125I-labeled transferrin internalization, but failed to inhibit CD14-mediated internalization of [3H]LPS monomers or aggregates. Immunogold electron microscope analysis found that CD14-bound LPS moved principally into noncoated structures (mostly tubular invaginations, intracellular tubules, and vacuoles), whereas relatively little moved into coated pits and vesicles. When studied using two-color laser confocal microscopy, internalized Texas Red-LPS and BODIPY-transferrin were found in different locations and failed to overlap completely even after extended incubation. In contrast, in THP-1 cells that expressed CD14 fused to the transmembrane and cytosolic domains of the low-density lipoprotein receptor, a much larger fraction of the cell-associated LPS moved into coated pits and colocalized with intracellular transferrin. These results suggest that CD14 (GPI)-dependent internalization of LPS occurs predominantly via noncoated plasma membrane invaginations that direct LPS into vesicles that are distinct from transferrin-containing early endosomes. A smaller fraction of the LPS enters via coated pits. Aggregation, which greatly increases LPS internalization, accelerates its entry into the nonclathrin-mediated pathway.     

Identification of the sheep homologue of the monocyte cell surface molecule--CD14

An ovine monocyte/macrophage cell surface antigen was recognized by three mouse monoclonal antibodies (mAbs) VPM65, VPM66 and VPM67. These mAbs also reacted with bovine cells. The antibodies immunoprecipitated a single, glycosyl-phosphatidylinositol-linked polypeptide of M(r) 55,000 which, when deglycosylated, was reduced to M(r) 53,000. They reacted strongly with peripheral blood monocytes, alveolar macrophages and peripheral blood granulocytes, and weakly with afferent lymph dendritic cells. They also reacted with macrophages in many different tissues but were non-reactive with lymphocytes. Competitive flow cytometry shows that these three mAbs recognize the same or a closely related epitope of a single antigen. An antigen-specific capture ELISA using the anti-human CD14 mAb (TUK4) revealed that all four mAbs associate with the same antigen. These data demonstrate that the mAbs react with the ovine homologue of the lipopolysaccharide (LPS)-LPS binding protein receptor, CD14.     

Amino-terminal sequencing of sheep CD1 antigens and identification of a sheep CD1D gene

The anti-CD1 monoclonal antibodies IAH-CC14 and SBU-T6 were used to immunopurify CD1 antigens from sheep thymocytes. The amino-terminal sequence of IAH-CC14 yielded 13 amino acids, and 29 amino acids were obtained from the SBU-T6 antigen. The sequence of the IAH-CC14 antigen was 100% identical to the predicted sequence of the sheep CD1B clone, SCD1B-42. The 29 amino acid sequence of the SBU-T6 antigen did not match identically with the derived amino acid sequence of any of the previously reported sheep CD1 genes but had closest similarity to the derived sequence of human CD1E. Degenerate polymerase chain reaction primers based on this sequence identified a group 2 sheep CD1 gene. The predicted amino acid sequence of this gene shows that it is not identical to the SBU-T6 peptide, indicating that a different, CD1D-like gene was cloned.     

Involvement of CD14 on human gingival fibroblasts in Porphyromonas gingivalis lipopolysaccharide-mediated interleukin-6 secretion

The lipopolysaccharides (LPS) of Porphyromonas gingivalis are implicated in the initiation and development of periodontal diseases. However, the mechanisms underlying P. gingivalis LPS-mediated periodontal destruction are still unknown. Here, it was found that P. gingivalis LPS activates human gingival fibroblasts (HGF) to release interleukin 6 (IL-6) via CD14. Flow-cytometric analysis showed that HGFs bind to fluorescein-isothiocyanate (FITC)-labelled LPS, and express CD14 on their surfaces. The binding of FITC LPS was competitively suppressed by unlabelled synthetic lipid A as well as by LPS. LPS-induced IL-6 production was inhibited by anti-CD14 monoclonal antibody in a dose-dependent manner. The binding of FITC LPS to HGF was abrogated by anti-CD14 monoclonal antibody. Engagement of LPS initiated the protein tyrosine phosphorylation of several intracellular proteins including extracellular signal-regulated kinase (ERK) 1 and 2, and these events were suppressed by the anti-CD14 monoclonal. These results suggest that CD14 is a cell surface binding site for LPS and is involved in the LPS-mediated activation of HGF.     

Human growth hormone-releasing hormone hGHRH(1-29)-NH2: systematic structure-activity relationship studies

Two complete and two partial structure-activity relationship scans of the active fragment of human growth hormone-releasing hormone, [Nle27]-hGHRH(1-29)-NH2, have identified potent agonists in vitro. Single-point replacement of each amino acid by alanine led to the identification of [Ala8]-, [Ala9]-, [Ala15]- (Felix et al. Peptides 1986 1986, 481), [Ala22]-, and [Ala28, Nle27]-hGHRH(1-29)-NH2 as being 2-6 times more potent than hGHRH(1-40)-OH (standard) in vitro. Nearly complete loss of potency was seen for [Ala1], [Ala3], [Ala5], [Ala6], [Ala10], [Ala11], [Ala13], [Ala14], and [Ala23], whereas [Ala16], [Ala18], [Ala24], [Ala25], [Ala26], and [Ala29] yielded equipotent analogues and [Ala7], [Ala12], [Ala17], [Ala20], [Ala21], and [Ala27] gave weak agonists with potencies 15-40% that of the standard. The multiple-alanine-substituted peptides [MeTyr1,Ala15,22,Nle27]-hGHRH(1-29)-NH2 (29) and [MeTyr1,Ala8,9,15,22,28,Nle 27]-hGHRH(1-29)-NH2 (30) released growth hormone 26 and 11 times, respectively, more effectively than the standard in vitro. Individual substitution of the nine most potent peptides identified from the Ala series with the helix promoter alpha-aminoisobutyric acid (Aib) produced similar results, except for [Aib8] (doubling vs [Ala8]), [Aib9] (having vs [Ala9]), and [Aib15] (10-fold decrease vs [Ala15]). A series of cyclic analogues was synthesized having the general formula cyclo(25-29)[MeTyr1,-Ala15,Xaa25,Nle27,Yaa29+ ++]-GHRH(1-29)-NH2, where Xaa and Yaa represent the bridgehead residues of a side-chain cystine or [i-(i + 4)] lactam ring. The ring size, bridgehead amino acid chirality, and side-chain amide bond location were varied in this partial series in an attempt to maximize potency. Application of lactam constraints in the C-terminus of GHRH(1-29)-NH2 identified cyclo(25-29)[MeTyr1,Ala15,DAsp25,Nle27,Orn29+ ++]-hGHRH(1-29)-NH2 (46) as containing the optimum bridging element (19-membered ring) in this region of the molecule. This analogue (46) was 17 times more potent than the standard. Equally effective was an [i-(i + 3)] constraint yielding the 18-membered ring cyclo(25-28)[MeTyr1,Ala15,Glu25,Nle,27Lys28]- hGHRH-(1-29)-NH2 (51) which was 14 times more potent than the standard. A complete [i-(i + 3)] scan of cyclo(i,i + 3)[MeTyr1,Ala15,Glui,Lys(i + 3),Nle27]-hGHRH(1-29)-NH2 was then produced in order to test the effects of a Glu-to-Lys lactam bridge at all points in the peptide. Of the 26 analogues in the series, 11 had diminished potencies of less than 10% that of the agonist standard, 4 were weak agonists (15-40% relative potency), and 4 analogues were equipotent to the standard. The 7 most potent analogues ranged in potency from 3 to 14 times greater than that of the standard and contained the [i-(i + 3)] cycles between residues 4-7, 5-8, 9-12, 16-19, 21-24, 22-25, and 25-28. The combined results from these systematic studies allowed for an analysis of structural features in the native peptide that are important for receptor activation. Reinforcement of the characteristics of amphiphilicity, helicity, and peptide dipolar effects, using recognized medicinal chemistry approaches including introduction of conformational constraints, has resulted in several potent GHRH analogues.     

Bile acids with differing hydrophilic-hydrophobic properties do not influence cytokine production by human monocytes and murine Kupffer cells

Bile acids have been proposed to exert immunological effects of potential pathogenic or therapeutic relevance, yet the experimental evidence remains preliminary. We reexamined the effects of a variety of bile salts with differing hydrophilic-hydrophobic properties on the production of interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF alpha) from monocytes and Kupffer cells. Monocytes from healthy human donors and Kupffer cells from 5-week-old mice were incubated for up to 18 hours with or without varying concentrations of bile salts and lipopolysaccharide (LPS). Monocyte viability was > or = 95% with up to 250 mumol/L sodium ursodeoxycholate and < or = 90% with 200 mumol/L chenodeoxycholate, decreasing sharply at higher concentrations. Kupffer cells were more vulnerable, particularly to chenodeoxycholate (viabilities of 25% and 0% at concentrations of 100 mumol/L and 200 mumol/L, respectively). In monocytes incubated in the presence of 20% fetal calf serum, neither ursodeoxycholate and chenodeoxycholate, nor a variety of other unconjugated and conjugated bile acids, tested up to their maximal noncytotoxic concentrations, influenced the IL-6 and TNF alpha production, at any level of LPS stimulation. Similar to monocytes, incubation of murine Kupffer cells with ursodeoxycholate and chenodeoxycholate did not influence cytokine release. In contrast, the addition of 10 nmol/L dexamethasone to monocytes significantly decreased TNF-alpha and IL-6 release (69 +/- 11% and 48 +/- 15%, respectively). When monocytes were incubated with 200 mumol/L chenodeoxycholate in the presence of lower concentrations of fetal calf serum (10% and 5%, respectively) a significant inhibition of cytokine release was observed, whereas incubation with ursodeoxycholate did not cause any effect. Flow cytometry using fluoresceinated LPS showed that chenodeoxycholate does not interact with the CD14 receptor, thus excluding the possibility of an interference with the LPS uptake by monocytes. Incubation with [14C]-chenodeoxycholate showed that the intracellular bile acid uptake was inversely related to the concentration of fetal calf serum, being negligible (< 3 fmol/cell) at the highest level. In conclusion, bile acids with widely different hydrophobicities are incapable of influencing the release of IL-6 and TNF alpha by monocytes and Kupffer cells, provided they are studied at noncytotoxic concentrations and in the presence of physiological amounts of proteins.     

Heparin-binding protein (CAP37) is internalized in monocytes and increases LPS-induced monocyte activation

Previous studies have shown that the neutrophil-derived heparin-binding protein (HBP), also known as CAP37 or azurocidin, potentiates the LPS-induced release of proinflammatory cytokines (TNF-alpha, IL-1, and IL-6) from isolated human monocytes. To date, the mechanisms by which HBP enhances LPS-induced monocyte activation have not been elucidated, and it is not known whether HBP also increases the LPS-induced production of other bioactive substances. We studied human monocytes activated by recombinant human HBP and LPS and their interaction with the LPS receptor CD14. We hypothesized that the stimulatory effect of HBP on the LPS-induced release of proinflammatory mediators from monocytes was mediated by specific binding of HBP to monocytes, which resulted in an up-regulation of CD14. Our results demonstrated that HBP alone (10 microg/ml) stimulated the production of TNF-alpha from isolated monocytes. In addition, HBP had an additive effect on LPS-induced production of TNF-alpha and PGE2, suggesting a generalized monocyte activation. We used flow cytometry to demonstrate that HBP had a high affinity to monocytes but not to the LPS receptor CD14, and experiments performed at 4 degrees C indicated an energy-dependent step in this process. Confocal microscopy showed that monocytes internalize HBP within 30 min. These data suggest that mechanisms other than increased CD14 expression are responsible for the enhanced release of TNF-alpha or PGE2 in response to HBP and LPS.     

Phosphatidylinositides bind to plasma membrane CD14 and can prevent monocyte activation by bacterial lipopolysaccharide

Although bacterial lipopolysaccharides (LPS) and several other microbial agonists can bind to mCD14 (membrane CD14), a cell-surface receptor found principally on monocytes and neutrophils, host-derived mCD14 ligands are poorly defined. We report here that phosphatidylinositol (PtdIns), phosphatidylinositol-4-phosphate, and other phosphatidylinositides can bind to mCD14. Phosphatidylserine (PS), another anionic glycerophospholipid, binds to mCD14 with lower apparent affinity than does PtdIns. LPS-binding protein, a lipid transfer protein found in serum, facilitates both PS- and PtdIns-mCD14 binding. PtdIns binding to mCD14 can be blocked by anti-CD14 monoclonal antibodies that inhibit LPS-mCD14 binding, and PtdIns can inhibit both LPS-mCD14 binding and LPS-induced responses in monocytes. Serum-equilibrated PtdIns also binds to mCD14-expressing cells, raising the possibility that endogenous PtdIns may modulate cellular responses to LPS and other mCD14 ligands in vivo.     

N-terminal and central regions of the human CD44 extracellular domain participate in cell surface hyaluronan binding

CD44 molecules are cell surface receptors for hyaluronan (HA). To define regions of the extracellular domain of CD44 that are important for HA binding, we have studied the ability of HA-blocking CD44 mAbs to bind to CD44 from a variety of sources. Five CD44 mAbs (5F12, BRIC235, 3F12, BU-75, and HP2/9) of 21 studied were identified that at least partially blocked FITC-labeled HA (HA-FITC) binding to the standard form of CD44 (CD44S) in CD44-transfected Jurkat cells. Analysis of reactivity of HA-blocking CD44 mAbs defined three distinct epitopes. Lack of reactivity of mAb 5F12 with a CD44 fusion protein (CD44-Rg) containing an N-terminal truncation of 20 amino acids (aa), as well as reactivity of mAb 5F12 with an N-terminal CD44 synthetic peptide (CD44-9A), demonstrated that the N-terminal proximal region of CD44 (aa 1 to 20) was involved in mAb 5F12 binding. A mutant cell line, CEM-NKR, derived from the T-ALL cell line, CEM, did not bind mAb 5F12 nor bind HA, whereas wild-type CEM did bind mAb 5F12 and HA. Sequence analysis of wild-type CEM and CEM-NKR CD44 cDNA demonstrated a G to A point mutation at position 575 in the CD44 cDNA of CEM-NKR, resulting in an arginine to histidine mutation at aa position 154. Taken together, our studies demonstrated that there are three epitopes to which HA-blocking mAbs bind in the extracellular domain of CD44, and that the CD44 N-terminal proximal and central regions are two regions in the extracellular domain of CD44 that may interact and either mediate or regulate HA binding to cell surface CD44.     

High-resolution HLA typing for the DQB1 gene by sequence-based typing

BACKGROUND: Monocytic tissue factor (TF), initiating the extrinsic blood coagulation pathway, is often upregulated under septic or inflammatory conditions. The complex activating mechanism remains largely unclear and no effective strategy has been firmly established. In this study, we used a model monocytic cell line (human leukemic THP-1 promonocytes) to address (1) the nature of TF activation in response to bacterial endotoxin and (2) the application of anti-inflammatory cytokines in relieving monocytic hypercoagulation. RESULTS: TF in THP-1 cells was substantially activated by exposure to bacterial endotoxin (LPS; 5 micrograms/ml) for 6 h. Human recombinant IL-4 (500 ng/ml) and IL-10 (500 ng/ml) inhibited TF activation induced by LPS. To determine if these cytokines depressed LPS recognition resulting in such inhibition, we employed an anti-CD14 mAb (UCHM-1; Sigma Chemical) to address the role of CD14 in LPS transmembrane signaling. LPS-induced TF activation was depressed by 35% upon inclusion of the anti-CD14 mAb (1:10 dilution). This antibody alone mimicked TF activation which accounted for 35% of the LPS-induced TF activation, suggesting the activating role of CD14 ligation. In addition, the anti-CD14 mAb elicited the production of nitric oxide (NO) which was found to be independent of TF activation. NO production could serve as an independent index for monitoring LPS recognition. IL-4 depressed the anti-CD14 mAb-induced TF activation as well as NO elicitation, indicating the blockade of CD14 ligation. In contrast, IL-10 showed differential inhibitory activities. TF activation induced by either LPS or anti-CD14 mAb was inhibited by IL-10 which did not show any inhibition on NO elicitation under these conditions. In a separate approach, neither IL-4 nor IL-10 inhibited phorbol ester-induced NO elicitation. More direct evidence came from an epifluorescent demonstration showing that IL-4 blocked binding of FITC-conjugated LPS and anti-CD14 mAb to THP-1 cells. CONCLUSIONS: Taken together, the results suggest that LPS action in relation to TF activation consists of CD14-independent and -dependent signaling including CD14 ligation. We also showed that anti-inflammatory cytokines (IL-4 and -10) significantly depressed TF activation. IL-4 antagonized CD14-dependent LPS recognition leading to the depression in TF activation.