Reduced T-cell receptor CD3zeta-chain protein and sustained CD3epsilon expression at the site of mycobacterial infection.

Control of mycobacterial infection by the cellular immune system relies both on antigen-presenting cells and on T lymphocytes. The quality of an effective cellular immune response is dependent on functional signal transduction residing in the cytoplasmic tails of the T-cell receptor CD3 components. In order to investigate potential effects of mycobacteria on T-cell receptor signalling, we examined the protein expression of T-cell signal transduction molecules (CD3zeta, ZAP-70, p59fyn, p56lck). In Western blots of peripheral blood mononuclear cells of Mycobacterium tuberculosis infected patients, only the CD3zeta-chain showed a marked reduction in protein expression. To investigate the situation in situ, immunoenzymatic and immunofluorescence stainings for CD3epsilon and CD3zeta expression were performed on sections of normal lymphoid tissue, M. leprae infected and sarcoid tissue. CD3epsilon and CD3zeta expression were similar with respect to intensity, localization and the number of cells stained in normal lymphoid tissue and in sarcoid granulomas. In contrast, the granulomas of M. leprae infected tissues showed a significantly reduced expression of CD3zeta compared to CD3epsilon. Using double immunofluorescence analysis, virtually no CD3zeta expression could be detected in comparison to the CD3epsilon expression in the lesions. Apparently, mycobacteria are capable of significantly reducing CD3zeta-chain expression, which may be restored by cytokines. IL-2-enhanced zeta-chain expression and T-cell effector functions, defined by interferon-gamma release, in M. tuberculosis-specific and human leucocyte antigen-DR restricted CD4+ T cells isolated from granuloma lesions from patients with pulmonary tuberculosis. Because CD3zeta is essential for CD3 signalling and for eliciting T-cell effector functions, reduced CD3zeta protein expression could result in altered signal transduction and inefficient T-cell effector functions. Alternatively, reduced CD3zeta-chain expression may protect T cells from repetitive TCR stimulation associated with anergy or apoptosis.

Mycobacteria-induced TNF-alpha and IL-10 formation by human macrophages is differentially regulated at the level of mitogen-activated protein kinase activity.

The clinical course of mycobacterial infections is linked to the capacity of pathogenic strains to modulate the initial antimycobacterial response of the macrophage. To elucidate some of the mechanisms involved, we studied early signal transduction events leading to cytokine formation by human monocyte-derived macrophages (MDM) in response to clinical isolates of Mycobacterium avium. TNF-alpha production induced by M. avium was inhibited by anti-CD14 mAbs, but not by Abs against the macrophage mannose receptor. Analysis of mitogen-activated protein (MAP) kinase activation (extracellular signal-regulated kinase 1/2, p38, and c-Jun NH(2)-terminal kinase) showed a rapid phosphorylation of all three subfamilies in response to M. avium, which was inhibited by anti-CD14 Abs. Using highly specific inhibitors of p38 (SB203580) and MAP kinase kinase-1 (PD98059), we found that activation of the extracellular signal-regulated kinase pathway, but not of p38, was essential for the M. avium-induced TNF-alpha formation. In contrast, IL-10 production was abrogated by the p38 inhibitor, but not by the MAP kinase kinase-1 inhibitor. In conclusion, M. avium-induced secretion of TNF-alpha and IL-10 by human macrophages is differentially regulated at the level of MAP kinase activity.

The involvement of CD14 in the activation of human monocytes by peptidoglycan monomers.

BACKGROUND: Cell-wall components of Gram-positive and Gram-negative bacteria induce the production of cytokines in human peripheral blood mononuclear cells. These cytokines are the main mediators of local or systemic inflammatory reaction that can contribute to the development of innate immunity. AIMS: This study was performed to analyze the involvement of CD14 molecule in the activation of human monocytes by peptidoglycan monomer (PGM) obtained by biosynthesis from culture fluid of penicillin-treated Brevibacterium divaricatum NRLL-2311. METHODS: Cytokine release of interleukin (IL)-1, IL-6 and tumor necrosis factor-alpha from human monocytes via soluble CD14 (sCD14) or membrane-associated (mCD14) receptor using anti-CD14 monoclonal antibody (MEM-18) or lipid A structure (compound 406) was measured in bioassays. RESULTS: The results demonstrated that PGM in the presence of human serum might induce the monokine release in a dose-dependent manner. The addition of sCD14 at physiologic concentrations enhanced the PGM-induced monokine release, while the monokine inducing capacity of PGM in the presence of sCD14 was inhibited by MEM-18. Effects of PGM were also blocked by glycolipid, compound 406, suggesting the involvement of binding structures similar to those for lipopolysaccharide. CONCLUSION: Activation of human monocytes by PGM involves both forms of CD14 molecule, sCD14 and mCD14.

Heterogeneity of human peripheral blood monocyte subsets.

In recent years the number of reports describing phenotypically and functionally distinct subsets of human blood leukocytes, and in particular of subtypes of antigen-presenting cells has continuously increased. A great diversity was described not only for dendritic cells (DC), but also for human blood monocytes (Mo) and macrophages (Mac). Similar to DC, the different types of Mo subsets could be defined by distinct phenotypes and immunoregulatory functions. The characterization of blood Mo subpopulations revealed that some of them exhibit common features with myeloid or lymphoid DC and tissue Mac, but also demonstrate the existence of novel unique cell populations. The generation of lymphoid and myeloid DC and their heterogeneity has been the subject of recent reviews. Here we focus on Mo from human peripheral blood and summarize the data (including our own) dealing with their phenotypic and functional, in particular immunoregulatory properties.

Fas (CD95)-Fas ligand interactions are responsible for monocyte apoptosis occurring as a result of phagocytosis and killing of Staphylococcus aureus.

Human peripheral blood monocytes become apoptotic following phagocytosis of Staphylococcus aureus. In this study, we investigated the mechanisms involved in this phenomenon. Cells exposed to bacteria were examined for the surface expression of Fas and Fas ligand (FasL). The level of soluble form of FasL was also measured in the culture supernatants. As Fas-mediated apoptosis involves the activation of caspases, the activities of caspase-8 and caspase-3 were determined. Finally, the involvement of oxidative stress in apoptosis of infected monocytes was investigated. The data indicated that as a consequence of phagocytosis of S. aureus, FasL is released from the monocyte surface and induces apoptosis of phagocytic monocytes and to some extent the bystander cells. The importance of this mechanism was confirmed by demonstrating that blockage of CD95 prevents S. aureus-induced apoptosis of monocytes. Cell death occurring after phagocytosis of S. aureus involves the activation of caspase-3-like proteases, as the specific caspase-3 inhibitor suppressed apoptosis of infected cells. The generation of reactive oxygen intermediates by phagocytic monocytes by itself is not sufficient as a death signal but rather acts in up-regulating FasL shedding and possibly in modulating caspase activity.

The interaction of human peripheral blood eosinophils with bacterial lipopolysaccharide is CD14 dependent.

Bacterial lipopolysaccharide (LPS, endotoxin) is a ubiquitous component of dust and air pollution and is suspected to contribute after inhalation to an activation of eosinophils in bronchial tissues of asthmatic patients, provoking inflammatory and allergic processes. We were therefore interested in the interaction of eosinophil granulocytes with LPS and have examined the activation of and uptake to human peripheral blood eosinophils by LPS. Eosinophils were stimulated by LPS and the endotoxic component lipid A and the release of tumor necrosis factor alpha (TNF-alpha) and of the eosinophil-specific granule protein eosinophil cationic protein (ECP) was estimated. The results show induction of TNF-alpha and ECP-release by LPS and lipid A in a dose-dependent manner. Anti-CD14 monoclonal antibody (moAb) (clone MEM-18) and the synthetic lipid A partial structure 406 blocked the release of TNF-alpha and ECP by LPS-stimulated eosinophils. Studies with radioactively labeled LPS showed dose-dependent uptake of (3)H-LPS to eosinophils. The (3)H-LPS uptake was found to be specific because preincubation with unlabeled LPS, compound 406 and also anti-CD14 antibodies inhibited uptake of (3)H-LPS to eosinophil granulocytes. By flow cytometry using anti-CD14 moAb and by reverse transcriptase-polymerase chain reaction (RT-PCR) technique, CD14 expression was detectable. Furthermore, messenger RNA (mRNA) expression of Toll-like receptors (TLR) 2 and TLR 4 was detected, indicating the presence of these CD14 coreceptors. The results indicate that eosinophils can take up LPS and can be stimulated by LPS in a CD14-dependent manner. Hence, in addition to allergens, eosinophils interact with endotoxin, a process that possibly exacerbates ongoing inflammatory and allergic processes.

Down-regulation of neutrophil functions by the ELR(+) CXC chemokine platelet basic protein.

The platelet-derived neutrophil-activating peptide 2 (NAP-2, 70 amino acids) belongs to the ELR(+) CXC subfamily of chemokines. Similar to other members of this group, such as IL-8, NAP-2 activates chemotaxis and degranulation in neutrophils (polymorphonuclear [PMN]) through chemokine receptors CXCR-1 and CXCR-2. However, platelets do not secrete NAP-2 as an active chemokine but as the C-terminal part of several precursors that lack PMN-stimulating capacity. As we have previously shown, PMN themselves may liberate NAP-2 from the precursor connective tissue-activating peptide III (CTAP-III, 85 amino acids) by proteolysis. Instead of inducing cell activation, continuous accumulation of the chemokine in the surroundings of the processing cells results in the down-regulation of specific surface-expressed NAP-2 binding sites and in the desensitization of chemokine-induced PMN degranulation. Thus, NAP-2 precursors may be regarded as indirect mediators of functional desensitization in neutrophils. In the current study we investigated the biologic impact of another major NAP-2 precursor, the platelet basic protein (PBP, 94 amino acids). We show that PBP is considerably more potent than CTAP-III to desensitize degranulation and chemotaxis in neutrophils. We present data suggesting that the high desensitizing capacity of PBP is based on its enhanced proteolytic cleavage into NAP-2 by neutrophil-expressed cathepsin G and that it involves efficient down-regulation of surface-expressed CXCR-2 while CXCR-1 is hardly affected. Correspondingly, we found PBP and, less potently, CTAP-III to inhibit CXCR-2- but not CXCR-1- dependent chemotaxis of neutrophils toward NAP-2. Altogether our findings demonstrate that the anti-inflammatory capacity of NAP-2 is governed by the species of its precursors.

Killing of Fas ligand-resistant renal carcioma cells by interleukin-2- and BCG-activated effector cells.

Activated cytolytic effector cells like lymphokine-activated killer (LAK) and the recently described bacillus-Calmette-Guerin-activated killer (BAK) cells are thought to mediate antitumor effects against metastatic renal cell carcinoma (RCC) and superficial bladder cancer respectively. Perforin and Fas ligand (FasL) have been described as the major lytic principles in cellular cytotoxicity. Using a radioactive-release assay and specific inhibitors, we investigated the molecular mechanisms used by LAK and BAK cells in the lysis of renal carcinoma cells. In addition, we evaluated the susceptibility of RCC cells to FasL-mediated cytotoxicity. LAK and BAK cells effectively lysed the renal cancer cell line SK-RC-35 upon cell-cell contact. Both effector cell populations were shown to produce perforin and FasL as determined by reverse transcriptase/polymerase chain reaction (RT-PCR). Using fluorescence-activated cell sorting analyses and RT-PCR, we detected a marked Fas receptor (Fas, CD95) expression on RCC cells. However, RCC cells were shown to be resistant to killing by recombinant FasL and lysis by BAK and LAK cells was not inhibited in the presence of anti-FasL antibody. In contrast, the cytotoxicity exerted by LAK and BAK cells was drastically reduced in the presence of the Ca2+-chelating agent EGTA as well as concanamycin A, a specific inhibitor of perforin-mediated lysis. These results demonstrate that cytolysis of FasL-resistant RCC cells by activated immune cells is mediated via perforin. Our findings give further insights into the molecular mechanisms involved in the elimination of RCC by cytotoxic lymphocytes activated with biological response modifiers.

Perforin-mediated lysis of tumor cells by Mycobacterium bovis Bacillus Calmette-Guérin-activated killer cells.

Immunotherapy with Bacillus Calmette-Guérin (BCG) is clinically established in the treatment of superficial bladder cancer. In our attempt to clarify the underlying immunological mechanism, we could previously show that stimulation of PBMC with BCG leads to the generation of cytotoxic BCG-activated killer (BAK) cells. Among others, these BAK cells as well as lymphokine-activated killer (LAK) cells have been suggested as possible effector cells during BCG therapy. To understand BCG-induced activation of effector lymphocytes more precisely, we investigated the lytic pathways of human BAK cells and compared BAK cell cytotoxicity with LAK cell cytotoxicity. Perforin and Fas ligand (FasL) are the major cytolytic molecules of cytotoxic lymphocytes. Our results demonstrate that BAK and LAK cells showed an increased expression of perforin and FasL as compared with unstimulated controls. Killing of T-24 bladder tumor as well as Jurkat cells by BAK and LAK cells was predominantly mediated via perforin as demonstrated by a drastically reduced lysis in the presence of concanamycin A and EGTA/MgCl2, respectively. In contrast, lysis (radioactive release assay) and membrane disintegration (Annexin V binding) of both targets by BAK and LAK cells could not be blocked with an inhibitory anti-FasL monoclonal antibody (NOK-1). Nevertheless, T-24 and Jurkat were susceptible to killing by recombinant soluble FasL and by Chinese hamster ovary cells expressing membrane-bound FasL. We conclude that cellular mediators of BCG effector mechanisms, such as BAK and LAK cells, kill their targets via perforin and independent of the FasL pathway. Because we also found increased numbers of perforin-expressing lymphocytes in patients after BCG therapy, our findings have potential clinical relevance because BCG therapy would not be impaired by FasL resistance of target cells, which recently has been described for some tumors.

In vitro generation of bacillus Calmette-Guérin-activated killer cells.

Tumor regression induced in cancer patients by local instillation of bacillus Calmette-Guérin (BCG) into the bladder is considered to be mediated by cellular immune and inflammatory reactions. In an attempt to elucidate which of these effects are relevant to tumoricidal activity, an in vitro system was employed in which the immunostimulatory effects of BCG could be studied. This report describes the induction of BCG-activated killer (BAK) cells, which effectively lyse bladder tumor cells. Human peripheral blood mononuclear cells (PBMC) were stimulated with viable and sonicated BCG (v-BCG and s-BCG, respectively) to generate BAK cells. Cytotoxicity of BAK cells was comparable with the cytotoxicity exerted by lymphokine-activated killer (LAK) cells generated by interferon (IFN)-gamma but did not reach the level of interleukin-2 (IL-2)-generated LAK cells. Induction of BAK cells was possible only with v-BCG and not with s-BCG. By depletion and enrichment of defined cell populations, the cytotoxic potential of BAK cells could be attributed to a population of CD8(+) and CD56(+) double-positive lymphocytes. Macrophages and CD4(+) cells were required for the induction of killing activity but had no such activity by themselves. Furthermore, the presence of IFN-gamma and IL-2 in the supernatants harvested during the generation of BAK cells was demonstrated. Monoclonal antibodies neutralizing these cytokines abolished BCG-mediated cytotoxicity. From these results, it is concluded that the known beneficial effect of local instillation of BCG on maintenance of the relapse-free state in superficial bladder cancer may be due to local generation of BAK cells.

Reduction of side effects of intravesical therapy with bacille Calmette-Guérin by pentoxifylline?--an in vitro approach.

Immunotherapy with intravesical bacille Calmette-Guérin (BCG) is the treatment of choice against superficial bladder cancer recurrences. However, this therapy is associated with side effects that are considered to be the result of inflammatory cytokines. Since pentoxifylline is known to interfere with the production of cytokines, this drug was tested in vitro with regard to a later clinical application in BCG-treated patients. The cytokine release and the cytotoxicity of interleukin-2 or BCG-stimulated mononuclear cells were analyzed, and the growth of BCG under the influence of pentoxifylline was assayed. The results showed an inhibition of cytokine release of stimulated mononuclear cells. The cytotoxicity of BCG-stimulated mononuclear cells but not of lymphokine-stimulated mononuclear cells against bladder carcinoma cells was significantly inhibited. Restimulation with fresh BCG restored cytotoxicity. Direct coincubation of BCG and pentoxifylline resulted in a reduction of mycobacterial metabolism. From these data, we conclude that the use of pentoxifylline to reduce BCG-related side effects should be tested further in a clinical study.

Human MO subsets as defined by expression of CD64 and CD16 differ in phagocytic activity and generation of oxygen intermediates.

Phagocytosis and killing of microorganisms by reactive oxygen radicals are important defence mechanisms of the immune system and it was shown that human monocytes (MO) are heterogeneous in exerting these functions. Previously, we described that human peripheral blood MO consist of a major subset of Fc gamma-receptor-I (CD64)-positive cells exhibiting low accessory cell capacity but high phagocytic activity, and a minor subset of CD64-negative cells with dendritic cell (DC)-like high T cell accessory cell capacity but low phagocytic capacity. Recently, we could show that each subset itself further differs in the expression of the Fc gamma-receptor-III (CD16) and T cell accessory activities resulting in four different subsets: two CD16+ subsets (CD64+ or CD64-) with high T cell stimulation capacity and two CD16- subsets (CD64+ or CD64-) with low accessory activities. In the present study we demonstrate that these subsets also differ in their ability to phagocytose opsonized bacteria (S. aureus and E. coli) and in the generation of reactive oxygen species. Both CD64+ subsets (CD16+ or CD16-) exhibit high phagocytic activity accompanied by intracellular superoxide induction. Luminol-dependent (mainly myeloperoxidase (MPO)-mediated) chemiluminescence (CL) response to latex and FMLP (formylmethionylleucylphenylalanine) was also high in these cell populations. Phagocytic activity and modest CL response was shown in CD64-/CD16+ but not in CD64-/CD16- cells, indicating that each subset except for CD64-/CD16- cells may engulf bacteria and exhibit MPO activity. Taken together, these data demonstrate further heterogeneity of peripheral blood MO in both, phagocytic activity and generation of reactive oxygen species indicating differences between the four subsets in this kind of defence mechanisms against pathogens.

Identification of distinct surface-expressed and intracellular CXC-chemokine receptor 2 glycoforms in neutrophils: N-glycosylation is essential for maintenance of receptor surface expression.

The G protein-coupled CXC-chemokine receptor CXCR-2 mediates activation of neutrophil effector functions in response to multiple ligands, including IL-8 and neutrophil-activating peptide 2 (NAP-2). Although CXCR-2 has been successfully cloned and expressed in several cell lines, the molecular properties of the native neutrophil-expressed receptor have remained largely undefined. Here we report on the identification and characterization of distinct CXCR-2 glycoforms and their subcellular distribution in neutrophils. Immunoprecipitation and Western blot analyses of surface-expressed receptors covalently linked to IL-8 or NAP-2 as well as in their unloaded state revealed the occurrence of a single CXCR-2 variant with an apparent size of 56 kDa. According to deglycosylation experiments surface-expressed CXCR-2 carries two N-linked 9-kDa carbohydrate moieties that are both of complex structure. In addition, two other CXCR-2 variants of 38 and 40 kDa were found to occur exclusively intracellular and to carry N-glycosylations of high mannose or hybrid type. These receptors did not participate in ligand-induced receptor trafficking, while surface-expressed CXCR-2 was internalized and re-expressed following stimulation with NAP-2. By enzymatic removal of one 9-kDa carbohydrate moiety in surface-expressed CXCR-2 we can show that neither NAP-2-induced trafficking nor signaling of the receptor is dependent on its full glycosylation. Instead, glycosylation was found to protect CXCR-2 from proteolytic attack, as even partial deglycosylation is associated with serine protease-mediated disappearance of the receptor from the neutrophil surface. Thus, although not directly involved in signaling, glycosylation appears to be required to maintain neutrophil responsiveness to CXC-chemokines during inflammation.

The beta-thromboglobulins and platelet factor 4: blood platelet-derived CXC chemokines with divergent roles in early neutrophil regulation.

The recruitment of neutrophil granulocytes to sites of tissue injury is one of the earliest events during host defense. Several chemotactic cytokines belonging to the CXC subfamily of chemokines are thought to be implicated in this kind of response. Especially those CXC chemokines that are stored in blood platelets and become immediately released upon activation are likely to dominate neutrophil-dependent host defense at the onset of inflammation. The major platelet-derived CXC chemokines are the beta-thromboglobulins and platelet factor 4 (PF-4), which are both released into the blood at micromolar concentrations. The availability as well as the functional activity of these mediators appear to be subject to tight control by diverse regulatory mechanisms. These include proteolytic processing of chemokine precursors, oligomer formation, and the differential usage of neutrophil-expressed receptors. Herein we review our work on early neutrophil regulation by PF-4, the beta-thromboglobulin neutrophil-activating peptide 2 (NAP-2) and its major precursor connective tissue-activating peptide III (CTAP-III). We moreover propose a model to assess the contribution by either of these chemokines to coordinated recruitment and activation of neutrophils in response to acute tissue injury.

The CXC-chemokine platelet factor 4 promotes monocyte survival and induces monocyte differentiation into macrophages.

Unstimulated monocytes rapidly undergo physiological changes resulting in programmed cell death (apoptosis) while stimuli promoting differentiation of these cells into macrophages were shown to inhibit apoptotic processes. In the present study, we report that the platelet-derived alpha-chemokine platelet factor 4 (PF4) induces the differentiation of monocytes into macrophages, as is evident from morphological changes as well as from the up-regulation of differentiation markers (carboxypeptidase M/MAX1 and CD71). Significant alterations of the phenotype were observed after 72 hours of culture in the presence of the chemokine and required a minimal concentration of 625 nmol/L PF4. PF4-induced macrophages were characterized by a lack of HLA-DR antigen on their surface but showed a strong increase in the expression of the CD28 ligand B7-2. Furthermore, PF4 stimulation prevented monocytes from undergoing spontaneous apoptosis during 72 hours of culture as determined in an annexin-V-binding assay. Although PF4 induced the secretion of relevant amounts of TNF-alpha, neutralizing antibodies directed against TNF-alpha or granulocyte-macrophage colony-stimulating factor (GM-CSF) did not revert PF4-induced rescue from programmed cell death, suggesting that PF4 exerts its antiapoptotic effects in a TNF-alpha- or GM-CSF-independent fashion. On the basis of these results, we propose a novel role for PF4 in the control of monocyte differentiation during an inflammatory process in vivo. (Blood. 2000;95:1158-1166)

Low in vitro production of interferon-gamma and tumor necrosis factor-alpha in HIV-seronegative patients with pulmonary disease caused by nontuberculous mycobacteria.

We studied 32 HIV-seronegative patients with pulmonary disease caused by nontuberculous mycobacteria (NTM). Immunologic studies included lymphocyte subset analysis by flow cytometry, measurement of interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha) production following in vitro stimulation of diluted whole blood (DWB) and peripheral blood mononuclear cells (PBMC) by phytohemagglutinin (PHA), anti-CD3 as well as purified protein derivative of tuberculin (PPD), and in four cases with different amounts of the very mycobacterium, which caused disease in these patients. Data were compared to those of 30 HIV-seronegative patients with disease by Mycobacterium tuberculosis (MTb). Following alpha-CD3-stimulation of PBMC, NTM patients showed lower IFN-gamma (P < 0.00005) and lower TNF-alpha (P < 0.02). For a subgroup of tuberculin skin test-positive NTM patients we found significantly lower PPD-induced IFN-gamma releases in cultured DWB (P < 0.0002) and PBMC (P < 0.0004) compared to MTb patients. Data for PPD-induced TNF-alpha release for this subgroup were also significant (P < 0.001 and P < 0.05, respectively). The four NTM patients with poor PPD-induced IFN-gamma response hardly showed increased cytokine production on stimulation with their specific mycobacterium. The lower production capacity of IFN-gamma and TNF-alpha of NTM patients compared to the MTb patients points to an immunologic imbalance forming the basis for their increased susceptibility to pulmonary infections by nontuberculous mycobacteria.

Platelet-derived CXC chemokines: old players in new games.

Although platelet factor 4 (PF-4) and the beta-thromboglobulin (beta-TG) proteins represent the first chemokines to be discovered, their functional roles in host defense became clear only recently. Residing in platelets as storage proteins and becoming released into the blood at very high concentrations, these mediators appear to fulfill different and complementary tasks as first-line mediators in the recruitment and activation of leukocytes, as well in the regulation of tissue repair. Whereas both proteins are structurally closely related members of the CXC chemokine subfamily, they are subject to quite dissimilar regulatory mechanisms controlling their generation and their spectrum of biological activities. Thus, proteolytic processing of inactive precursors plays a decisive role in whether the beta-TG proteins will act as stimulatory or inhibitory agents in neutrophil activation via the G protein-coupled receptors CXCR-1 and 2. PF-4, existing as a single molecular form, is largely resistant to proteolytic modification, but its interaction with an unusual receptor(s) on leukocytes (a proteoglycan) appears to depend on its oligomeric state. There is growing evidence that both chemokines may interfere with each other at various regulatory levels to promote coordinated cell activation. Moreover, recent findings suggest novel and unexpected activities for these chemokines, which may extend our view on early host defense.

Binding of lipopolysaccharide (LPS) to CHO cells does not correlate with LPS-induced NF-kappaB activation.

Activation of myeloid cells by lipopolysaccharide (LPS) is a key event in the development of gram-negative sepsis. One crucial step within this process is the binding of LPS to CD14. CD14 is a glycosylphosphatidylinositol (GPI)-anchored membrane protein requiring at least one additional membrane-spanning molecule for signal transduction. It is not clear whether the function of CD14 is to merely catalyze LPS binding, followed by the interaction of LPS with the signal transducer, or whether CD14 has a more specific function and may be a part of the signaling complex. To address this question we generated Chinese hamster ovary (CHO) cells expressing a human GPI-anchored form of LPS-binding protein (mLBP) to substitute for CD14 as LPS acceptor molecule. By comparison of CHO / mLBP with CHO / vector and CHO / CD14 cells we found that expression of GPI-linked LBP results in an enhanced binding of LPS but not in an increase in cell activation as determined by translocation of NF-kappaB. Furthermore, excess of recombinant soluble LBP resulted also in increased LPS binding without affecting NF-kappaB translocation. These data show that LPS binding alone is not sufficient to induce signaling. We conclude that CD14 is more than a catalyst for LPS binding: it seems to be directly involved in LPS signaling and thus appears to be an essential part of the signaling complex.