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.     

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.     

Mutation of amino acids 39-44 of human CD14 abrogates binding of lipopolysaccharide and Escherichia coli

As a key receptor for lipopolysaccharide (LPS) on the surface of monocytes and macrophages, the CD14 molecule is primarily involved in non-specific host defense mechanisms against gram-negative bacteria. To delineate the structural basis of LPS binding, 23 mutants in the N-terminal 152 amino acids of human CD14 were generated and stably transfected into CHO cells. In each mutant, a block of five amino acids was substituted by alanine. Reactivity of the mutants with anti-CD14 mAbs, and their ability to interact with LPS and Escherichia coli were tested. 4 of 21 expressed CD14 mutants, ([Ala9-Ala13]CD14, [Ala39-Ala41, Ala43, Ala44]CD14, [Ala51-Ala55]CD14 and [Ala57, Ala59, Ala61-Ala63]CD14), are not recognized by anti-CD14 mAbs that interfere with the binding of LPS to human monocytes. However, only [Ala39-Ala41, Ala43, Ala44]CD14 is unable to react with fluorescein-isothiocyanate-labeled LPS or with FITC-labeled E. coli (055:B5). In addition, [Ala39-Ala4l, Ala43, Ala44]CD14 does not mediate LPS (E. coli 055:B5; 10 ng/ml)-induced translocation of nuclear factor kappaB in CHO-cell transfectants. The results indicate that the region between amino acids 39 and 44 forms an essential part of the LPS-binding site of human CD14.     

V2 vasopressin receptor dysfunction in nephrogenic diabetes insipidus caused by different molecular mechanisms

B cells and macrophages both activate NF-kappaB/Rel in response to lipopolysaccharide (LPS), but differ in sensitivity to LPS and in downstream genes that are activated. CD14 is a high-affinity receptor for LPS found on macrophages, but not B cells. We expressed human CD14 (hCD14) in the mouse B lymphoma, 70Z/3, and a mutant, 1B8, which responds slowly to LPS, to test whether expression of hCD14 could correct or bypass the defect in 1B8 cells. We compared the timing and extent of known responses to LPS in 70Z/3 cells and the 1B8 mutants. The hCD14+ 1B8 and 70Z/3 cells responded more rapidly and were sensitive to 100-fold lower levels of LPS than their untransfected counterparts. Degradation of the IkappaB-alpha and -beta molecules and translocation of the NF-kappaB/Rel complexes into the nucleus were more rapid and the steady-state levels of Igk mRNA and mIgM on the cell surface were markedly increased in cells that expressed hCD14. The LPS response of the hCD14+ 1B8 and 70Z/3 cells showed subtle differences. In the 1B8 hCD14 cells, the p50/p50 complexes were never abundant in nuclear extracts, and degradation of IkappaB-beta was slower than in hCD14 70Z/3 cells. This partial correction of the 1B8 phenotype suggests that the defective component in 1B8 participates in the CD14 signaling pathway and could include the B-cell LPS receptor itself.     

In vivo and in vitro evidence for the involvement of tumor necrosis factor-alpha in the induction of leptin by lipopolysaccharide

To examine the role of tumor necrosis factor-alpha (TNF alpha) in mediating leptin secretion during an immunological challenge, we studied the effects of lipopolysaccharide (LPS) and TNF alpha on leptin secretion in endotoxin-sensitive C3H/HeOuJ (OuJ) mice, endotoxin-insensitive C3H/HeJ (HeJ) mice, and primary adipocytes cultured from both. Intraperitoneal injection of LPS increased plasma concentrations of TNF alpha and leptin in OuJ mice, but not in HeJ mice, suggesting a causal relationship between the induction of TNF alpha and leptin. Consistent with this idea, i.p. injection of recombinant murine TNF alpha increased plasma leptin in both OuJ and HeJ mice. To determine whether TNF alpha induces leptin secretion by acting directly on fat cells, primary adipocytes from OuJ and HeJ mice were cultured in the presence of TNF alpha or LPS. Whereas LPS was without effect on leptin secretion by adipocytes, TNF alpha induced a marked increase in the cell supernatant leptin concentration. These data demonstrate that TNF alpha plays a role in regulating the increase in leptin caused by LPS. Moreover, they show that TNF alpha can act directly on adipocytes to stimulate leptin secretion. Our results are consistent with the emerging view that leptin is a key hormone coupling immune system activity to energy balance.     

Prostaglandin E2 primes naive T cells for the production of anti-inflammatory cytokines

In addition to their capacity to induce pain, vasodilatation and fever, prostaglandins E (PGE) exert anti-inflammatory activities by inhibiting the release of pro-inflammatory cytokines by macrophages and T cells, and by increasing interleukin (IL)-10 production by macrophages. We here report that PGE2, the major arachidonic acid metabolite released by antigen-presenting cells (APC), primes naive human T cells for enhanced production of anti-inflammatory cytokines and inhibition of pro-inflammatory cytokines. Unfractionated as well as CD45RO- CD31+ sort-purified neonatal CD4 T cells acquire the capacity to produce a large spectrum of cytokines after priming with anti-CD3 and anti-CD28 monoclonal antibodies (mAb), in the absence of both APC and exogenous cytokines. PGE2 primes naive T cells in a dose-dependent fashion for production of high levels of IL-4, IL-10 and IL-13, and very low levels of IL-2, interferon (IFN)-gamma, tumor necrosis factor (TNF)-alpha, and TNF-beta. PGE2 does not significantly increase IL-4 production in priming cultures, whereas it suppresses IL-2 and IFN-gamma. Addition of a neutralizing mAb to IL-4 receptor in primary cultures, supplemented or not with PGE2, prevents the development of IL-4-producing cells but does not abolish the effects of PGE2 on IL-10 and IL-13 as well as T helper (Th)1-associated cytokines. Addition of exogenous IL-2 in primary cultures does not alter the effects of PGE2 on naive T cell maturation. Thus PGE2 does not act by increasing IL-4 production in priming cultures, and its effects are partly IL-4 independent and largely IL-2 independent. Together with the recent demonstration that PGE2 suppresses IL-12 production, our results strongly suggest that this endogenously produced molecule may play a significant role in Th subset development and that its stable analogs may be considered for the treatment of Th1-mediated inflammatory diseases.     

Role of CR4 in Mycobacterium tuberculosis-human macrophages binding and signal transduction in the absence of serum

The beta2 integrin CR4 is involved in Mycobacterium tuberculosis phagocytosis by human mononuclear phagocytes through the opsonin C3bi. In this study, we demonstrate that M. tuberculosis can bind directly to monocyte-derived macrophages via CR4 in the absence of any opsonins. CR4-transfected CHO cells gave similar results, suggesting recognition by CR4 of bacterial structure. Furthermore, binding of M. tuberculosis transduced a potent signal, resulting in tyrosine phosphorylation of macrophage proteins, which was in part mediated by CR4.     

Soluble tumour necrosis factor receptor release after anti-CD3 monoclonal antibody treatment in mice is independent of tumour necrosis factor-alpha release

The involvement of TNF-alpha in the release of soluble TNF receptors was assessed in mice, treated with anti-CD3 monoclonal antibodies. After treatment with three different anti-CD3 mAb, we simultaneously studied serum levels of TNF-alpha, soluble TNF receptor P55 and P75. All three anti-CD3 mAb triggered the release of both of the soluble TNF receptors, whereas only one of the anti-CD3 mAb triggered TNF-alpha release. These data demonstrate that in our model soluble TNF receptor release is independent of TNF-alpha release.     

Effect of B-cell receptor engagement on CD40-stimulated B cells

Activation of human B cells in vitro either by cross-linking of surface immunoglobulins (sIg) or by triggering CD40 antigen, in the presence of interleukin-10 (IL-10) and interleukin-2 (IL-2), may result in high levels of immunoglobulin secretion in vitro. We studied the combined effects of ligation of the B-cell receptor (BCR) and CD40 [with anti-CD40 monoclonal antibody (mAb)] on B-cell proliferation and production of human immunoglobulin. For this purpose highly purified splenic B cells were cultured with various combinations of anti-CD40 and IL-10/IL-2 or IL-4 in the presence of CD32-transfected L cells. Simultaneous cross-linking of the BCR was achieved by mAb held on CD32-L cells or Staphylococcus aureus (SA). We found that dual BCR and CD40 ligation with IL-10/IL-2 leads to reduced immunoglobulin G (IgG) secretion compared with B cells stimulated with either anti-CD40 and IL-10/IL-2, or compared with B cells stimulated with SA or anti-BCR mAb and IL-10/IL-2. Dual BCR and CD40 ligation with anti-immunoglobulin mAb (anti-kappa + anti-lambda light chains) but not with SA induced a similar reduction in IgM production. The reduced immunoglobulin secretion found during dual ligation is accompanied by increased proliferation. This was independent of cytokine stimulation but SA/CD40-induced proliferation was increased in the presence of IL-10/IL-2, although not with IL-4. The combination anti-kappa and anti-lambda with anti-CD40 showed a long-term suppression of IgG and IgM production (at least 14 days), while anti-kappa or anti-lambda alone, or SA, allowed a moderate recovery of immunoglobulin production by day 14. These results suggest that simultaneous B-cell antigen receptor cross-linking and CD40 engagement via CD40L on T cells induces strong initial proliferation. This may be followed later by antibody production depending on the strength of the BCR signal and the presence of the appropriate cytokines.     

Reduction of fecal contamination of street-vended beverages in Guatemala by a simple system for water purification and storage, handwashing, and beverage storage

CD4+ cells from young (3 months) and old (19 months) mice were stimulated by plate-bound anti-CD3 monoclonal antibody (mAb) alone or also by soluble anti-CD28 mAb. Supernatants were analysed by enzyme-linked immunosorbent assay (ELISA) to determine cytokine concentrations. Total RNA was extracted from cells, reverse transcribed and the cDNA amplified by polymerase chain reaction (PCR) to evaluate the amount of specific mRNA. The results indicate that anti-CD3 alone is not sufficient to induce interleukin-2 (IL-2) production in CD4+ cells from both young and old mice. However, anti-CD28, together with anti-CD3 mAb, induces a much higher production of IL-2 in CD4+ cells from young as compared with old mice. Conversely, interferon-gamma (IFN-gamma) production is also induced by anti-CD3 alone and is higher in CD4+ cells from old as compared with young mice. Upon addition of anti-CD28 mAb, IFN-gamma production increases in both groups, but it remains much higher in old than in young mice. Also the production of IL-4 and IL-10 is induced by anti-CD3 mAb but it is increased by the addition of anti-CD28 mAb. CD4+ cells from old mice produce more IL-4 and IL-10 as compared with cells from young mice. The amounts of cytokine specific mRNA in CD4+ cells from young and old mice parallel the cytokine levels in culture supernatants. Results on the mRNA turnover indicate that when CD4+ cells are stimulated by anti-CD3 or costimulated also by anti-CD28 mAb, the IFN-gamma, IL-4 and IL-10 specific mRNAs are more stable in old than in young mice, suggesting that mRNA stability has a relevant role in the different patterns of cytokine production.     

Human CD14 mediates recognition and phagocytosis of apoptotic cells

Cells undergoing programmed cell death (apoptosis) are cleared rapidly in vivo by phagocytes without inducing inflammation. Here we show that the glycosylphosphatidylinositol-linked plasma-membrane glycoprotein CD14 on the surface of human macrophages is important for the recognition and clearance of apoptotic cells. CD14 can also act as a receptor that binds bacterial lipopolysaccharide (LPS), triggering inflammatory responses. Overstimulation of CD14 by LPS can cause the often fatal toxic-shock syndrome. Here we show that apoptotic cells interact with CD14, triggering phagocytosis of the apoptotic cells. This interaction depends on a region of CD14 that is identical to, or at least closely associated with, a region known to bind LPS. However, apoptotic cells, unlike LPS, do not provoke the release of pro-inflammatory cytokines from macrophages. These results indicate that clearance of apoptotic cells is mediated by a receptor whose interactions with 'non-self' components (LPS) and 'self' components (apoptotic cells) produce distinct macrophage responses.     

The induction of acute phase proteins by lipopolysaccharide uses a novel pathway that is CD14-independent

LPS (endotoxin) and proinflammatory cytokines (IL-6, IL-1, and TNF-alpha) are potent inducers of acute phase proteins (APP). Since LPS induces high levels of these cytokines after its interaction with CD14, a protein expressed on the surface of monocytes and neutrophils, it has been assumed that CD14 mediates the LPS induction of APP expression. To test this hypothesis, CD14-deficient and control mice were injected with low doses of LPS, and the expression of several APP that are normally up-regulated by LPS was measured. CD14-deficient mice showed no alteration in the induction of APP, including serum amyloid A, LPS-binding protein, fibrinogen, or ceruloplasmin; in contrast, C3H/HeJ mice, which carry a mutation in the Lps gene, do not up-regulate the expression of these proteins. These studies show that the up-regulation of APP by LPS utilizes a non-CD14 receptor and requires a functional Lps gene.     

Apoptosis of CD4+ T cells induced after contact with HIV1-infected or non-infected macrophages

The hallmark of human immunodeficiency virus type 1 (HIV1) infection is the relentless destruction of CD4+ T lymphocytes. Indirect cell killing mechanisms are thought to play an outstanding role in lymphocyte depletion. One such proposed mechanism is the induction of apoptosis through cross-linking of the CD4 receptor by anti-CD4 antibodies or by the HIV1 envelope protein expressed at the surface of infected cells. Here we provide evidence that apoptosis is triggered in CD4+ lymphoblastoid cells (MT4) following cocultivation with monocyte-derived macrophages (MDMs) productively infected with the monocytotropic isolate HIV1 PAR. Blocking virus replication by AZT abrogates apoptosis of MT4 cells. Infected MDMs do not transmit virus infection to target cells. DNA nucleosomal fragmentation occurs at 46-66 h after starting cocultures. It is inhibited by the addition of neutralizing anti-gp120 monoclonal antibody (mAb), implying that the gp120/CD4 interaction triggers the apoptotic process. Cocultivating with MDMs, either infected or not, in the presence of anti-CD4 mAb Leu-3a, also leads to MT4 cell death, with DNA fragmentation being detected at 24-40 h. Leu-3a induced apoptosis does not require cross-linking of CD4 by anti-immunoglobulin, showing that MDMs provide an alternative to conventional cross-linking. Both the infected and the non-infected MDMs were found to stimulate 3H-thymidine incorporation in cocultivated MT4 cells.     

Role of stress-activated mitogen-activated protein kinase (p38) in beta 2-integrin-dependent neutrophil adhesion and the adhesion-dependent oxidative burst

Bacterial LPS elicits both rapid activation of the stress-activated MAP kinase p38 in polymorphonuclear leukocytes (PMN) and rapid adhesion of the PMN to ligands for the leukocyte integrin CD11b/CD18. The functional correlation between these two events was examined. The time course for tyrosine phosphorylation of p38 in PMN in response to 10 ng/ml LPS in 1% normal human serum was consistent with participation in signaling for leukocyte integrin-dependent adhesion, with transient phosphorylation peaking at 10 to 20 min. The concentration dependence of p38 phosphorylation also resembled that for PMN adhesion, with <1 ng/ml LPS eliciting a response. Phosphorylation was inhibited by mAb 60b against CD14, but not by mAb 26ic, a nonblocking anti-CD14. The function of p38 in integrin-dependent adhesion and the adhesion-dependent oxidative burst was tested using a specific inhibitor of p38, SB203580. SB203580 inhibited adhesion by diminishing the initial rate of adherence in response to both LPS and TNF, with a half-maximal concentration in the range of 0.1 to 0.6 microM. It did not, however, block adhesion in response to formyl peptide or PMA. The p38 inhibitor also blocked the adhesion-dependent oxidative burst with a half-maximal concentration similar to that for adhesion. Timed delivery of the compound during the lag phase preceding H2O2 production suggested that p38 kinase activity was required throughout the lag but not after the oxidase was assembled. These results suggest that p38 functions in PMN to signal leukocyte integrin-dependent adhesion and the subsequent massive production of reactive oxygen intermediates.     

Anti-CD45 and anti-CD52 (Campath) monoclonal antibodies effectively eliminate systematically disseminated human non-Hodgkin's lymphoma B cells in Scid mice

Severe combined immunodeficient (Scid) mice inoculated with the human (t(14;18)-positive B cell lines DoHH2 and BEVA develop lethal systemically disseminated lymphoma (de Kroon et al., Leukemia 8:1385, and Blood 80 [suppl 1]:436). These models were used to study the therapeutic effect of rat-anti-human CD52 (Campath-1G) or CD45 monoclonal antibodies (mAbs) on systemically disseminated tumor cells and on tumor cells present in solid tumor masses. Both mAbs were effective in inhibiting growth of systemically disseminated malignant cells. When treatment with anti-CD52 or anti-CD45 mAbs at a dose of 30 micrograms/mouse/d for 4 days was started 24 hours after intravenous inoculation of human DoHH2 or BEVA cells, a 3-log kill of tumor cells was observed as measured by prolonged survival. After treatment, surviving animals injected with high numbers of BEVA cells showed tumor masses in liver, kidney, and mesenteric lymph nodes. In contrast to nontreated animals, however, only low numbers of malignant cells were found in peripheral blood, and bone marrow was free of tumor cells. Similarly, after mAb treatment of mice inoculated subcutaneously (sc) with DoHH2 cells, no tumor cells could be found in the bone marrow, and few DoHH2 cells could be detected in the peripheral blood, spleen, liver, kidney, or lung. In contrast, tumor cells present in subcutaneous tumors and axillary lymph nodes were relatively unaffected by mAb therapy. The presence of rat immunoglobulin (Ig) could be demonstrated on surviving tumor cells. The presence of murine macrophages in areas in these tumors that were depleted of DoHH2 cells suggested that the mAb-mediated antitumor effect observed in the Scid mouse model is mediated by cellular mechanisms. Apparently these mechanisms were not sufficient to eliminate the fast-growing tumor cells present in the protected sites. Our results indicate that treatment with anti-CD52 or anti-CD45 mAbs potentially may be useful as adjuvant immunotherapy for systemically disseminated B cell lymphoma.