HLA Class II Antibody Activation of Endothelial Cells Promotes Th17 and Disrupts Regulatory T Lymphocyte Expansion.

Kidney transplantation is the most successful treatment option for patients with end-stage renal disease, and chronic antibody-mediated rejection is the principal cause of allograft loss. Predictive factors for chronic rejection include high levels of HLA alloantibodies (particularly HLA class II) and activation of graft endothelial cells (ECs). The mechanistic basis for this association is unresolved. We used an experimental model of HLA-DR antibody stimulation of microvascular ECs to examine the mechanisms underlying the association between HLA class II antibodies, EC activation and allograft damage. Activation of ECs with the F(Ab')2 fragment of HLA-DR antibody led to phosphorylation of Akt, ERK and MEK and increased IL-6 production by ECs cocultured with allogeneic peripheral blood mononuclear cells (PBMCs) in an Akt-dependent manner. We previously showed that HLA-DR-expressing ECs induce polarization of Th17 and FoxP3(bright) regulatory T cell (Treg) subsets. Preactivation of ECs with anti-HLA-DR antibody redirected EC allogenicity toward a proinflammatory response by decreasing amplification of functional Treg and by further increasing IL-6-dependent Th17 expansion. Alloimmunized patient serum containing relevant HLA-DR alloantibodies selectively bound and increased EC secretion of IL-6 in cocultures with PBMCs. These data contribute to understanding of potential mechanisms of antibody-mediated endothelial damage independent of complement activation and FcR-expressing effector cells.

Alteration of CD1 expression in multiple sclerosis.

Studies of multiple sclerosis (MS) have concentrated mainly on antigen presentation of peptides derived from the myelin sheath, while the implication of lipid antigen has been less explored in this pathology. As the extracellular environment regulates expression of the lipid antigen-presenting molecule CD1, we have examined whether sera from patients alters CD1 surface expression in monocyte-derived dendritic cells. We have shown that: (i) CD1 group 1 proteins were highly expressed in the presence of MS sera; (ii) sera from MS patients differentially regulated CD1 group 1 versus CD1 group 2 molecular expression; and (iii) CD1 was expressed strongly in monocytes from MS patients under immunosuppressive treatment. Overall, these results reveal that CD1 expression is modified in MS and provide novel information on the regulation of lipid antigen presentation in myeloid cells.

Identification of Novel Human Monocyte Subsets and Evidence for Phenotypic Groups Defined by Interindividual Variations of Expression of Adhesion Molecules.

Monocytes contribute to immune responses as a source for subsets of dendritic cells and macrophages. Human blood monocytes are classified as classical, non-classical and intermediate cells. However, the particular functions of these subsets have been hard to define, with conflicting results and significant overlaps. One likely reason for these ambiguities is in the heterogeneity of these monocyte subsets regrouping cells with divergent functions. To better define monocyte populations, we have analysed expression of 17 markers by multicolour flow cytometry in samples obtained from 28 control donors. Data acquisition was tailored to detect populations present at low frequencies. Our results reveal the existence of novel monocyte subsets detected as larger CD14+ cells that were CD16+ or CD16neg. These large monocytes differed from regular, smaller monocytes with respect to expression of various cell surface molecules, such as FcR, chemokine receptors, and adhesion molecules. Unsupervised multidimensional analysis confirmed the existence of large monocytes and revealed interindividual variations that were grouped according to unique patterns of expression of adhesion molecules CD62L, CD49d, and CD43. Distinct inflammatory responses to TLR agonists were found in small and large monocytes. Overall, refining the definition of monocyte subsets should lead to the identification of populations with specific functions.

Search for Dislocation Free Helium 4 Crystals.

The giant plasticity of [Formula: see text]He crystals has been explained as a consequence of the large mobility of their dislocations. Thus, the mechanical properties of dislocation free crystals should be quite different from those of usual ones. In 1996-1998, Ruutu et al. published crystal growth studies showing that, in their helium 4 crystals, the density of screw dislocations along the c-axis was less than 100 per cm[Formula: see text], sometimes zero. We have grown helium 4 crystals using similar growth speeds and temperatures, and extracted their dislocation density from their mechanical properties. We found dislocation densities that are in the range of 10[Formula: see text]-10[Formula: see text] per cm[Formula: see text], that is several orders of magnitude larger than Ruutu et al. Our tentative interpretation of this apparent contradiction is that the two types of measurements are somewhat indirect and concern different types of dislocations. As for the dislocation nucleation mechanism, it remains to be understood.

Despite lack of monospecific anti-DRw13 sera, a corresponding class II determinant can be defined by a DRw13 restricted anti-influenza virus T cell clone.

The study of a T3+ T4+ T8- human T cell clone COTC2 with both specific proliferative response and cytolytic activity for influenza A virus infected cells reveals that: the restricting element of this clone is strongly associated with DRw13 molecule(s) as seen by the study of a large panel of antigen presenting cells (APC) and by the observation that monoclonal antibodies (MoAb) specific for DR molecules inhibit its proliferative activity while anti-DQ MoAb do not. These results indicate that there exists a DRw13 associated determinant that can be defined at the functional level by COTC2 recognition despite the absence of monospecific anti-DRw13 serum. In contrast to the results found by other groups, the restriction of this DRw13 restricted clone follows the DRw13 serological definition irrespective of the DW type of the APC. These results indicate that the polymorphism of HLA class II molecules can be further defined at the functional level by monoclonal populations of T cells in conjunction with molecular definition.

Accelerated in vitro differentiation of blood monocytes into dendritic cells in human sepsis.

Summary Sepsis-induced immune depression is characterized by infection susceptibility and monocyte early deactivation. Because monocytes are precursors for dendritic cells (DC), alterations in their differentiation into DC may contribute to defective immune responses in septic patients. We therefore investigated the ability of monocytes to differentiate into functional DC in vitro in patients undergoing surgery for peritonitis. Monocytes from 20 patients collected immediately after surgery (D0), at week 1 and at weeks 3-4 and from 11 control donors were differentiated into immature DC. We determined the phenotype of monocytes and derived DC, and analysed the ability of DC to respond to microbial products and to elicit T cell responses in a mixed leucocyte reaction (MLR). We show that, although monocytes from septic patients were deactivated with decreased responses to lipopolysaccharide (LPS) and peptidoglycan and low human leucocyte antigen D-related (HLA-DR) expression, they expressed the co-stimulatory molecule CD80, CD40 and CCR7. Monocytes collected from patients at D0 and week 1 differentiated faster into DC with early loss of CD14 expression. Expression of HLA-DR increased dramatically in culture to reach control levels, as did responses of DC to LPS and peptidoglycan. However, although patient and control immature DC had similar abilities to induce T cell proliferation in MLR, maturation of DC derived from patients did not increase T cell responses. These results show that circulating monocytes from septic patients express markers of activation and/or differentiation despite functional deactivation, and differentiate rapidly into phenotypically normal DC. These DC fail, however, to increase their T cell activation abilities upon maturation.

Neutrophil CD14: biochemical properties and role in the secretion of tumor necrosis factor-alpha in response to lipopolysaccharide.

CD14 is a myeloid cell differentiation Ag expressed primarily by monocytes and macrophages. CD14 has recently been shown to function as a receptor for a complex of LPS and LPS binding protein (LBP), an acute phase serum protein also present in normal serum in trace amounts. In the presence of LBP, LPS strongly activates monocytes via CD14 as measured by TNF secretion. This pathway of monocyte activation is thought to be a major contributor to the symptoms of endotoxin shock. Another major cell type involved in the response to Gram-negative infection is the neutrophil. Recent studies have shown that neutrophils also express CD14 and suggest that they can respond to LPS through a similar pathway. However, the biochemical nature of neutrophil CD14 has not previously been described. In this report, we have analyzed several biochemical characteristics of neutrophil CD14. We show that CD14 is actively synthesized by neutrophils as a glycosylphosphatidyl-inositol-anchored protein, indistinguishable in size from monocyte CD14. Furthermore, neutrophils, like monocytes, shed a smaller soluble form of CD14 into culture supernatants. In addition, like monocytes, neutrophils respond to LPS/LBP complexes via CD14 by releasing TNF-alpha. The described properties and function of neutrophil CD14 suggest that it may directly participate in the acute inflammatory response and in endotoxin shock.

Phosphorylation of class I HLA antigens in U-937 monocyte-like cells. Role of protein kinase C.

Phorbol esters and 1-oleoyl-2-acetylglycerol induce an increase in phosphorylation of a series of 44-kD proteins in the U-937 human monocyte-like cell line. The phenomenon is rapid at 37 degrees C, starting 0.5 min after adding the inducer, increasing to a maximum of 5-10 min and decreasing to the basal level at 30 min. It is also observed at 4 degrees C, though much more slowly. Immunoprecipitation and two-dimensional electrophoresis demonstrated that these phosphorylated proteins belong to the class I HLA antigens.

Molecular localization to a unique DR beta-chain of the restriction element of an anti-influenza T cell clone.

The DR beta-chains of a panel of DRw13 cells were characterized by two-dimensional polyacrylamide gel electrophoresis in order to identify the molecule bearing the restriction element of a DR-restricted proliferative and cytotoxic T cell clone COT C2 specific for DRw13 and the influenza A virus. Because in different DRw13 haplotypes one DRw13 beta-chain can be associated with other distinct DRw13 beta-chains, such complex associations allowed us to determine the ability of a given DR beta-chain to present the antigen or COT C2. The presence of the DR beta-chain 6B5, and only this chain, is associated with the ability to induce the proliferation of clone COT C2 whatever the second DR beta-chain associated with 6B5 is, namely 6B4 or 6B3. The DRw13 cells that express 6B4 or 6B3 but not 6B5 could not present the antigen to COT C2. Moreover, ILR2, a monoclonal antibody that precipitated 6B5, blocked the proliferation of COT C2, whereas 7.3.19.1, a monoclonal antibody that precipitated only 6B4 and 6B3, could not block the proliferation of this clone. Thus, the DRw13 beta-chain 6B5 appears as the unique class II element that restricts the response of the T cell clone COT C2.

The monocyte differentiation antigen, CD14, is anchored to the cell membrane by a phosphatidylinositol linkage.

CD14 is a myeloid differentiation Ag expressed primarily on peripheral blood monocytes and macrophages. Although its function is unknown, the CD14 gene maps to a region encoding several myeloid growth factors and receptors. Analysis of the CD14 protein sequence deduced from the cDNA shows that although the CD14 protein contains a characteristic leader peptide, it lacks a characteristic transmembrane region, suggesting that CD14 may be anchored to the membrane via glycosylphosphatidylinositol (PI). Treatment of monocytes as well as a CD14-expressing neuroglioma cell line with PI-phospholipase C removed CD14 from the cell surface. Furthermore, monocytes from a patient with paroxysmal nocturnal hemoglobinuria, a disease characterized by lack of expression of other PI-linked proteins, failed to express CD14. Interestingly, the CD14-expressing neuroglioma cell line, which had been transfected with a single CD14 cDNA, released a soluble form of CD14 into the supernatant. Soluble forms of CD14 have previously been observed in serum of normal individuals and in culture supernatants of CD14+ cells. Biosynthetic experiments reveal that this soluble form of CD14 (48 kDa), which is smaller than the form released from the membrane by PI-phospholipase C (53 kDa), does not contain ethanolamine, the first constitutent of the PI-anchoring system. These studies demonstrate that CD14 is a member of the family of PI-anchored proteins and suggest that soluble forms of CD14 represent molecules that completely lack the PI-anchoring system.

CD14-dependent and CD14-independent signaling pathways in murine macrophages from normal and CD14 knockout mice stimulated with lipopolysaccharide or taxol.

The antitumor agent, Taxol, shares with bacterial LPS the ability to activate murine macrophages, and its LPS-mimetic effects are blocked by LPS analogue antagonists. Since CD14 is central to the recognition of LPS by macrophages, we sought to examine a role for CD14 in the response to Taxol vs LPS. A comparison of responses of macrophages from wild-type mice with those from mice lacking CD14 due to a targeted disruption of the CD14 gene (CD14-deficient knockout (CD14KO)) revealed that like LPS, Taxol induces both CD14-dependent and -independent pathways of gene activation, although the CD14 dependency of Taxol stimulation is much less striking than that observed with LPS. The macrophage interaction with low concentrations of LPS (< or = 10 ng/ml) is largely CD14 dependent, as evidenced by the lack of induction of TNF-alpha, IL-1beta, and interferon-inducible protein-10 (IP-10) genes by CD14KO macrophages cultured in the absence of soluble CD14 (i.e., in autologous CD14KO -/- mouse serum). However, at high concentrations of LPS or Taxol, a CD14-independent pathway of activation is observed: this pathway leads to minimal IP-10 gene induction, even though induction of TNF-alpha and IL-1beta occurs. Measurements of TNF secretion followed a similar pattern to that observed at the level of steady state mRNA. These data suggest the existence of two pathways of activation by both LPS and Taxol: one that is CD14 dependent and leads to induction of TNF-alpha, IL-1beta, and IP-10 gene induction, and a CD14-independent pathway that results in the induction of TNF-alpha and IL-1beta, with minimal induction of IP-10.

Recombinant soluble CD14 mediates the activation of endothelial cells by lipopolysaccharide.

Recent studies have suggested that soluble CD14 found in serum is involved in the LPS-induced activation of endothelial cells (EC). To more fully investigate the relevance of sCD14 to LPS-induced activation of EC, we have used recombinant soluble CD14 (rsCD14) and have examined, under serum-free conditions, its role in the LPS-induced EC response in the presence of LPS alone as well as in the presence of LPS-binding protein. Our studies show that EC can be activated by high concentrations of LPS in the presence of rsCD14 alone. However, at low concentrations of LPS (5 and 10 ng/ml), the rsCD14-stimulated activation is strongly enhanced by LPS-binding protein. In addition, we show that LPS binds to rsCD14 directly; in the presence of low concentrations of LPS this binding is enhanced by the presence of LPS-binding protein. These results show that while the membrane form of CD14 can function as a receptor, its soluble form can function as a co-ligand with LPS in the EC-LPS response.

Biochemistry of HLA-DRw6: evidence for seven distinct haplotypes.

The DRw6 specificity, which has a frequency of 11% in the Caucasian population, cannot be positively defined, since no monospecific allo-antiserum is available. This particular status among DR specificities led us to study the DRw6 haplotypes at the molecular level. We performed 2D-PAGE analysis of HLA-DR molecules in 44 different DRw6 haplotypes. The data were obtained from six homozygous typing cells, eight families informative for the segregation of the DRw6 haplotype, and 15 unrelated donors. Five unique beta-chain electrophoretic patterns were detected, indicating the existence of five structurally distinct DRw6 beta-chains. Each haplotype expresses one or two beta-chains. The different combinations of the DR beta-chains present in a single haplotype allow to characterize seven unique DRw6 haplotypes. In contrast to what has been previously found for DR2 and DR4, there is no DR beta-chain common to all the DRw6 cells. Correlation of the biochemical data with the recent serologic (DRw13 vs DRw14) and cellular (Dw9, Dw18, Dw19) splits of the DRw6 specificity will be discussed.

Recombinant soluble CD14 prevents mortality in mice treated with endotoxin (lipopolysaccharide).

Endotoxic shock is a life-threatening condition mediated by cytokines released after exposure to bacterial LPS/endotoxin. Activation of monocytes and neutrophils by the binding of LPS to the membrane receptor, CD14, plays a key role in this response. Furthermore, a soluble form of the CD14 receptor enhances the endothelial cell response to LPS. We show here that despite the agonist effects of soluble CD14 on the endothelial cell response to LPS, recombinant soluble CD14 is able to protect mice from LPS-induced lethality. This protection appears to be associated with the inhibition of TNF-alpha release. These results suggest that the soluble CD14 receptor may represent a new form of therapy for endotoxic shock in humans.

Induction of a novel mechanism of accelerated bacterial clearance by lipopolysaccharide in CD14-deficient and Toll-like receptor 4-deficient mice.

Despite the lack of a proinflammatory response to LPS, CD14-deficient mice clear Gram-negative bacteria (Escherichia coli 0111) at least 10 times more efficiently than normal mice. In this study, we show that this is due to an early and intense recruitment of neutrophils following the injection of Gram-negative bacteria or LPS in CD14-deficient mice; in contrast, neutrophil infiltration is delayed by 24 h in normal mice. Similar results of early LPS-induced PMN infiltration and enhanced clearance of E. coli were seen in Toll-like receptor (TLR) 4-deficient mice. Furthermore, the lipid A moiety of LPS induced early neutrophil infiltration not only in CD14-deficient and TLR-4-deficient mice, but also in normal mice. In conclusion, the lipid A component of LPS stimulates a unique and critical pathway of innate immune responses that is independent of CD14 and TLR4 and results in early neutrophil infiltration and enhanced bacterial clearance.

Recombinant soluble CD14 inhibits LPS-induced tumor necrosis factor-alpha production by cells in whole blood.

CD14 functions as a cell surface receptor for LPS in the activation of monocytes/macrophages and neutrophils by endotoxin. To assess the utility of soluble forms of the CD14 receptor as a possible therapeutic for endotoxin shock, we have produced recombinant human soluble CD14 using a baculovirus expression system. We find that the recombinant protein is not only expressed on the surface of the insect cells as a glycosyl phosphatidylinositol (GPI)-anchored protein, but is also released into the culture medium as a soluble form that lacks the GPI anchor. Functional analyses of recombinant human soluble CD14 show that it binds specifically to LPS and can inhibit the LPS-induced release of TNF-alpha by macrophages and mononuclear cells as well as by cells in whole human blood when used at concentrations of approximately 70 micrograms/ml. Thus, soluble CD14 may be useful as an adjunct in the treatment of endotoxin shock.

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.

Lipopolysaccharide structure influences the macrophage response via CD14-independent and CD14-dependent pathways.

CD14, a protein expressed on the surface of monocytes and neutrophils, is a major receptor for lipopolysaccharide (LPS). Studies with normal and CD14-deficient macrophages show that responses to low concentrations of LPS require expression of CD14, whereas responses to high concentrations of LPS are CD14-independent. Since LPS isolated from different bacterial species shows structural variability, studies were performed to determine whether differences in LPS structure influence CD14-dependent and CD14-independent responses. Studies with LPS purified from Escherichia coli, Salmonella abortus subspecies equi, Salmonella minnesota, Pseudomonas aeruginosa, Neisseria meningitidis, Bacteroides fragilis, and Rhodobacter sphaeroides show that the strongest CD14-dependent responses require a typical O-antigen, long carbohydrate chains, at least 6 acyl chains in their lipid A, and 2-phosphorylated Kdo moieties; wild-type LPS lacking a typical O-antigen and containing short carbohydrate chains and 2-phosphorylated Kdo moieties induces the strongest CD14-independent response.

CD14 plays no major role in shock induced by Staphylococcus aureus but down-regulates TNF-alpha production.

Recent in vitro studies have suggested that CD14, a major receptor for LPS, may also be a receptor for cell wall components of Gram-positive bacteria and thus play a role in Gram-positive shock. To analyze the in vivo role of CD14 in responses to Gram-positive bacteria, CD14-deficient and control mice were injected with Staphylococcus aureus, and the effects on lethality, bacterial clearance, and production of cytokines were analyzed. Survival of CD14-deficient and control mice did not differ significantly after administration of various doses of either unencapsulated or encapsulated S. aureus; furthermore, mice in both groups displayed similar symptoms of shock. In addition, inflammatory cytokines such as TNF-alpha and IL-6 were readily detectable in the serum of CD14-deficient mice injected with live or antibiotic-killed S. aureus. Surprisingly, the serum concentration of TNF-alpha in CD14-deficient mice was at least threefold higher than in control mice after injection of either unencapsulated or encapsulated S. aureus, suggesting that CD14 down-regulates TNF-alpha. A similar increase in serum TNF-alpha occurred when CD14-deficient animals were injected with gentamicin-killed bacteria even though no symptoms of shock were observed. These studies indicate that CD14, in contrast to its key function in responses to the Gram-negative bacterium, Escherichia coli 0111, does not play a prominent role in septic shock induced by S. aureus, and that the symptoms of S. aureus shock are not due solely to TNF-alpha.

Lipopolysaccharide binding protein and CD14 interaction induces tumor necrosis factor-alpha generation and neutrophil sequestration in lungs after intratracheal endotoxin.

It has been proposed that lipopolysaccharide (LPS) bound to the 60-kD LPS binding protein (LBP) forms an LPS/LBP complex that, in turn, binds to the CD14 receptor on monocytes/macrophages and stimulates the release of cytokines. We examined the role of LBP and CD14 in tumor necrosis factor-alpha (TNF-alpha) production and neutrophil (polymorphonuclear leukocyte [PMN]) sequestration in lungs induced by intratracheal instillation of LPS using rabbit lungs perfused at constant flow with lactated Ringer-albumin solution. LPS alone (Salmonella minnesota, wild type; 20 ng) or in the presence of LBP (500 ng) was injected intratracheally. In some experiments, human PMNs (5 x 10(7)) were added to the perfusate after a 2-hour period of perfusion. Samples of lung perfusate were collected every 30 minutes for 180 minutes when bronchoalveolar lavage was also performed. TNF-alpha concentrations in the perfusate and bronchoalveolar lavage fluid were determined by use of a bioassay with L-929 fibroblasts, and PMN accumulation in lungs was determined by myeloperoxidase assay of lung homogenates. LPS alone did not significantly increase TNF-alpha production or lung PMN accumulation, whereas the LPS/LBP complex increased TNF-alpha concentration in perfusate twofold and PMN accumulation twofold compared with the effect of LPS alone. Intratracheal instillation of anti-CD14 monoclonal antibody MY4 (40 micrograms) with the LPS/LBP complex prevented TNF-alpha release and PMN sequestration, whereas an isotype-matched control monoclonal antibody was ineffective. Therefore, LBP in the airspace enhances the LPS effect on TNF-alpha production via a CD14-dependent pathway, and as a result, CD14 activation can contribute to lung PMN sequestration.(ABSTRACT TRUNCATED AT 250 WORDS)