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.

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.

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.

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.

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.

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.

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 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.

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.

Evidence that the receptor for soluble CD14:LPS complexes may not be the putative signal-transducing molecule associated with membrane-bound CD14.

Membrane-bound CD14 acts as a receptor for lipopolysaccharide (LPS) on monocytes/macrophages and neutrophils. Studies have suggested that the activation of monocytes/macrophages by the binding of LPS to membrane-bound CD14 may require the association of a signal-transducing molecule with membrane-bound CD14. The observation that non-CD14 expressing cells, such as endothelial cells, can nevertheless be activated by a complex of LPS and a soluble form of CD14 (sCD14) suggests that the receptor for this complex may be identical to the signal transducing molecule associated with membrane-bound CD14. The studies described show that two CD14-specific MoAb are able to block the LPS-induced activation of endothelial cells but do not affect the response of monocytes to LPS. This suggests that the interaction of the sCD14:LPS complex with endothelial cells is distinct from the interaction of membrane-bound CD14 with its putative signal-transducing molecule.

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.

Resistance to endotoxin shock and reduced dissemination of gram-negative bacteria in CD14-deficient mice.

Endotoxin shock is the result of activation of the immune system by endotoxin/LPS, a component of Gram-negative bacteria. CD14, a GPI-anchored glycoprotein expressed strongly by monocyte/macrophages, is one of several receptors for endotoxin/LPS. The role of CD14 in bacterial-induced and LPS-induced shock was tested in CD14-deficient mice produced by gene targeting in embryonic stem cells. CD14-deficient mice were found to be highly resistant to shock induced by either live Gram-negative bacteria or LPS; however, at very high concentrations of LPS or bacteria, responses through non-CD14 receptors could be detected. Surprisingly, CD14-deficient mice also showed dramatically reduced levels of bacteremia, suggesting an unexpected role for CD14 in the dissemination of Gram-negative bacteria.

CD14-independent responses to LPS require a serum factor that is absent from neonates.

Monocytes/macrophages and neutrophils can respond to endotoxin via a high-affinity receptor (CD14), requiring low levels of LPS (< 1 ng/ml) as well as through another pathway(s) that requires high levels of LPS (> 10 ng/ml). Both pathways result in the secretion of high levels of cytokines, such as TNF-alpha, and the up-regulation of various other effector molecules. To further define the activation of cells by LPS via a pathway that does not involve CD14, we have used an experimental model that distinguishes CD14-dependent from CD14-independent responses using saturating amounts of an anti-CD14 Ab to block the CD14-dependent response. Analysis of the ability of various individuals to respond to LPS via via both the CD14-dependent and CD14-independent pathways shows that adults can respond via both pathways; furthermore, in the presence of 100 ng of LPS/ml, the primary response is CD14 independent. In contrast to the response by adults, neonates can only respond via the CD14-dependent pathway. Further analysis has shown that the CD14-independent pathway requires a non-CD14 plasma protein present in adult plasma that is either missing or nonfunctional in neonate (cord) plasma.

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.

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.

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.