Innate immune responses to TLR2 and TLR4 agonists differ between baboons, chimpanzees and humans.

BACKGROUND: African catarrhine primates differ in bacterial disease susceptibility. METHODS: Human, chimpanzee, and baboon blood were stimulated with TLR-detected bacterial agonists and cytokine/chemokine induction assessed by real-time PCR. RESULTS: Humans and chimpanzees shared similar cytokine/chemokine responses, while baboon cytokine/chemokine induction differed. Generally, responses were agonist independent. CONCLUSIONS: These primates tend to generate species rather than agonist-specific responses to bacterial agonists.

Biochemical characterization of a second family of human Ia molecules, HLA-DS, equivalent to murine I-A subregion molecules.

In mice, two families of structurally distinct Ia molecules, one designated I-A and the other I-E, have been identified and characterized. The HLA-DR molecules represent one family of human Ia molecules equivalent to the murine I-E molecules on the basis of amino acid sequence homology. We describe the isolation and biochemical characterization of a second family of human Ia molecules, designated HLA-DS for second D-region locus, equivalent to the murine I-A molecules. The human HLA-DS molecules consist of two polypeptide chains, DS alpha (37,000 mol wt) and DS beta (29,000 mol wt), with 73% amino acid sequence identity to the murine I-A molecules. Furthermore, the HLA-DS molecules are closely linked genetically to HLA-DR molecules, a situation analogous to that observed in mice. The similarity in molecular weights of the DR and DS molecules might explain why others have failed to identify the latter in man.

The complexity of HLA-DS molecules. A homozygous cell line expresses multiple HLA-DS alpha chains.

Ia molecules expressed by an HLA-DRw6 homozygous cell line were immunoprecipitated with anti-Ia allosera and monoclonal antibodies and analyzed by 2-D gel electrophoresis. The DRw6 homozygous cell line was shown to express two DS beta chains; this observation extends our previous finding that a DR5 homozygous cell line expresses two DS beta chains and suggests that the expression of at least two DS beta chains by DR homozygous cell lines is a generalized phenomenon. The data presented here document for the first time that a DR homozygous cell line expresses at least two DS alpha chains. Therefore, this cell line expresses at least two DS molecules with the potential for the expression of four DS molecules. In agreement with previous reports, the cell line was shown to express two DR beta chains and one DR alpha chain that combine to form two DR molecules. The molecular specificities of two MB1 allosera and two MB1 -like monoclonal antibodies were also compared in these studies. Both MB1 allosera isolated a single DS molecule, while the MB1 -like monoclonal antibodies isolated at least two DS molecules. Therefore, these studies document for the first time that anti-Ia reagents which are specific for the MB1 or MB1 -like determinants in population studies do not recognize the same Ia molecules in immunochemical studies. The data presented here for the expression of at least two DS alpha chains and the location of the MB1 allodeterminant on only one of multiple DS molecules are in agreement with recent studies at the gene level.

An HLA-DR5 homozygous cell line expresses two DS (I-A-like) molecules.

Previous studies have indicated that LLA-DR homozygous cell lines express two DR molecules but only a single DS (I-A-like) molecule. This report demonstrates that an HLA-DR5 homozygous cell line expresses at least two distinct DS molecules. These two DS molecules are formed by the association of a single DS alpha chain with either of two DS beta chains. Four distinct Ia molecules have now been identified from this DR5 homozygous cell line.

The HLA-DR4 family of haplotypes consists of series of distinct DR and DS molecules.

Among DR4-associated HLA-D antigens, distinct and consistent structural variations were found for the products of two human "Ia-like" loci, DR and DS. Analysis of neuraminidase-treated immunoprecipitated DR molecules from 15 HLA-DR4-associated HLA-D homozygous B-lymphoblastoid cell lines by two dimensional polyacrylamide gel electrophoresis identified five distinct DR beta chains. In addition, gel analysis of immunoprecipitated DS molecules identified three distinct DS beta chains. Altogether, five distinct DR4 haplotypes were defined according to the observed structural diversity of the DR and DS beta chains. These gene products presumably contribute the dominant polymorphisms recognized by T cells in mixed lymphocyte reaction (MLR). Thus, these studies indicate that the serologic specificity known as HLA-DR4 is not a single haplotype, but a determinant present on products of individual loci arrayed into distinctly different haplotypes. These findings suggest that distinct products of individual loci, rather than conventional HLA specificities defined by alloimmune sera, may represent the genetic markers relevant to HLA-D/DR associated diseases.

The CD14 monocyte differentiation antigen maps to a region encoding growth factors and receptors.

CD14 is a myelomonocytic differentiation antigen expressed by monocytes, macrophages, and activated granulocytes and is detectable with the monoclonal antibodies MO2, MY4, and LeuM3. Analyses of complementary DNA and genomic clones of CD14 show that it has a novel structure and that it maps to chromosome 5 within a region containing other genes encoding growth factors and receptors; it may therefore represent a new receptor important for myeloid differentiation. In addition, the CD14 gene is included in the "critical" region that is frequently deleted in certain myeloid leukemias.

The role of Kupffer cell oxidant production in early ethanol-induced liver disease.

Considerable evidence for a role of Kupffer cells in alcoholic liver disease has accumulated and they have recently been shown to be a predominant source of free radicals. Several approaches including pharmacological agents, knockout mice, and viral gene transfer have been used to fill critical gaps in understanding key mechanisms by which Kupffer cell activation, oxidant formation, and cytokine production lead to liver damage and subsequent pathogenesis. This review highlights new data in support of the hypothesis that Kupffer cells play a pivotal role in hepatotoxicity due to ethanol by producing oxidants via NADPH oxidase.

Peptide map comparisons of similar serologically defined HLA-DR antigens isolated from different lymphoblastoid cell lines.

In these studies, we have examined the possibility that DR subtypes, closely related to each other in structure, compose the major DR allotypic groups. The structures of DR molecules, isolated from pairs of cell lines that had the same serologically defined HLA-DR type, were compared by peptide mapping. HLA-DR molecules isolated from pairs of cell lines in which both members were either DR1, DR2, or DR7 were identical. However, DR molecules isolated from cell lines LG-29 (DR5) and LG-38 (DR5) displayed two distinct differences in their small (beta) subunits. This number of differences, two of 15 peptides or 15%, is much fewer than is observed between DR allotypes (approximately 50%) and suggests that at least two subtypes exist within the DR5 allotype family.

Microheterogeneity of HLA-DR4 haplotypes: DNA sequence analysis of LD"KT2" and LD"TAS" haplotypes.

We have isolated and sequenced cDNA clones corresponding to the polymorphic alpha and beta chains encoded by the DR and DQ subregions of two HLA-DR4 haplotypes, LD"KT2" and LD"TAS". These two haplotypes are distinguished on the basis of mixed lymphocyte culture typing. The data indicate that the designation of LD"TAS" as a distinct subtype from Dw13 is very likely due to amino acid differences in the DQ beta chain. In contrast, LD"KT2" differs from TAS and Dw13 by a single amino acid substitution at position 37 of the DR beta 1 chain. The functional and evolutionary significance of these polymorphisms is discussed.

A novel molecule expressing HLA-DR antigenic determinants.

Our previous studies suggested that the polymorphism of HLA-DR antigens (the human equivalent of murine I-E antigens) was a result of structural variation in the small (beta) subunit. In order to more accurately define this polymorphism we have expanded these studies to include HLA-DR antigens isolated with monoclonal cells derived from genotypically HLA-homozygous DRw2, DR2w5, and DRw7 lymphoblastoid cells derived from offspring of consanguineous relationships. Our results indicate the large (alpha) subunits of DRw2 and DRw7 antigens are nearly identical, while their beta subunits show many differences. In contrast, both the alpha and beta subunits of the DRw5 antigen differ strikingly from the respective subunits of the DRw2 and DRw7 antigens. The significance of the variability of the DRw5 alpha subunit is in question at this point. One intriguing possibility is that DRw5 actually represents the human counterpart of the mouse I-A subregion antigen and that the monoclonal antibody is reacting with a determinant which is shared by the human equivalents of murine I-A and I-E antigens.

HLA-DS molecules and their relation to supertypic specificities.

We have analyzed the structures of HLA-DS molecules from DR1, 2, 3, 4, 5, and 7 cell lines by two-dimensional gel electrophoresis and examined the relationship of HLA-DS molecules to those expressing the supertypic specificities MT1, MB3, and MT3. These studies have allowed us to conclude that the HLA-DS locus is as polymorphic as HLA-DR and that the MT1 and MB3 supertypic specificities reside on DS molecules. Furthermore, MT1 molecules from DR1, DR2, and DRw6 cell lines are structurally different as are MB3 molecules from DR4 and DR5 cell lines. In addition, studies using two MT3 specific reagents, a monoclonal antibody, 109d6, and an alloantiserum, Hon, suggest that MT3 is a cross-reactive determinant present on products of two different loci: a DS4 molecule and a DR7-like molecule.

Parameters influencing membrane CD14 expression and soluble CD14 levels in sepsis.

INTRODUCTION: Membrane (mCD14) and soluble (sCD14) CD14 are pattern recognition receptors for bacterial cell wall fragments. They play an important role in the generation of the innate immune response against bacterial pathogens. Differential expression of these receptors may be relevant for the clinical course of patients with sepsis. PATIENTS AND METHODS: 32 patients with an early onset of sepsis (duration of symptoms < 24h) were examined repeatedly by flow cytometry for expression of mCD14, and by ELISA for levels of sCD14, leukocyte elastase and C-reactive Protein (CRP). RESULTS: At study entry, mCD14 expression was reduced in all patients with sepsis, but returned to normal levels during the course of the disease in survivors only. mCD14 was found to be inversely correlated with severity of disease, leukocyte elastase, and C-reactive protein. Among patients with severe disease and Apache II scores >or= 20, sCD14 levels at study entry were significantly higher in those who survived by day 28, as compared to non-survivors (p = 0.02). CONCLUSION: The data presented are compatible with a recently published hypothesis derived from in vitro experiments suggesting that leukocyte elastase may be responsible for cleavage of mCD14 from the monocyte surface. The data also suggest that higher sCD14 levels may be beneficial in sepsis. Persistently reduced mCD14 expression seems to be a marker for severity of disease in patients with sepsis.

Further characterization of HLA-DS molecules: implications for studies assessing the role of human Ia molecules in cell interactions and disease susceptibility.

HLA-DS molecules, the human homologs of murine I-A molecules, were analyzed and compared to DR molecules by two-dimensional gel analysis. These analyses allowed us to define six forms of DS molecules, suggesting that DS molecules were as polymorphic as DR molecules. The supertypic specificities, MT1 and MB3 were localized to DS molecules, whereas MT3 appeared to represent a crossreactive determinant present on products of two different loci encoding Ia-like molecules in man, a DS4 molecule and a DR7-like molecule. These studies also revealed that MT1 molecules isolated from DR1 and DR2 cell lines were different, as were MB3 molecules isolated from DR4 and DR5 cell lines. Extensive crossreactions between DR and DS molecules were observed by using several monoclonal antibodies. This sharing of epitopes between products of different loci for human Ia molecules has important functional implications.

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.

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.

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.

Mouse placental macrophages have a decreased ability to present antigen.

Large numbers of macrophages can be found in an animal's uteroplacental unit. This high concentration of macrophages suggests they must play an important role during placental development. To gain a better understanding of the functional capacity of placental macrophages, we have obtained a highly enriched placental macrophage culture and have derived several cell lines from this population. Both placental macrophages and cell lines show colony-stimulating factor 1-dependent growth, express Fc receptors, and can perform Fc-receptor-mediated phagocytosis. In addition, they express macrophage markers Mac-1, F4/80, and CD14. Although placental macrophages express major histocompatibility complex class II molecules constitutively, they display a decreased ability to present protein antigens to T cells. Since primary fetal liver macrophages of the same gestational stage also show a decreased ability to present antigens, this phenomenon may reflect a developmental stage of macrophages.

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.

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.