Susceptibility to Coccidioides species in C57BL/6 mice is associated with expression of a truncated splice variant of Dectin-1 (Clec7a).

Coccidioides posadasii spherules stimulate macrophages to make cytokines via TLR-2 and Dectin-1. We used formalin-killed spherules and 1,3-beta-glucan purified from spherules to stimulate elicited peritoneal macrophages and myeloid dendritic cells (mDCs) from susceptible (C57BL/6) and resistant (DBA/2) mouse strains. DBA/2 macrophages produced more TNF-alpha and IL-6 than macrophages from C57BL/6 mice, and the amount of TNF-alpha made was dependent on both TLR2 and Dectin-1. DCs from C57BL/6 mice made more IL-10 and less IL-23p19 and IL-12p70 than did DBA/2 DC. These responses were inhibited by a monoclonal antibody to Dectin-1. DBA/2 mice expressed full-length Dectin-1, whereas C57BL/6 mice spliced out exon 3, which encodes most of the stalk. RAW cells transduced to express the full-length Dectin-1 responded better to FKS than cells expressing truncated Dectin-1. We compared the isoform of Dectin-1 expressed by 34 C57BL/6 X DBA/2 recombinant inbred (BXD RI) lines with their susceptibility to Coccidioides immitis. In 25 of 34 RI lines susceptibility or resistance corresponded to short or full-length isoforms, respectively. These results suggest that alternative splicing of the Dectin-1 gene contributes to susceptibility of C57BL/6 mice to coccidioidomycosis, and affects the cytokine responses of macrophages and mDCs to spherules.

Plasma CD14 decreases monocyte responses to LPS by transferring cell-bound LPS to plasma lipoproteins.

CD14, a myeloid cell-surface receptor and soluble plasma protein, binds LPS and other microbial molecules and initiates the innate immune response to bacterial invasion. The blood concentration of soluble CD14 (sCD14) increases during the systemic response to infection. Although high sCD14 blood levels have correlated with increased risk of dying from severe sepsis, sCD14 can diminish cell responses to LPS. We show here that in human serum, sCD14 increases the rate at which cell-bound LPS is released from the monocyte surface and binds to plasma lipoproteins. This enhanced rate of LPS efflux is associated with a significant reduction in the ability of monocytes to produce cytokines in response to LPS. Serum from septic patients reduced the LPS-monocyte interaction by as much as tenfold, and depletion of sCD14 from the serum restored LPS-monocyte binding and release kinetics to near normal levels. In serum from septic patients, monocyte-bound LPS also moved more rapidly into lipoproteins, which completely neutralized the biologic activity of the LPS that bound to them. In human plasma, sCD14 thus diminishes monocyte responses to LPS by transferring cell-bound LPS to lipoproteins. Stress-related increases in plasma sCD14 levels may help prevent inflammatory responses within the blood.

MD-2 binds to bacterial lipopolysaccharide.

Many LPS binding proteins have been described, but the exact nature of the LPS receptors that signal cells remains unclear. MD-2 is a molecule that is found in association with Toll-like receptor 4, which has been shown to be a receptor for LPS. We have produced human MD-2 in baculovirus and tested it for LPS binding. MD-2 binds the lipid A region of LPS without the need for LPS binding protein. These data suggest that MD-2 may be binding LPS as part of the TLR4 receptor complex.

The use of a DNA probe for the differentiation of rodent malaria strains and species.

A clone, PCsv4, derived from a partial genomic library of the rodent malaria Plasmodium chabaudi chabaudi AS strain, contains an insert which when used as a probe at low stringency in Southern blotting of genomic DNA from a variety of strains, subspecies and species of rodent malaria parasites, results in a pattern of hybridisation which is specific to each of the DNA samples used, thus providing an accurate method to define a rodent malaria line. The insert only hybridises to DNA derived from malaria parasites of rodent species. The insert also hybridises to a small number of RNA transcripts.

Identification of the four human malaria parasite species in field samples by the polymerase chain reaction and detection of a high prevalence of mixed infections.

Genus- and species-specific sequences are present within the small subunit ribosomal RNA genes of the four human malaria parasites. Oligonucleotide primer pairs specific to each species were designed for specific amplification by the Polymerase Chain Reaction (PCR), to detect each malaria species. DNA equivalent to 5 microliters of blood was sufficient for the detection of each of the species. Blood samples obtained from 196 patients attending a malaria clinic in Trad province (Thailand) were analyzed. Detection and identification of the parasites, solely by electrophoretic analysis of the PCR products, has proven to be more sensitive and accurate than by routine diagnostic microscopy. A high proportion of mixed species infections were brought to light by the PCR assay. Implications for medical treatment and epidemiological studies are discussed.

Cloning of genomic fragment from Plasmodium chabaudi expressing a 105 kilodalton antigen epitope.

The 105 kDa antigen of Plasmodium chabaudi has many characteristics of the P. falciparum Pf 155 (RESA) molecule. A clone (pPC105e) from a P. chabaudi genomic library was isolated using immune screening with a 105 kDa antigen specific monoclonal antibody (B7E10). Southern, Northern and Western blotting analyses provide evidence for a lack of variability at the protein and DNA levels. A subclone of the insert in the expression vector pEX2, synthesises a fusion peptide which contains the epitope recognized by B7E10. Sequences homologous to the insert were detected in the genome of three other rodent and two primate malarias.

Use of a DNA probe to analyse the dynamics of infection with rodent malaria parasites confirms that parasite clearance during crisis is predominantly strain- and species-specific.

A DNA probe, PCsv4.1, isolated from Plasmodium chabaudi chabaudi AS, generates in Southern blotting experiments restriction fragment length polymorphisms specific to a particular rodent malaria parasite line. It was used to develop an assay which allows identification and semi-quantitative compositional analysis of sample parasite populations in which one or more strains, subspecies or species were present. In experiments where mechanisms effecting parasite clearance during crisis were studied, the assay was used to determine the composition of parasite populations present in P. c. chabaudi AS infected mice challenged during crisis with homologous or heterologous parasites. It was thus confirmed that clearance mechanisms during crisis operate in a predominantly specific manner.

Cross-reactive asparagine-rich determinants shared between several blood-stage antigens of Plasmodium falciparum and the circumsporozoite protein.

From a Plasmodium falciparum cDNA expression library derived from mRNA of the asexual blood stages, we isolated and sequenced five different cDNA clones whose predicted protein products were unusually rich in asparagine (Asn). Two of the clones, R5 and G5, contain tandem imperfectly repeated sequences based on Asn-Asn-Thr (NNT) and Asn-Asn-Met (NNM) respectively. The other three, E4, C5 and R13, as well as G5, contain stretches of polyasparagine varying in length from 2 to 26 residues. Results of DNA blotting experiments with the individual cDNA sequences as probes suggest that each of the five clones corresponds to a different P. falciparum gene. The fragments of P. falciparum proteins expressed by the cDNA clones shared cross-reactive antigenic determinants which were present on multiple P. falciparum proteins. In immunoblotting experiments, owl monkey antibodies selected for binding to the polypeptide expressed by clone E4, C5 or G5 reacted with the expressed proteins from all 5 clones, and with at least 10 proteins from schizont infected erythrocytes. The cross-reactive epitopes could be modeled by two Asn-rich peptide structures: (1) (NNT)8, whose sequence was based on the R5 repeat; and (2) (NPNA)6, whose sequence was based on the Asn-rich repeat of the P. falciparum circumsporozoite protein (CSP). Antibodies that bound to each peptide were selected from sera of immune monkeys that had never been exposed to sporozoites. The selected antibodies bound all 5 expressed proteins in immunoblotting assays and also bound to several proteins from parasitized erythrocytes. Such cross reactivity between the CSP repeating unit and several blood-stage antigens has not been previously reported.

Biased distribution of msp1 and msp2 allelic variants in Plasmodium falciparum populations in Thailand.

Plasmodium falciparum isolates were obtained from Thai patients attending a malaria clinic on the Thai-Kampuchean border over 4 cross-sectional surveys carried out at 3-monthly intervals. The genetic structure of the parasite populations was determined by nested polymerase chain reaction (PCR) amplification of polymorphic regions of 3 P. falciparum antigen genes: msp1, msp2 and glurp. Although a high degree of diversity characterized these isolates, the overall population structure of the parasites associated with patent malaria infections was observed to remain relatively stable over time. The highest degree of polymorphism was observed with msp2, and the mean number of lines per infection (multiplicity of infection) calculated with this marker was higher than that obtained using msp1 or glurp alone, or combined. Infections with > or = 2 parasite lines were seen in 76% of the samples, and were proportionally more numerous at the start and end of the rainy season. Two interesting exceptions to the random distribution were observed and involved 2 allelic variants which in one case were found dissociated (msp1 MAD20-family) and in the other were associated (msp2 FC27-family). The epidemiological significance of these types of data is discussed.

Knowledge of cellular receptors for bacterial endotoxin--1995.

Septic shock due to infections with gram-negative bacteria remains a major clinical problem for infectious disease specialists, although our understanding of the pathophysiology of this syndrome has improved greatly over the past 5 years. The discovery of lipopolysaccharide (LPS) binding protein, a serum protein that catalyses the transfer of LPS to cellular receptors for LPS, was a major breakthrough. The finding that CD14, a glycophosphatidylinositol-linked membrane protein expressed by macrophages, is a receptor for LPS has made a major difference in our understanding of cellular activation by LPS. We will review studies dealing with LPS's binding to these proteins and its activation of cells. A better understanding of septic shock at the molecular level should lead to the development of new treatments for this lethal disease.

A region of human CD14 required for lipopolysaccharide binding.

CD14, a glycosylphosphatidylinositol-anchored protein on the surface of monocytes, macrophages, and polymorphonuclear leukocytes, is a receptor for lipopolysaccharide (LPS). CD14 binding of LPS is enhanced by serum proteins, especially lipopolysaccharide binding protein. The serum-dependent binding of LPS to CD14 stimulates macrophages to make cytokines, which can cause septic shock in humans and animals. Here, we identify a region in human CD14 which is important in serum-dependent LPS binding and LPS-induced cellular activation. Four small regions (4-5 amino acids long) within the N-terminal 65 amino acids of CD14 were deleted singly or in combination. The deletion mutants were stably expressed in Chinese hamster ovary (CHO) cells. The mutants were characterized in three assays: reactivity with anti-CD14 monoclonal antibody, serum-dependent LPS binding, and LPS-induced activation of NF-kappa B. Some of the mutants selectively lost reactivity with the anti-CD14 monoclonal antibody that inhibited serum-dependent LPS binding and cellular activation. All of the mutants bound much less LPS than wild type CD14 in the presence of serum. None of the mutants bound more LPS than control CD14-CHO cells in the absence of serum. CD14-CHO cells respond to LPS by activation of NF-kappa B. All of the deletion mutants were less active LPS receptors than wild type CD14-CHO cells. The delta AVEVE mutant, the delta DDED and delta PQPD double mutant, and the delta DDED, delta PQPD, delta AVEVE, and delta DPRQY quadruple deletion mutants were essentially inactive LPS receptors in CHO cells. These studies suggest that the 65 N-terminal amino acids of CD14 are critical for serum-dependent binding of LPS to CD14 and subsequent signal transduction in CHO cells.

The N-terminal half of membrane CD14 is a functional cellular lipopolysaccharide receptor.

CD14, a glycosylphosphatidylinositol-anchored protein on the surface of monocytes, macrophages, and polymorphonuclear leukocytes, is a receptor for lipopolysaccharide (LPS). It was recently reported that an N-terminal 152-amino-acid fragment of soluble CD14 was an active soluble lipopolysaccharide receptor (T. S. -C. Juan, M. J. Kelley, D. A. Johnson, L. A. Busse, E. Hailman, S. D. Wright, and H. S. Lichenstein, J. Biol. Chem. 270:1382-1387, 1995). To determine whether the N-terminal half of the membrane CD14 was a functional LPS receptor on the cell membrane, we engineered a chimeric gene coding for amino acids 1 to 151 of CD14 fused to the C-terminal region of decay-accelerating factor and expressed it in Chinese hamster ovary cells and 70Z/3 cells. We found that the chimeric, truncated CD14 is a fully functional LPS receptor in both cell lines.

Soluble CD14 enhances membrane CD14-mediated responses to peptidoglycan: structural requirements differ from those for responses to lipopolysaccharide.

The purpose of this study was to identify the functional significance of the binding of soluble CD14 (sCD14) to bacterial peptidoglycan (PGN) and to compare the structural requirements of sCD14 for the binding to PGN and lipopolysaccharide (LPS) and for sCD14-mediated enhancement of PGN- and LPS-induced cell responses. sCD14 did not facilitate the responses of membrane CD14 (mCD14)-negative pre-B 70Z/3 cells to PGN, although it facilitated the responses of these cells to LPS and although mCD14 facilitated the responses of 70Z/3 cells to PGN. sCD14 enhanced mCD14-mediated cell activation by both PGN and LPS, but only the responses to LPS, and not to PGN, were enhanced by LPS-binding protein. Four 4- or 5-amino-acid-long sequences within the 65-amino-acid N-terminal region of sCD14 were needed for binding to both PGN and LPS and for enhancement of cell activation by both PGN and LPS. However, deletions of individual sequences had different effects on the ability of sCD14 to bind to PGN and to LPS and on the ability to enhance the responses to PGN and to LPS. Thus, there are different structural requirements of sCD14 for binding to PGN and to LPS and for the enhancement of PGN- and LPS-induced cell activation.

CD14 is a cell-activating receptor for bacterial peptidoglycan.

The hypothesis that CD14 (an endotoxin receptor present on macrophages and neutrophils) acts as a cell-activating receptor for bacterial peptidoglycan was tested using mouse 70Z/3 cells transfected with human CD14. 70Z/3 cells transfected with an empty vector were unresponsive to insoluble and soluble peptidoglycan, as well as to low concentrations of endotoxin. 70Z/3-CD14 cells were responsive to both insoluble and soluble peptidoglycan, as well as to low concentrations of endotoxin, as measured by the expression of surface IgM, activation of NF-kappaB, and degradation of IkappaB-alpha. Peptidoglycan also induced activation of NF-kappaB and degradation of IkappaB-alpha in macrophage RAW264.7 cells. These peptidoglycan-induced effects (in contrast to endotoxin-induced effects) were not inhibited by polymyxin B. Both peptidoglycan- and endotoxin-induced activation of NF-kappaB were inhibited by anti-CD14 mAb. The N-terminal 151 amino acids of CD14 were sufficient for acquisition of full responsiveness to both peptidoglycan and endotoxin, but CD14 deletion mutants lacking four small regions within the N-terminal 65 amino acids showed differentially diminished responses to peptidoglycan and endotoxin. These results identify CD14 as the functional receptor for peptidoglycan and demonstrate that similar, but not identical sequences in the N-terminal 65-amino acid region of CD14 are critical for the NF-kappaB and IgM responses to both peptidoglycan and endotoxin.

Assessment of parasite population dynamics in mixed infections of rodent plasmodia.

Cloned lines of the four rodent Plasmodium species can be differentiated by the RFLP pattern generated following Southern blotting and probing with PCsv4.1, a probe derived from a P. chabaudi chabaudi genomic library. Groups of CBA/Ca mice were inoculated simultaneously with cloned lines from two parasite species or strains. Six mixed species and three mixed strain infections using rodent malaria lines were initiated. The composition of the parasite population in each group was determined qualitatively and semi-quantitatively by analysis of the DNA purified from daily blood samples, thereby providing a dynamic representation of each mixed infection. Effects on the course of parasitaemias are presented and discussed.

Identification and quantification of rodent malaria strains and species using gene probes.

A DNA probe PCsv4 and a subclone thereof PCsv4.1, hybridize specifically to rodent malaria DNA. DNA purified from a small volume (10 microliters) of infected mouse blood was used to determine the composition of the parasite population present. The hybridization signal following PCsv4 probing of slot-blotted DNA correlated directly with parasitaemia. The hybridization pattern and intensity, resulting from probing restriction enzyme digested and Southern-blotted genomic DNA, determined the identity of the infecting parasite line(s), and provided a semi-quantitative measure of parasite burden. Fifteen parasite lines representative of all four Plasmodium species infecting rodents can be differentiated in this way.

MD-2 binds to bacterial lipopolysaccharide.

The exact roles and abilities of the individual components of the lipopolysaccharide (LPS) receptor complex of proteins remain unclear. MD-2 is a molecule found in association with toll-like receptor 4. We produced recombinant human MD-2 to explore its LPS binding ability and role in the LPS receptor complex. MD-2 binds to highly purified rough LPS derived from Salmonella minnesota and Escherichia coli in five different assays; one assay yielded an apparent KD of 65 nm. MD-2 binding to LPS did not require LPS-binding proteins LBP and CD14; in fact LBP competed with MD-2 for LPS. MD-2 enhanced the biological activity of LPS in toll-like receptor 4-transfected Chinese hamster ovary cells but inhibited LPS activation of U373 astrocytoma cells and of monocytes in human whole blood. These data indicate that MD-2 is a genuine LPS-binding protein and strongly suggest that MD-2 could play a role in regulation of cellular activation by LPS depending on its local availability.

Helicobacter pylori lipopolysaccharide can activate 70Z/3 cells via CD14.

Helicobacter pylori persistently colonizes the human gastrointestinal tract and is associated with chronic gastritis and, in some cases, peptic ulcer disease or gastric neoplasms. One factor in the persistence of this organism may be its inability to elicit a strong inflammatory response. Lipopolysaccharide (LPS) is a proinflammatory substance found in the cell walls of all gram-negative bacteria. H. pylori LPS has been found by several different measures to be less active than LPS from Enterobacteriaceae. This study addresses the role of CD14 and LPS-binding protein in the cellular response to H. pylori LPS. We report that H. pylori LPS activates mammalian cells expressing CD14 at much lower LPS concentrations than those for control cells not expressing CD14. The maximal activation of CD14-70Z/3 cells by H. pylori LPS also requires LPS-binding protein. H. pylori LPS at concentrations as high as 30 microg/ml does not elicit an interleukin-8 (IL-8) response from the epithelial cell line SW620 in the presence of CD14; 10 ng of Escherichia coli LPS per ml elicits a maximal IL-8 response. Furthermore, in contrast to some other types of LPS with little activity, H. pylori LPS does not inhibit the CD14-70Z/3 cell response to E. coli LPS. From these studies, we conclude that H. pylori LPS, though much less active than E. coli LPS, stimulates cells via CD14.

Structure-function analysis of CD14 as a soluble receptor for lipopolysaccharide.

CD14 is a glycophosphatidylinositol-linked protein expressed by myeloid cells and also circulates as a plasma protein lacking the glycophosphatidylinositol anchor. Both membrane and soluble CD14 function to enhance activation of cells by lipopolysaccharide (LPS), which we refer to as receptor function. We have previously reported the LPS binding and cell activation functions of a group of five deletion mutants of CD14 (Viriyakosol, S., and Kirkland, T.N. (1995) J. Biol. Chem. 270, 361-368). We have now studied the functional impact of these mutations on soluble CD14. We found that some deletions that abrogated LPS binding in membrane CD14 have no effect on LPS binding in soluble CD14. In fact, some of the soluble CD14 deletion mutants bound LPS with an apparent higher affinity than wild-type CD14. Furthermore, we found that all five deletions essentially ablated soluble CD14 LPS receptor function, whereas only two of the deletions completely destroyed membrane CD14 LPS receptor function. Some of the mutants were able to compete with wild-type CD14 in soluble CD14-dependent assays of cellular activation. We concluded that the soluble and membrane forms of CD14 have different structural determinants for LPS receptor function.