Role of chemokines and formyl peptides in pneumococcal pneumonia-induced monocyte/macrophage recruitment

Host-derived chemoattractant factors are suggested to play crucial roles in leukocyte recruitment elicited by inflammatory stimuli in vitro and in vivo. However, in the case of acute bacterial infections, pathogen-derived chemoattractant factors are also present, and it has not yet been clarified how cross-talk between chemoattractant receptors orchestrates diapedesis of leukocytes in this context of complex chemoattractant arrays. To investigate the role of chemokine (host-derived) and formyl peptide (pathogen-derived) chemoattractants in leukocyte extravasation in life-threatening infectious diseases, we used a mouse model of pneumococcal pneumonia. We found an increase in mRNA expression of eight chemokines (RANTES, macrophage-inflammatory protein (MIP)-1alpha, MIP-1beta, MIP-2, IP-10, monocyte chemoattractant protein (MCP)-1, T cell activation 3, and KC) within the lungs during the course of infection. KC and MIP-2 protein expression closely preceded pulmonary neutrophil recruitment, whereas MCP-1 protein production coincided more closely than MIP-1alpha with the kinetics of macrophage infiltration. In situ hybridization of MCP-1 mRNA suggested that MCP-1 expression started at peribronchovascular regions and expanded to alveoli-facing epithelial cells and infiltrated macrophages. Interestingly, administration of a neutralizing Ab against MCP-1, RANTES, or MIP-1alpha alone did not prevent macrophage infiltration into infected alveoli, whereas combination of the three Abs significantly reduced macrophage infiltration without affecting neutrophil recruitment. The use of an antagonist to N-formyl peptides, N-t-Boc-Phe-D-Leu-Phe-D-Leu-Phe, reduced both macrophages and neutrophils significantly. These data demonstrate that a complex chemokine network is activated in response to pulmonary pneumococcal infection, and also suggest an important role for fMLP receptor in monocyte/macrophage recruitment in that model.     

Sensing of environmental signals: classification of chemoreceptors according to the size of their ligand binding regions

Central to the different forms of taxis are methyl‐accepting chemotaxis proteins (MCPs). The increasing number of genome sequences reveals that MCPs differ enormously in sequence, topology and genomic abundance. This work is a one‐by‐one bioinformatic analysis of the almost‐totality of MCP genes available and a classification of motile bacteria according to their lifestyle. On average, motile archaea have 6.7 MCP genes per genome whereas motile bacteria have more than twice as much. We show that the number of MCPs per genome depends on bacterial lifestyle and metabolic diversity, but weakly on genome size. Signal perception at an MCP occurs at the N‐terminal ligand binding region (LBR). Here we show that around 88% of MCPs possess an LBR that remains un‐annotated in SMART. MCPs can be classified into two clusters according to the size of the LBR. Cluster I receptors have an LBR between 120 and 210 amino acids whereas cluster II receptors have larger LBRs of 220–299 amino acids. There is evidence that suggests that some cluster II LBRs are composed of two cluster I LBRs. Further evidence indicates that other cluster II LBRs might harbour novel sensor domains. Cluster II receptors are dominant in archaea whereas cluster I receptors are prevalent in bacteria. MCPs can be classified into six different receptor topologies and this work contains a first estimation of the relative abundance of different receptor topologies in bacteria and archaea. Topologies involving extracytoplasmic sensing are prevalent in bacteria whereas topologies with cytosolic signal recognition are abundant in archaea.     

Molecular cloning of human macrophage capping protein cDNA. A unique member of the gelsolin/villin family expressed primarily in macrophages.

Macrophage capping protein (MCP) is a Ca(2+)-sensitive protein which reversibly blocks the barbed ends of actin filaments but does not sever preformed actin filaments. The human cDNA for MCP has been cloned and sequenced. The derived amino acid sequence predicts a polypeptide of 38.4 kDa. Human MCP expressed in Escherichia coli using a pET12a vector was functionally identical to the native protein purified from rabbit alveolar macrophages with respect to Ca2+ sensitivity and ability to block monomer exchange at the barbed end of actin filaments. Sequence comparison with other actin-binding protein sequences indicates that MCP is a member of the gelsolin/villin family of barbed end blocking proteins. Unlike gelsolin, this protein has a limited tissue distribution being detected primarily in macrophages where it was abundant, representing 0.9-1% of the total cytoplasmic protein. Northern blot analysis of U937 and HL60 cells differentiated to macrophage-like cells demonstrated that MCP message increases to 2.6 and greater than 7 times initial levels, respectively. Human MCP displays a 93% amino acid sequence identity with two recently described mouse proteins, gCap39 and Mbh1. Its abundance in macrophages and the corresponding increases in mRNA levels upon promyelocyte and monocyte development into macrophages indicate that MCP may play an important role in macrophage function.     

Isolation of cDNA clones coding for the alpha-subunit of human beta-hexosaminidase. Extensive homology between the alpha- and beta-subunits and studies on Tay-Sachs disease

The lysosomal beta-hexosaminidases (N-acetyl-beta-glucosaminidase, EC 3.2.1.30) occur as two major isozymes, hexosaminidase A (alpha beta a beta b) and hexosaminidase B (2(beta a beta b)). To facilitate the investigations of the biosynthesis and structure of the enzymes and the nature of mutation in Tay-Sachs disease, we have isolated cDNA clones coding for the alpha-subunit. The polypeptide chains of hexosaminidase A (30 mg) were digested with trypsin, and peptides were isolated by reverse phase high pressure liquid chromatography and their amino acid sequences determined. One of alpha-chain peptides contained a string of seven amino acids from which two sets of oligonucleotides were specified. They were used to screen the SV40-transformed human fibroblast cDNA library of Okayama and Berg. Three cDNA clones, designated pHexA, identified from among 5 X 10(5) clones screened, contained the deduced amino-acid sequences of five alpha-chain peptides. Genomic DNA homologous to pHexA cDNA mapped to human chromosome 15 in somatic cell hybrids, as expected for the pre-alpha-polypeptide. Two of the clones contained identical polyadenylation sites, while the third was polyadenylated about 450 base pairs downstream. The two types of clones were found to correspond to a major 2.0-kilobase pair and a minor 2.3-kilobase pair mRNA species. Blot hybridizations of mRNA and DNA from Tay-Sachs variant fibroblasts revealed absence or reduction of levels of both mRNA species among infantile and juvenile variants, but no observable DNA alterations. Alignment of the pre-alpha- and pre-beta-polypeptides revealed 55% nucleotide and 57% amino acid homology. These data suggest a common origin of the HEXA and HEXB genes and account for the similar substrate specificities of the alpha-dimer subunit, hexosaminidase S, and hexosaminidase B.     

The effect of MCP-1 depletion on chemokine and chemokine-related gene expression: evidence for a complex network in acute inflammation

The expression of chemokines has been suggested to involve an interdependent network, with the absence of a single chemokine affecting the expression of multiple other chemokines. Monocyte chemoattractant protein (MCP-1), a member of C-C chemokine superfamily, plays a critical role in the recruitment and activation of leukocytes during acute inflammation. To examine the effect of the loss of MCP-1 on expression of the chemokine network, we compared the mRNA expression profiles of MCP-1(-/-) and wild type mice during the acute inflammatory phase of excisional wounds. Utilizing a mouse cDNA array containing 514 chemokine and chemokine related genes, the loss of MCP-1 was observed to cause a significant upregulation of nine genes (Decorin, Persephin, IL-1beta, MIP-2, MSP, IL1ra, CCR5, CCR3, IL-11) and significant downregulation of two genes (CCR4 and CD3Z) in acute wounds. The array data was confirmed by semi-quantitative RT-PCR. The effect of MCP-1 deletion on chemokine expression was further examined in isolated macrophages. Compared to wild type, LPS-stimulated peritoneal macrophages from MCP-1(-/-) mice showed a significant increase in the expression of RANTES, MIP-1beta, MIP-1alpha and MIP-2 mRNA. The data suggest that loss of a single chemokine perturbs the chemokine network not only in the setting of acute inflammation but even in an isolated inflammatory cell, the macrophage.     

Metallothionein cDNA, promoter, and genomic sequences of the tropical green mussel, Perna viridis.

The primary structure of the cDNA and metallothionein (MT) genomic sequences of the tropical green mussel (Perna viridis) was determined. The complete cDNA sequences were obtained using degenerate primers designed from known metallothionein consensus amino acid sequences from the temperate species Mytilus edulis. The amino acid sequences of P. viridis metallothionein deduced from the coding region consisted of 72 amino acids with 21 cysteine residues and 9 Cys-X-Cys motifs corresponding to Type I MT class of other species. Two different genomic sequences coding for the same mRNA were obtained. Each putative gene contained a unique 5'UTR and two unique introns located at the same splice sites. The promoters for both genes were different in length and both contained metal responsive elements and active protein-binding sites. The structures of the genomic clones were compared with those of other species. J. Exp. Zool. 284:445-453, 1999.     

Human monocyte chemoattractant protein-1 (MCP-1). Full-length cDNA cloning, expression in mitogen-stimulated blood mononuclear leukocytes, and sequence similarity to mouse competence gene JE

The purpose of this work was to analyze cDNA encoding human monocyte chemoattractant protein-1 (MCP-1), previously isolated from glioma cell line culture fluid. Screening of a cDNA library from total poly(A) RNA of glioma cell line U-105MG yielded a clone that coded for the entire MCP-1. Nucleotide sequence analysis and comparison with the amino acid sequence of purified MCP-1 showed that the cDNA clone comprises a 53-nucleotide 5'-non-coding region, an open reading frame coding for a 99-residue protein of which the last 76 residues correspond exactly to pure MCP-1, and a 389-nucleotide 3'-untranslated region. The hydrophobicity of the first 23 residues is typical of a signal peptide. Southern blot analysis of human and animal genomic DNA showed that there is a single MCP-1 gene, which is conserved in several primates. MCP-1 mRNA was induced in human peripheral blood mononuclear leukocytes (PBMNLs) by PHA, LPS and IL-1, but not by IL-2, TNF, or IFN-gamma. Among proteins with similar sequences, the coding regions of MCP-1 and mouse JE show 68% identity. This suggest that MCP-1 is the human homologue of the mouse competence gene JE.     

A novel murine beta -defensin expressed in tongue, esophagus, and trachea

beta-Defensins are broad spectrum antimicrobial peptides expressed at epithelial surfaces. Two human beta-defensins, HBD-1 and HBD-2, have been identified. In the lung, HBD-2 is an inducible product of airway epithelia and may play a role in innate mucosal defenses. We recently characterized rat homologs (RBD-1, RBD-2) of the human genes and used these sequences to identify novel mouse genes. Mouse beta-defensin-4 (MBD-4) was amplified from lung cDNA using polymerase chain reaction primers designed from conserved sequences of RBD-2 and HBD-2. A full-length cDNA was cloned which encodes a putative peptide with the sequence MRIHYLLFTFLLVLLSPLAAFTQIINNPITCMTNGAICWGPCPTAFRQIGNCGHFKVRCCKIR. The peptide shares approximately 40% identity with HBD-2. MBD-4 mRNA was expressed in the esophagus, tongue, and trachea but not in any of 20 other tissues surveyed. Cloning of the genomic sequence of MBD-4 revealed two nearly (>99%) identical sequences encoding MBD-4 and the presence of numerous additional highly similar genomic sequences. Radiation hybrid mapping localized this gene to a region of chromosome 8 near several other defensins, MBD-2, MBD-3, and alpha-defensins (cryptdins)-3 and -17, consistent with a gene cluster. Our genomic cloning and mapping data suggest that there is a large beta-defensin gene family in mice. Identification of murine beta-defensins provides an opportunity to understand further the role of these peptides in host defense through animal model studies and the generation of beta-defensin-deficient animals by gene targeting.     

Molecular cloning, nucleotide sequence, and tissue distribution of malonyl-CoA decarboxylase

Malonyl-CoA decarboxylase was purified from goose uropygial gland, reduced, carboxymethylated, and digested with trypsin. Several peptides were purified by high performance liquid chromatography and their amino acid sequences determined. Oligonucleotide probes were prepared based on their amino acid sequences. Size-selected RNA from the goose uropygial gland was used to construct cDNA libraries in lambda gt11 and pUC9 vectors. Immunological screening of the lambda gt11 cDNA library yielded one clone, lambda DC1, which contained a 2.2-kilobase pair insert; hybridization with the synthetic oligonucleotide probes confirmed its identity as malonyl decarboxylase. Screening of the pUC9 cDNA library with the insert of lambda DC1 as a probe detected one clone, pDC2, with an insert of 2.9 kilobase pairs. The nucleotide sequences of the two cDNAs revealed an open reading frame encoding a polypeptide of 462 amino acids. The deduced amino acid sequence was confirmed as malonyl-CoA decarboxylase by matching it to the amino acid sequences of three tryptic peptides derived from mature enzyme. Northern blot analysis of mRNA from goose brain, kidney, liver, lung, and gland revealed malonyl-decarboxylase mRNA of 3000 nucleotides. Since clone pDC2 contains a 2928-nucleotide insert, it represents nearly the full length of mRNA. Brain, kidney, lung, and liver contained less than 1% of the malonyl-CoA decarboxylase mRNA in the gland. Southern blot analysis of genomic DNA showed a single band in both liver and gland, suggesting that malonyl-CoA decarboxylase is a single copy gene.     

Characterization of lipopolysaccharide-induced macrophage gene expression

A cDNA library from LPS-treated murine peritoneal macrophages has been screened by differential hybridization with radiolabeled cDNA from untreated and LPS-treated macrophages. Six clones hybridizing with mRNA sequences present in LPS-treated cells but not in controls were selected for further characterization. When the recombinant bacteriophage DNA from each clone was used as a probe in Northern analysis of total RNA from LPS-treated macrophages, inducible mRNA ranging from 1.45 to 6.4 kb were seen. In five of six cases, the mRNA expression was undetectable in untreated macrophage cultures. All but one clone identified mRNA that were inducible even in the presence of cycloheximide, indicating the independence of such gene expression from protein synthesis; none of the genes were superinduced by this treatment. The time course of expression differed among the individual genes. Four were induced transiently, whereas two showed stable increasing accumulation through an 8-h period after stimulation. In addition, four of the genes were seen within 30 min of stimulation, whereas two were seen only after 2 to 4 h. Two genes were induced only by treatment with LPS, whereas four were also induced in response to other agents, including IFN-gamma, macrophage CSF, and PMA. The insert sequences from these recombinant clones did not hybridize with a set of cDNA encoding other inducible gene products, including TNF, IL-1, ornithine decarboxylase, c-myc, c-fos, JE, or KC. Thus, these six cDNA appear to encode inducible macrophage genes that are distinct from one another as well as from a selection of previously described early genes. Although their functional identity remains indeterminate, they may encode previously described early proteins induced in macrophages treated with LPS.     

Identification and characterization of a new gene family induced during macrophage activation.

In this report, we describe the primary structure and regulation of two novel IFN-gamma-inducible genes expressed during the process of macrophage activation. We used the RAW 264.7 cell line to prepare a cDNA library, from which inducible genes were selected by differential hybridization. Two cDNA clones, mag-1 and mag-2 (for macrophage-activation gene-1 and -2), were induced by IFN-gamma treatment in both RAW 264.7 cells and thioglycolate-elicited peritoneal macrophages, but not in the noncytolytic cell line, WEHI-3. A comparison of the nucleotide and deduced amino acid sequences of clones mag-1 and mag-2 with sequences in available data bases revealed no homologs. However, comparison of mag-1 and mag-2 sequences with each other revealed that these genes are homologous, with conserved residues concentrated at the amino terminus. Kinetic analyses revealed similar temporal patterns of induction of mRNA expression for these genes after IFN-gamma treatment. In addition, the genes showed distinct response patterns to the macrophage-activating stimuli IFN-gamma and LPS used either alone or in combination. Analysis of a panel of cell types of various lineages demonstrated that expression of these genes was associated with cellular activation in multiple cell types. As a result of the sequence similarities between these genes, we propose that they define a new family of IFN-gamma-regulated genes in macrophages.     

Monocyte chemotactic protein-1 and -2 messenger ribonucleic acids in the ovine uterus: regulation by pregnancy, progesterone, and interferon-tau

Endometrial leukocytes may play important roles during pregnancy. Because chemokines are regulators of immune cell activity and trafficking, this study determined if mRNAs for monocyte chemotactic proteins (MCP) were present in the ovine uterus and regulated by progesterone (P) and/or recombinant ovine interferon tau (roIFN-tau). Uteri of normal cycling and pregnant ewes (experiment 1) and uteri of ovariectomized ewes receiving intrauterine infusions of IFN-tau and/or i.m. injections of P (experiment 2) were used to detect MCP-1 and MCP-2 mRNA. In experiment 1, slot-blot hybridization analysis of endometrial total RNA revealed that MCP-1 and MCP-2 mRNA levels did not change during the estrous cycle but increased between Days 13 and 19 of pregnancy. Using in situ hybridization, MCP-1 and MCP-2 mRNA were localized to immune cells in the subepithelial compact stroma. Histomorphological studies and in situ hybridization for major basic protein (MBP) indicated that MCP-positive immune cells were eosinophils. In experiment 2, treatment with P and roIFN-tau increased (P < 0.05) the number of MCP-1- and MCP-2-expressing eosinophils in the endometrium compared to ewes treated with P alone. Injection of the P receptor antagonist (ZK 137,316) inhibited effects of P and/or roIFN-tau to recruit eosinophils expressing MCP-1 and MCP-2 mRNAs. Endometrial production of MCPs by eosinophils during early pregnancy may play a role(s) in central implantation and/or placentation in ewes that is crucial for successful establishment of pregnancy.     

Differential expression of the human thymosin-beta 4 gene in lymphocytes, macrophages, and granulocytes

A cDNA clone encoding human thymosin-beta 4 was isolated from a cDNA library prepared from peripheral blood leukocytes of a patient with acute lymphocytic leukemia. This clone contained the entire coding sequence of 43 amino acid residues of thymosin-beta 4 and had an initiation codon and two termination codons. The amino acid and nucleotide sequences in the coding region were well conserved between rat and human. Nine of 132 nucleotides were different in the coding sequences (93% homology), but the deduced amino acid sequences were identical. No signal peptide was found in the deduced protein sequence. Human thymosin-beta 4 mRNA, approximately 830 nucleotides in length, was about 30 nucleotides larger than rat thymosin-beta 4 mRNA. Expression of the human thymosin-beta 4 gene in various primary myeloid and lymphoid malignant cells and in a few human hemopoietic cell lines was studied. Northern blot analyses of different neoplastic B lymphocytes revealed that steady state levels of thymosin-beta 4 mRNA varied as a function of differentiation stage. Thymosin-beta 4 mRNA levels were decreased in myeloma cells as are class II human leukocyte antigen, Fc receptor, and complement receptor, suggesting a relationship between thymosin-beta 4 and the immune response. Thymosin-beta 4 mRNA was more highly expressed in mature granulocytes than in immature blastic cells. Treatment of THP-1 cells, a human monocytic cell line, with recombinant human interferon-lambda reduced the levels of thymosin-beta 4 mRNA. Its level decreased after differentiation of THP-1 cells into Ia+ macrophages, but increased after differentiation of HL-60 cells into Ia- macrophages. The pattern of thymosin-beta 4 gene expression suggests that it may play a fundamental role in the host defense mechanism.     

Construction and characterization of normalized cDNA library of maize inbred Mo17 from multiple tissues and developmental stages

Comprehensive complementary DNA (cDNA) library is a valuable resource for functional genomics. In this study, we set up a normalized cDNA library of Mo17 (MONL) by saturation hybridization with genomic DNA, which contained expressed genes of eight tissues and organs from inbred Mo17 of maize (Zea mays L.). In this library, the insert sizes range from 0.4 kb to 4 kb and the average size is 1.18 kb. 10.830 clones were spotted on nylon membrane to make a cDNA microarray. Randomly picked 300 clones from the cDNA library were sequenced. The cDNA microarry was hybridized with pooled tissue mRNA probes or housekeeping gene cDNA probes. The results showed the normalized cDNA library comprehensively includes tissue-specific genes in which 71% are unique ESTs (expressed sequence tags) based on the 300 sequences analyzed. Using BLAST program to compare the sequences against online nucleotide databases, 88% sequences were found in ZmDB or NCBI, and 12% sequences were not found in existing nucleotide databases. More than 73% sequences are of unknown function. The library could be extensively used in developing DNA markers, sequencing ESTs, mining new genes, identifying positional cloning and candidate gene, and developing microarrays in maize genomics research.     

Primary structures of MCP-1 and MCP-2, natural peptide antibiotics of rabbit lung macrophages

The microbicidal peptides, MCP-1 and MCP-2, of rabbit alveolar macrophages were purified by an improved procedure that employed preparative gel electrophoresis and high performance liquid chromatography. The peptides were arginine- and cystine-rich and lacked free sulfhydryl groups and detectable levels of carbohydrate. Complete sequence determinations revealed that MCP-1 differed from MCP-2 only by the substitution of arginine for leucine at residue 13 from the NH2 terminus and that the molecules were each single chain polypeptides of 33 amino acid residues containing three intramolecular disulfide bonds. The complete amino acid sequences of MCP-1 and MCP-2 are: (sequence in text)     

Isolation and Identification of Novel Terpene Lactones from Quince Fruit (Cydonia oblonga Mill., Marmelo)

A glucoamylase gene has been cloned from a Rhizopus genomic DNA library using synthetic oligonucleotides corresponding to the amino acid sequence of the glucoamylase. Since this glucoamylase gene was not expressed in yeast cells, we have cloned a glucoamylase gene from a cDNA library prepared from Rhizopus mRNA. Sequence analysis of both glucoamylase genes revealed that the genomic gene contained 4 intervening sequences and the cDNA gene lacked 145 nucleotides corresponding to the N-terminal region. The glucoamylase consists of 604 amino acids including a putative signal peptide and its molecular weight was calculated to be 65,000. The glucoamylase gene to be expressed in yeast cells was constructed by recombination of both genes. The yeast cells containing this constructed glucoamylase gene secreted the glucoamylase into the culture fluid and grew at almost the normal rate on a medium containing starch as the sole carbon source.     

Genomic organization of four novel nondisulfide-bridged peptides from scorpion Mesobuthus martensii Karsch: gaining insight into evolutionary mechanism

At least 25 nondisulfide-bridged peptides (NDBPs) have been identified and characterized from scorpions. However, the genomic organization of the genes that encode these peptides have not been reported yet. BmKa1, BmKa2 and BmKb1 are three novel genes that code for NDBPs identified by our group from Mesobuthus martensii Karsch. Based on their cDNA sequences, the genomic DNA sequences encoding these peptides were obtained using the PCR method. Sequence analysis showed that three distinct genomic structural patterns are used to encode these three peptides. The BmKa1 gene is not interrupted by any introns. However, the BmKa2 gene is composed of two exons, interrupted by a 67 bp intron that is located in the DNA region encoding the mature peptide. Two genomic homologues of the BmKb1 cDNA sequence, named BmKb1′ and BmKb2, respectively, were obtained. The BmKb1′ gene contains one intron of 593 bp, inserted into the DNA region that encodes the signal peptide. Similarly, the BmKb2 gene also contains an intron that interrupts the exon that encodes the NDBP signal peptide. The amino acid sequences deduced for BmKb2 and BmKb1′ differ only at one position. The data suggest that the genomic organizational pattern of NDBPs displays more divergence than that exhibited by the genes that encode disulfide-bridged peptides from scorpions.