A hypothesis for the existence of two types of tuberculosis, reflecting two distinct types of immune failure to control the pathogen, based upon prevalence of mycobacterium-specific IgG subclasses.

Most people infected by Mycobacterium tuberculosis, about 90%, contain the pathogen and are healthy. Most investigators have concluded that pathogen-specific Th1 cells contribute to protection. Pulmonary tuberculosis, the most prevalent form of disease, is associated with destructive granulomas, the formation of which also appears to involve Th1 cells. In what sense then do the two Th1 components of the response, in healthy infected individuals and patients, differ? An insight into this question might provide clues for attaining effective vaccination and better treatment. We approached this question by examining the relative prevalence of different IgG isotypes among anti-mycobacterium-specific antibodies in patients and healthy infected individuals as a surrogate marker for the Th1/Th2 phenotype of the response. Our observations lead us to agree that healthy infected individuals generate a predominant Th1 response. Our observations also lead us to propose that many patients make a similar kind of response as healthy infected individuals, but that this response is too weak to contain the infection. We refer to such individuals as having type I tuberculosis. Other patients appear to have a greater and detrimental Th2 component to their immune response than that of healthy infected individuals. We refer to these individuals as having type II tuberculosis. This proposal that there are two types of tuberculosis, reflecting two distinct types of failure by the immune system, will, if correct, be pertinent to vaccine design, treatment of tuberculosis and in making further progress in our understanding the genetics of susceptibility to M. tuberculosis.

CC chemokine receptor (CCR)4 and the CCR10 ligand cutaneous T cell-attracting chemokine (CTACK) in lymphocyte trafficking to inflamed skin.

The chemokine thymus and activation-regulated chemokine (TARC; CCL17) is displayed by cutaneous (but not intestinal) venules, and is thought to trigger vascular arrest of circulating skin homing memory T cells, which uniformly express the TARC receptor CC chemokine receptor (CCR)4. Cutaneous T cell-attracting chemokine (CTACK; CCL27), expressed by skin keratinocytes, also attracts cutaneous memory T cells, and is hypothesized to assist in lymphocyte recruitment to skin as well. Here we show that chronic cutaneous inflammation induces CD4 T cells expressing E-selectin binding activity (a marker of skin homing memory cells) in draining lymph node, and that these E-selectin ligand+ T cells migrate efficiently to TARC and to CTACK. In 24 h in vivo homing assays, stimulated lymph node T cells from wild-type mice or, surprisingly, from CCR4-deficient donors migrate efficiently to inflamed skin; and an inhibitory anti-CTACK antibody has no effect on wild-type lymphocyte recruitment. However, inhibition with anti-CTACK monoclonal antibody abrogates skin recruitment of CCR4-deficient T cells. We conclude that CTACK and CCR4 can both support homing of T cells to skin, and that either one or the other is required for lymphocyte recruitment in cutaneous delayed type hypersensitivity.

Bacterial lipopolysaccharide rapidly inhibits expression of C-C chemokine receptors in human monocytes.

The present study was designed to investigate the effect of bacterial lipopolysaccharide (LPS) on C-C chemokine receptors (CCR) expressed in human mononuclear phagocytes. LPS caused a rapid and drastic reduction of CCR2 mRNA levels, which binds MCP-1 and -3. CCR1 and CCR5 mRNAs were also reduced, though to a lesser extent, whereas CXCR2 was unaffected. The rate of nuclear transcription of CCR2 was not affected by LPS, whereas the mRNA half life was reduced from 1.5 h to 45 min. As expected, LPS-induced inhibition of CCR2 mRNA expression was associated with a reduction of both MCP-1 binding and chemotactic responsiveness. The capacity to inhibit CCR2 expression in monocytes was shared by other microbial agents and cytokines (inactivated Streptococci, Propionibacterium acnes, and to a lesser extent, IL-1 and TNF-alpha). In contrast, IL-2 augmented CCR2 expression and MCP-1 itself had no effect. These results suggest that, regulation of receptor expression in addition to agonist production is likely a crucial point in the regulation of the chemokine system.

A member of the dendritic cell family that enters B cell follicles and stimulates primary antibody responses identified by a mannose receptor fusion protein.

Dendritic cells (DCs) are known to activate naive T cells to become effective helper cells. In addition, recent evidence suggests that DCs may influence naive B cells during the initial priming of antibody responses. In this study, using three-color confocal microscopy and three-dimensional immunohistograms, we have observed that in the first few days after a primary immunization, cells with dendritic morphology progressively localize within primary B cell follicles. These cells were identified by their ability to bind a fusion protein consisting of the terminal cysteine-rich portion of the mouse mannose receptor and the Fc portion of human immunoglobulin (Ig)G1 (CR-Fc). In situ, these CR-Fc binding cells express major histocompatibility complex class II, sialoadhesin, and CD11c and are negative for other markers identifying the myeloid DC lineage, such as (CD11b), macrophages (F4/80), follicular DCs (FDC-M2), B cells (B220), and T cells (CD4). Using CR-Fc binding capacity and flow cytometry, the cells were purified from the draining lymph nodes of mice 24 h after immunization. When injected into naive mice, these cells were able to prime T cells as well as induce production of antigen-specific IgM and IgG1. Furthermore, they produced significantly more of the lymphocyte chemoattractant, macrophage inflammatory protein (MIP)-1alpha, than isolated interdigitating cells. Taken together, these results provide evidence that a subset of DCs enters primary follicles, armed with the capacity to attract and provide antigenic stimulation for T and B lymphocytes.

A key role for CC chemokine receptor 4 in lipopolysaccharide-induced endotoxic shock.

CC chemokine receptor (CCR)4, a high affinity receptor for the CC chemokines thymus and activation-regulated chemokine (TARC) and macrophage-derived chemokine (MDC), is expressed in the thymus and spleen, and also by peripheral blood T cells, macrophages, platelets, and basophils. Recent studies have shown that CCR4 is the major chemokine receptor expressed by T helper type 2 (Th2) polarized cells. To study the in vivo role of CCR4, we have generated CCR4-deficient (CCR4(-/-)) mice by gene targeting. CCR4(-/-) mice developed normally. Splenocytes and thymocytes isolated from the CCR4(-/-) mice failed to respond to the CCR4 ligands TARC and MDC, as expected, but also surprisingly did not undergo chemotaxis in vitro in response to macrophage inflammatory protein (MIP)-1alpha. The CCR4 deletion had no effect on Th2 differentiation in vitro or in a Th2-dependent model of allergic airway inflammation. However, CCR4(-/-) mice exhibited significantly decreased mortality on administration of high or low dose bacterial lipopolysaccharide (LPS) compared with CCR4(+/+) mice. After high dose LPS treatment, serum levels of tumor necrosis factor alpha, interleukin 1beta, and MIP-1alpha were reduced in CCR4(-/-) mice, and decreased expression of MDC and MIP-2 mRNA was detected in peritoneal exudate cells. Analysis of peritoneal lavage cells from CCR4(-/)- mice by flow cytometry also revealed a significant decrease in the F4/80(+) cell population. This may reflect a defect in the ability of the CCR4(-/-) macrophages to be retained in the peritoneal cavity. Taken together, our data reveal an unexpected role for CCR4 in the inflammatory response leading to LPS-induced lethality.

Cloning and characterization of a specific receptor for the novel CC chemokine MIP-3alpha from lung dendritic cells.

Dendritic cells are potent antigen-presenting cells involved in the initiation of immune responses. The trafficking of these cells to tissues and lymph nodes is mediated by members of the chemokine family. Recently, a novel CC chemokine known as MIP-3alpha or liver and activation-regulated chemokine has been identified from the EMBL/GenBank/DDBJ expressed sequence tag database. In the present study, we have shown that the messenger RNA for MIP-3alpha is expressed predominantly in inflamed and mucosal tissues. MIP-3alpha produced either synthetically or by human embryonic kidney 293 cells is chemotactic for CD34(+)-derived dendritic cells and T cells, but is inactive on monocytes and neutrophils. MIP-3alpha was unable to displace the binding of specific CC or CXC chemokines to stable cell lines expressing their respective high affinity receptors, namely CCR1-5 and CXCR1 and CXCR2, suggesting that MIP-3alpha acts through a novel CC chemokine receptor. Therefore, we used degenerate oligonucleotide-based reverse transcriptase PCR to identify candidate MIP-3alpha receptors in lung dendritic cells. Our results show that the orphan receptor known as GCY-4, CKRL-3, or STRL-22 is a specific receptor for MIP-3alpha, and that its activation leads to pertussis toxin-sensitive and phospholipase C-dependent intracellular Ca2+ mobilization when it is expressed in HEK 293 cells.

A broad-spectrum chemokine antagonist encoded by Kaposi's sarcoma-associated herpesvirus.

Kaposi's sarcoma-associated herpesvirus encodes a chemokine called vMIP-II. This protein displayed a broader spectrum of receptor activities than any mammalian chemokine as it bound with high affinity to a number of both CC and CXC chemokine receptors. Binding of vMIP-II, however, was not associated with the normal, rapid mobilization of calcium from intracellular stores; instead, it blocked calcium mobilization induced by endogenous chemokines. In freshly isolated human monocytes the virally encoded vMIP-II acted as a potent and efficient antagonist of chemotaxis induced by chemokines. Because vMIP-II could inhibit cell entry of human immunodeficiency virus (HIV) mediated through CCR3 and CCR5 as well as CXCR4, this protein may serve as a lead for development of broad-spectrum anti-HIV agents.

Definition, function and pathophysiological significance of chemokine receptors.

Chemokines and their receptors are at the core of many processes in biology, from routine immunosurveillance and the inflammatory process, through to the infection of cells by HIV. In the past two years, various bioinformatic and cloning strategies have led to an explosion in the number of chemokines and receptors that have been identified. Although the picture is far from complete, several themes are emerging. In particular, there are important differences between observations in vitro, where there appears to be much redundancy, and studies in vivo, where distinct roles are clearer. In this review, Timothy Wells, Christine Power and Amanda Proudfoot discuss the chemokines and their receptors and recent data from immunological and virology studies, and speculate on the potential of interfering with the chemokine network as a useful approach to ameliorating disease.

Crystallization and preliminary X-ray diffraction studies of human RANTES.

The chemotactic cytokine RANTES (Regulated on Activation, Normal T-cell Expressed and Secreted) is a potent chemoattractant and activator of a number of leukocytes, with a molecular mass of 8 kDa. Crystals of this protein have been grown from 100 mM sodium acetate buffer (pH 4.6) containing 200 mM magnesium acetate, with 20% (w/v) PEG 4000 and 6% (v/v) glycerol. The crystals grow as thick rods, which diffract to at least 1.8 A resolution on a rotating anode X-ray source. The crystals belong to space group p2(1)2(1)2(1) with unit cell dimensions a = 95.14 A, b = 57.58 A and c = 24.01 A with alpha = beta = gamma = 90 degrees. The asymmetric unit contains two molecules of the RANTES monomer, with a VM of 2.0 A(3)/Da.

Chemokine receptors - the new frontier for AIDS research.

CD4 is widely known as the HIV receptor, but is insufficient to allow viral infection. Recently, members of the family of chemokine receptors have been identified as the missing co-receptors, which act with CD4 to allow the virus to enter cells. These discoveries open up the possibilities of novel therapeutic strategies to combat HIV infection and AIDS.

The chemokine system: novel broad-spectrum therapeutic targets.

Chemokines are cytokines that specifically direct the trafficking of immune cells in the body. They offer a novel point of therapeutic intervention, as inhibiting specific chemokines and receptors could prevent the excessive recruitment of leukocytes to sites of inflammation. This approach could be considered to act upstream of the therapies used today which, for the most part, act on the cells already at the site of inflammation. The receptors for chemokines are G-protein-coupled seven-transmembrane receptors, which are particularly tractable for the pharmaceutical industry. The search for small-molecule inhibitors of these receptors has been fruitful and the numbers of patents and, more recently, peer-reviewed publications are growing rapidly. The first clinical trial was initiated this year, so although it is too soon to be able to report these results we hope to see the outcome of this research in the near future.

Selectivity and antagonism of chemokine receptors.

The chemokine superfamily can be subdivided into two groups based on their amino terminal cysteine spacing. The CXC chemokines are primarily involved in neutrophil-mediated inflammation and, so far, two human receptors have been cloned. The CC chemokines tend to be involved in chronic inflammation, and recently we have cloned a fourth leukocyte receptor for this group of ligands. Understanding what makes one receptor bind its range of agonists is important if we are to develop potent selective antagonist. We have started to investigate the molecular basis of this receptor selectivity by looking at why CC chemokines do not bind to the CXC receptors in several ways. First, we looked at the role of the three-dimensional structure of the ligand, and have solved the three dimensional structure of RANTES using nuclear magnetic resonance spectroscopy. The structure is similar to that already determined for the CC chemokine macrophage inflammatory protein-1 beta, and it has a completely different dimer interface to that of the CXC chemokine interleukin-8 (IL-8). However, the monomer structures of all the chemokines are very similar, and at physiological concentrations the proteins are likely to be monomeric. Second, by examining all the known CC and CXC chemokines, we have found a region that differs between the two subfamilies. Mutations of one of the residues in this region, Leu-25 in IL-8, to tyrosine (which is conserved at this position in CC chemokines) enables the mutant IL-8 to bind CC chemokine receptor-1 (CC-CKR-1) and introduces monocyte chemoattractant activity. Using other mutations in this region, we can show a direct interaction with the N-terminus of CC-CKR-1. Third, we have found that modification of the amino terminus of RANTES by addition of one amino acid makes it into an antagonist with nanomolar potency. Taken together, this data suggests a two-site model for receptor activation and for selectivity between CC and CXC chemokines, with an initial receptor contact provided by the main body of the chemokine, and activation provided by the amino terminal region.

A chimeric MIP-1alpha/RANTES protein demonstrates the use of different regions of the RANTES protein to bind and activate its receptors.

Human RANTES (CCL5) and MIP-1alpha (CCL3) bind and activate several CC chemokine receptors. RANTES is a high-affinity ligand for CCR1 and CCR5, and it binds CCR3 with moderate affinity and CCR4 with low affinity. MIP-1alpha has similar binding characteristics to RANTES except that it does not bind to CCR3. Here we have generated a chimera of human MIP-1alpha and RANTES, called MIP/RANTES, consisting of the eight amino terminal residues of MIP-1alpha preceding the CC motif, and the remainder of the sequence is RANTES. The chimera is able to induce chemotaxis of human monocytes. MIP/RANTES has >100-fold reduction in binding to CCR1 and does not bind to CCR3 but retains full, functional binding to CCR5. It has equivalent affinity for CCR5 to MIP-1alpha and RANTES, binding with an IC(50) of 1.12 nM, and is able to mobilize calcium and induce endocytosis of CCR5 in PBMC in a manner equi-potent to RANTES. It also retains the ability to inhibit R5 using HIV-1 strains. Therefore, we conclude that the amino terminus of RANTES is not involved in CCR5 binding, but it is essential for CCR1 and CCR3.

Cloning of a full-length cDNA encoding the neutrophil-activating peptide ENA-78 from human platelets.

Here we describe the cloning of a full-length cDNA encoding a neutrophil chemoattractant peptide, ENA-78, from human platelets. The cDNA encodes a predicted sequence of 114 amino acids and contains the Cys motif C-X-C found in other members of the alpha-chemokine family which also includes interleukin 8 (IL-8). ENA-78 has a high degree of sequence identity with other platelet-derived chemokines which also share overlapping chemotactic activities such as GRO alpha and the neurophil-activating peptide 2 (NAP-2; derived by proteolytic cleavage of the connective-tissue-activating peptide III (CTAP-III)).

Characterisation of the RANTES/MIP-1 alpha receptor (CC CKR-1) stably transfected in HEK 293 cells and the recombinant ligands.

The CC chemokines RANTES and MIP-1 alpha are known to activate certain leucocytes and leucocytic cell lines. We have produced and fully characterised the recombinant proteins expressed in E. coli. They induce chemotaxis of the pro-monocytic cell line, THP-1 and T cells. THP-1 cells express three of the known CC chemokine receptors. In order to study the activation of a single receptor, we have expressed the shared receptor (CC CKR-1) for RANTES and MIP-1 alpha stably in the HEK 293 cell line. We have examined the effects of RANTES and MIP-1 alpha on the CC CKR-1 transfectants by equilibrium binding studies and in a chemotaxis assay. RANTES competes for [125I]RANTES with an IC50 of 0.6 +/- 0.23 nM, whereas MIP-1 alpha competes for its radiolabelled counterpart with an IC50 of 10 +/- 1.6 nM in the transfectants. These affinities are the same as those measured on the THP-1 cell line. The stably transfected HEK 293 cells respond to both these chemokines in the chemotaxis assay with the same EC50 values as those measured for THP-1 cells. This indicates that this cellular response can be mediated through the CC CKR-1 receptor.

Identification of the horse epidermal growth factor (EGF) coding sequence and its use in monitoring EGF gene expression in the endometrium of the pregnant mare.

The PCR technique and highly degenerate oligonucleotide primers were used to amplify a 282 bp fragment of the horse (Equus caballus) epidermal growth factor (EGF) cDNA. The clone corresponded to 94 amino acids of the EGF precursor molecule. The deduced amino acid sequence of the 53 residue EGF mitogenic peptide within the precursor sequence showed 60-70% identity with five other published EGF sequences. The PCR cDNA fragment hybridized to a 4.9 kb transcript in horse kidney and endometrial RNA which was of a similar size to the mature EGF transcript found in other mammalian species. The horse cDNA clone was used in Northern blots to monitor EGF expression in the endometrium of pregnant mares up to day 83 of gestation (term = 330-340 days). The level of expression increased from day 33 and showed a further dramatic increase between days 35 and 45, which coincides with the onset of implantation and placentation in this species. Levels remained elevated up to day 83. The horse DNA sequence was used to design sense and antisense oligonucleotide probes (45-mers) for in situ hybridization studies. The antisense probe showed specific hybridization to the glandular, but not lumenal, epithelial cells of the endometrium and there was no signal in fetal membranes. The in situ hybridization signal increased between days 35 and 45 to a similar degree to that observed in the Northern blot analysis. This dramatic increase in EGF expression in the glandular epithelium of the mare's endometrium during pregnancy may provide a mitogenic stimulus to the endometrium and/or trophoblast to facilitate placental differentiation and attachment. Alternatively, the precursor could be involved in the endometrial gland secretory process which is necessary to produce uterine milk for fetal sustenance. The PCR cloning methods used in this study should be generally applicable to the cloning of EGF cDNAs from other species.

A valid ELISPOT assay for enumeration of ex vivo, antigen-specific, IFNgamma-producing T cells.

We describe an ELISPOT technique for the detection of antigen specific IFNgamma-producing T cells. The technique is performed on spleen cells plated directly ex vivo into ELISPOT trays without an in vitro pre-culture step. Thus, the assay is likely to reflect the in vivo activity of the cells. We have found that very high cell densities (at least 10(6) cells/well) are required for optimal detection of spot forming cells, and only at a high density of cells is the number of spots detected linearly related to the number of primed cells plated. If lower numbers of antigen primed cells are used, then unprimed spleen cells from syngeneic mice can be added to the well to increase the cell density. Under these conditions, we find that the number of spots is linearly proportional to the number of primed cells plated, even if these are well below a million cells/well. Experiments with MHC congenic mice indicate that the high density of spleen cells required to obtain optimal spot formation reflects a requirement for an MHC restricted function, probably efficient antigen presentation to T cells. The formation of IFNgamma spots is antigen dependent and abrogated by depleting the antigen primed cells of T cells. We conclude that this linear assay can be used to efficiently detect ex vivo antigen-specific IFNgamma-producing T cells.

Chemokine receptors and their role in leukocyte activation.

Chemokines were originally isolated based on their abilities to selectively attract and recruit leukocyte populations. Over the last few years there has been an explosion in the number of new chemokines identified, and as a result many receptors previously considered to be orphans have now been paired up with their ligands. Here we review some of the latest results in this area, illustrating with data from our laboratory. The central question from a drug discovery perspective, is to show whether inhibiting chemokine receptors leads to a change in disease status. Although we are still a long way from having candidate molecules to take into the clinic, a flavour of what may be possible can be inferred from mutant chemokines with antagonistic properties. We discuss recent data using two of these proteins, Met-RANTES which has anti-inflammatory properties, and AOP-RANTES which has been shown to prevent infection of macrophages and T-cells by M-tropic HIV strains.

Effect of a CC chemokine receptor antagonist on collagen induced arthritis in DBA/1 mice.

Chemokines are small proteins that selectively activate and recruit leukocytes to sites of inflammation. Several of them, including the CC chemokines RANTES, MIP-1 alpha, MIP-1 beta, MCP-1, and the CXC chemokines IL-8, GRO-alpha, ENA-78 have been identified in rheumatoid synovium, implicating a potential role for these molecules in rheumatoid arthritis. We have investigated the expression patterns of CC chemokine receptors in the joints of mice with collagen-induced arthritis, a model for human rheumatoid arthritis. In addition, we have investigated the incidence and severity of arthritis in mice receiving administration of MetRANTES, a modified chemokine which is a nanomolar antagonist of certain CC chemokine receptors. The mRNA expression pattern of the chemokines and their receptors in the joints of arthritic mice was investigated using reverse transcriptase-PCR and in situ hybridization. An upregulation of the CC chemokine receptors mCCR1, mCCR2; mCCR3 and mCCR5 was found in the joints from arthritic mice, compared to control animals. In addition, injections of MetRANTES reduced the incidence of disease in a dose dependent manner. Furthermore, in MetRANTES-treated mice that did develop arthritis a significantly lower severity of disease was observed compared with control animals. Our data clearly demonstrate a role for CC chemokines and their receptors in inflammatory joint destruction and support the use of chemokine receptor antagonists as potential tools to control inflammatory diseases such as rheumatoid arthritis.

Repeatability of the Petrifilm HEC test and agreement with a hydrophobic grid membrane filtration method for the enumeration of Escherichia coli O157:H7 on beef carcasses.

The Petrifilm HEC test (3M Canada Inc., London, Ontario), a quantitative microbiological test for Escherichia coli O157:H7, was evaluated for its performance as a beef-carcass monitoring test. Test repeatability and agreement and agreement with an E. coli O157:H7 detection method using a hydrophobic grid membrane filter (HGMF) overlaid onto cefixime-tellurite-sorbitol MacConkey agar (CT-SMAC) followed by a latex agglutination test for the O157 antigen were determined by using pure cultures of E. coli O157:H7, beef samples experimentally contaminated with bovine feces containing E. coli O157:H7, and naturally contaminated beef carcasses of unknown E. coli O157:H7 status from a local abattoir. The Petrifilm HEC test showed excellent repeatability and excellent agreement with the HGMF-CT-SMAC method when test samples were obtained from pure cultures and experimentally contaminated meat. All 125 naturally contaminated beef carcasses surveyed were negative for E. coli O157:H7 with both microbial methods. The Petrifilm HEC test, however, demonstrated a significantly lower proportion of cross-reactive organisms (false-positive reactions) than the HGMF-CT-SMAC method. Given the performance of this test coupled with its ease of use and compact size, it shows considerable promise for carcass testing where abattoir laboratory facilities are limited and as a substitute for more complex laboratory testing methods used in established laboratories.