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Segmental copy-number polymorphisms (CNPs) represent a significant component of human genetic variation and are likely to contribute to disease susceptibility. These potentially multiallelic and highly polymorphic systems present new challenges to family-based genetic-analysis tools that commonly assume codominant markers and allow for no genotyping error. The copy-number quantitation (CNP phenotype) represents the total number of segmental copies present in an individual and provides a means to infer, rather than to observe, the underlying allele segregation. We present an integrated approach to meet these challenges, in the form of a graphical model in which we infer the underlying CNP phenotype from the (single or replicate) quantitative measure within the analysis while assuming an allele-based system segregating through the pedigree. This approach can be readily applied to the study of any form of genetic measure, and the construction permits extension to a wide variety of hypothesis tests. We have implemented the basic model for use with nuclear families, and we illustrate its application through an analysis of the CNP located in gene CCL3L1 in 201 families with asthma.

Recent advances in the field of innate immunity have driven an important reappraisal of the role of these processes in airway disease. Various strands of evidence indicate that resident cells, such as macrophages and epithelial cells, have central importance in the initiation of inflammation. Macrophage activation has the potential to regulate not just typical aspects of innate immunity but also, via a variety of intricate cell-cell networks, adaptive responses and responses characterized by Th2-type cytokine production. In turn, such adaptive immune processes modify the phenotype and function of the innate immune system. Cooperative responses between monocytic cells and tissue cells are likely to be crucial to the generation of effective inflammatory responses, and a realization of the importance of these networks is providing a new way of identifying antiinflammatory therapies. Importantly, the repeated cycles of allergic and nonallergic inflammation that comprise chronic human airway disease are not necessarily well described by current terminology, and we propose and describe a concept of contiguous immunity, in which continual bidirectional cross-talk between innate and adaptive immunity describes disease processes more accurately.

Traditionally, PGD(2) has been considered to be a pro-inflammatory mediator, acting via classical PG receptors, such as the PGD(2) receptor (DP). PGD(2) is degraded rapidly in vitro and in vivo to a variety of metabolites, the majority of which were thought, until recently, to be physiologically inactive. Several "inactive" metabolites, particularly 15d-PGJ(2), have been shown to have wide-ranging effects on leukocytes and other cell types, however, and a potentially important anti-inflammatory role for PGD(2) has now been recognized, and the complexity of PGD(2) signaling is beginning to be elucidated. PGD(2) and its metabolites are biologically active over a broad concentration range, and, intriquingly, it appears that there are marked concentration-dependent variations in the consequences of signaling by these eicosanoids, which have the potential to exert pro- and anti-inflammatory effects. For example, the actions of PGD(2) can influence multiple stages in the life of the mature eosinophil, from causing its release from the bone marrow to inducing its recruitment and activation and, ultimately, regulating its apoptosis. This review is concerned with the diverse responses induced in leukocytes by PGD(2) and its metabolites and the signaling mechanisms which are thought to be responsible for them.

Although there is overwhelming pressure from funding agencies and the general public for scientists to bridge basic and translational studies, the fact remains that there are significant hurdles to overcome in order to achieve this goal. The purpose of this Opinion article is to examine the nature of these hurdles and to provide food for thought on the main obstacles that impede this process.

Viral and bacterial pathogens cause inflammation via Toll-like receptor (TLR) signaling. We have shown that effective responses to LPS may depend on cooperative interactions between TLR-expressing leukocytes and TLR-negative tissue cells. The aim of this work was to determine the roles of such networks in response to agonists of TLRs associated with antiviral and autoimmune responses. The TLR3 agonist poly(I:C) activated epithelial cells, primary endothelial cells, and two types of primary human smooth muscle cells (airway [ASMC] and vascular) directly, while the TLR7/8 agonist R848 required the presence of leukocytes to activate ASMC. In keeping with these data, ASMC expressed TLR3 but not TLR7 or TLR8. Activation of ASMC by poly(I:C) induced a specific cytokine repertoire characterized by induction of CXCL10 generation and the potential to recruit mast cells. We subsequently explored the ability of TLR agonists to cooperate in the induction of inflammation. Dual stimulation with LPS and poly(I:C) caused enhanced cytokine generation from epithelial and smooth muscle cells when in the presence of leukocytes. Thus, inflammatory responses to pathogens are regulated by networks in which patterns of TLR expression and colocalization of tissue cells and leukocytes are critical.

The identification of Toll-like receptors has revolutionised our understanding of innate immunity. TLR4 transduces the LPS signal and that of a number of structurally and functionally unrelated agonists. However, recent evidence adds to longstanding concerns that endotoxin contamination of bacterially derived recombinant TLR4 agonists is responsible for effects attributed to these molecules. We highlight key factors in differentiating specific agonist effects from those of endotoxin and emphasize why conventional methods of detecting and eliminating LPS may lead to erroneous results. We propose that considerable caution is needed in the investigation of TLR4 agonists, particularly when using proteins produced in a bacterium that also houses the most ideal TLR4 agonist, LPS.

The potential for proteases to regulate mammalian TLR signalling is controversial. We found that inhibition of extracellular serine proteases did not reduce activation of TLR4, but observed that the protease plasmin, an important fibrinolytic plasma enzyme that also exerts proinflammatory functions in monocytes, potentiated TLR2 and TLR4 signalling in RAW264.7 macrophages. Plasmin enhanced endogenous production of TNFalpha and activation of an NF-kappaB reporter plasmid. These actions were prevented by inhibition of its proteolytic activity and were not recapitulated by agonists of protease-activated receptors. These studies link fibrinolysis and TLR signalling, identifying further mechanisms potentially involved in activation of innate immunity.

Apoptosis, programmed cell death, of neutrophil and eosinophil granulocytes is a potential control point in the physiological resolution of innate immune responses. There is also increasing evidence that cellular processes of apoptosis can be dysregulated by pathogens as a mechanism of immune evasion and that delayed apoptosis, resulting in prolonged inflammatory cell survival, is important in persistence of tissue inflammation. The identification of cell-type specific pathways to apoptosis may allow the design of novel anti-inflammatory therapies or agents to augment the innate immune responses to infection. This review will explore the physiological roles of granulocyte apoptosis and their importance in infectious and non-infectious lung disease.

CRTH2 is a recently described chemoattractant receptor for the prostaglandin, PGD(2), expressed by Th2 cells, eosinophils and basophils, and believed to play a role in allergic inflammation. Here we describe the potency of several PGD(2) metabolites at the receptor to induce cell migration and activation. We report for the first time that the PGD(2) metabolite, 9alpha,11beta-PGF(2), and its stereoisomer, PGF(2alpha), are CRTH2 agonists. 9alpha,11beta-PGF(2) is a major metabolite produced in vivo following allergen challenge, whilst PGF(2alpha) is generated independently of PGD synthetase, with implications for CRTH2 signalling in the presence or absence of PGD(2) production.

Many host cell types, including endothelial and epithelial cells, neutrophils, monocytes, natural killer cells, dendritic cells and macrophages, initiate the first line of defense against infection by sensing conserved microbial structures through Toll-like receptors (TLRs). Recognition of microbial ligands by TLRs induces their oligomerization and triggers intracellular signaling pathways, leading to production of pro- and anti-inflammatory cytokines. Dysregulation of the fine molecular mechanisms that tightly control TLR signaling may lead to hyperactivation of host cells by microbial products and septic shock. A prior exposure to bacterial products such as lipopolysaccharide (LPS) may result in a transient state of refractoriness to subsequent challenge that has been referred to as 'tolerance'. Tolerance has been postulated as a protective mechanism limiting excessive inflammation and preventing septic shock. However, tolerance may compromise the host's ability to counteract subsequent bacterial challenge since many septic patients exhibit an increased incidence of recurrent bacterial infection and suppressed monocyte responsiveness to LPS, closely resembling the tolerant phenotype. Thus, by studying mechanisms of microbial tolerance, we may gain insights into how normal regulatory mechanisms are dysregulated, leading ultimately to microbial hypo-responsiveness and life-threatening disease. In this review, we present current theories of the molecular mechanisms that underlie induction and maintenance of 'microbial tolerance', and discuss the possible relevance of tolerance to several infectious and non-infectious diseases.

Endotoxin tolerance has the potential to limit phagocyte responses to Toll-like receptor (TLR) agonists, but the role of tolerance in regulating neutrophil responses is unknown. We investigated neutrophil responses to prolonged lipopolysaccharide (LPS) exposure and observed induction of tolerance in intracellular signaling pathways and respiratory burst. These effects were not prevented by granulocyte macrophage-colony stimulating factor (GM-CSF) pretreatment, and tolerized neutrophils retained the ability to respond to GM-CSF and other survival factors with a delay in apoptosis. In addition, LPS-exposed neutrophils showed continued generation of CXC chemokine ligand 8, which was not reduced in tolerized cells. Induction of tolerance was associated with a loss of TLR4 surface expression. Tolerance, therefore, induces a selective reprogramming of neutrophil function, but cells retain a predominantly proinflammatory phenotype.

Toll-like receptors (TLRs) play an essential role in the detection of invading pathogens and in the induction of host antimicrobial defenses. TLR4, the major endotoxin receptor, and TLR2, with agonists derived principally from gram-positive organisms, are likely to be important in the pathogenesis of sepsis. Both TLR2 and TLR4 agonists regulate important neutrophil functions, including adhesion, generation of reactive oxygen species, and release of chemokines, and activate major proinflammatory signaling pathways, including the nuclear factor- kappa B pathway. TLR stimulation produces only a modest direct inhibition of neutrophil apoptosis, although this signal is greatly amplified by the presence of monocytes, suggesting that regulation of the life span of neutrophils by TLR agonists may be principally mediated by responses of other endotoxin-responsive cells. We suggest that activation of neutrophils by TLRs is highly regulated, permitting acute neutrophil antimicrobial responses to TLR activation while providing a "brake" on inflammation by requiring the presence of mononuclear cells to significantly extend neutrophil survival.

Inappropriate platelet activation is a feature of acute and chronic diseases such as disseminated intravascular coagulation (DIC) and atherosclerosis. Since proinflammatory microbial-derived agonists can be involved in the pathogenesis of these diseases, we examined the potential role of TLR4 (mediating responses to LPS) and TLR2 (which responds to bacterial lipopeptides) in platelet activation. Our data suggested low-level expression of TLR2 and TLR4 on platelets, determined by flow cytometry, and we also observed expression of TLR4 on a megakaryocytic cell line by both flow cytometry and immunohistochemistry. Stimulation of the platelets with the TLR4 agonist LPS, and the synthetic TLR2 agonist Pam3CSK4, resulted in no platelet aggregation, no increase in CD62P surface expression and no increase in the cytosolic concentration of Ca2+. The TLR agonists were also unable to directly activate platelets primed with epinephrine, or pretreated with a low concentration of ADP or PAF. Pretreatment of platelets with LPS or Pam3CSK4 also failed to modulate the platelet response to submaximal concentrations of the classical platelet agonists ADP and PAF. We conclude that the TLR agonists LPS and Pam3CSK4 have no direct effect on platelet activation and that platelet TLRs may be a remnant from megakaryocytes. TLR2 and TLR4 agonists are thought to have a significant role in diseases such as atherosclerosis and DIC, but our research suggests that this is through a mechanism other than direct platelet activation or by modification of platelet responses to other agonists.

TLRs (Toll-like receptors) comprise a family of proteins whose function is principally to facilitate the detection of, and response to, pathogens. Protozoa, helminths, viruses, bacteria and fungi can all activate TLR signalling, and these signals have important roles in the activation of host defence. TLRs may also respond to products of tissue damage, providing them with roles in infective and sterile inflammation. Their role as detectors of pathogens and pathogen-associated molecules provides molecular mechanisms to underpin the observations leading to the hygiene hypothesis. Targeting of TLR signalling has implications in the control of infection, vaccine design, desensitization to allergens and down-regulation of inflammation. This review will explore TLR history, molecular signalling and the potential roles of TLRs in chronic lung disease.

Neutrophils are amongst the first immune cells to arrive at sites of infection, where they initiate antimicrobial and proinflammatory functions, which serve to contain infection. Sensing and defeating microbial infections are daunting tasks as a result of their molecular heterogeneity; however, Toll-like receptors (TLRs) have emerged as key components of the innate-immune system, activating multiple steps in the inflammatory reaction, eliminating invading pathogens, and coordinating systemic defenses. Activated neutrophils limit infection via the phagocytosis of pathogens and by releasing antimicrobial peptides and proinflammatory cytokines and generating reactive oxygen intermediates. Through the production of chemokines, they additionally recruit and activate other immune cells to aid the clearance of the microbes and infected cells and ultimately, mount an adaptive immune response. In acute inflammation, influx of neutrophils from the circulation leads to extremely high cell numbers within tissues, which is exacerbated by their delayed, constitutive apoptosis caused by local inflammatory mediators, potentially including TLR agonists. Neutrophil apoptosis and safe removal by phagocytic cells limit tissue damage caused by release of neutrophil cytotoxic granule contents. This review addresses what is currently known about the function of TLRs in the biology of the human neutrophil, including the regulation of TLR expression, their roles in cellular recruitment and activation, and their ability to delay apoptotic cell death.

Neutrophil chemokine receptor expression can be altered by exposure to Toll-like receptor (TLR) agonists, a process that is thought to have the potential to localize neutrophils to sites of infection. In order to investigate this process in more detail, we examined the regulation of highly pure neutrophil CXCR1 and CXCR2 expression and function by selective agonists of TLR2 (Pam(3)CSK(4)) and TLR4 (lipopolysaccharide, LPS). CXCR1 and CXCR2 were down-regulated by TLR engagement. CXCR2 loss was more rapid and showed a dependence upon soluble helper molecules (LPS binding protein and CD14) that was not evident for CXCR1, suggesting differential coupling of LPS signalling to CXCR1 and CXCR2 loss. However, TLR engagement in highly pure neutrophils did not result in complete loss of chemokine receptors, and LPS-treated neutrophils remained able to mount a respiratory burst to CXCL8 and CXCL1, and were able to migrate towards CXCL8 in assays of under-agarose chemotaxis. Thus, although treatment of purified human neutrophils with TLR2 and TLR4 agonists modifies chemokine receptor expression, remaining receptors remain functionally competent.

Rationale: Toll-like receptors 2 and 4 (TLR2, TLR4) enable cellular responses to bacterial lipoproteins, LPS, and endogenous mediators of cell damage. They have an established role in the activation of leukocytes, endothelial cells, and some smooth muscle cell types, but their roles in airway smooth muscle are uncertain., Objectives: To determine the roles of TLRs in activation of airway smooth muscle., Methods: Airway smooth muscle cells were cultured with TLR agonists, in the presence or absence of mononuclear leukocytes., Measurements and Main Results: We observed expression of TLR2 and TLR4 mRNAs, which could be upregulated by treatment with proinflammatory cytokines in primary human airway smooth muscle, but no important functional responses to agonists of these TLRs were seen. Coincubation of airway smooth muscle with peripheral blood mononuclear cells, at concentrations as low as 250 mononuclear cells/ml, resulted in a marked cooperative response to TLR stimuli, and synergistic production of cytokines, including chemokines (interleukin [IL-]-8) and IL-6. This cooperative response was greater when monocytes were enriched and was transferable using supernatants from LPS-stimulated peripheral blood mononuclear cells. Activation of cocultures required IL-1 generation from mononuclear cells, and was blocked by IL-1 receptor antagonist, though IL-1 generation alone was not sufficient to account for the magnitude of mononuclear cell-dependent coculture activation., Conclusions: These data indicate that potent amplification of inflammation induced by TLR agonists, such as LPS, may be achieved by cooperativity between airway smooth muscle and leukocytes involved in immune surveillance or inflammation.

The chemokine receptor CCR3 regulates the chemotaxis of leukocytes implicated in allergic disease, such as eosinophils. Incubation of eosinophils with CCL11, CCL13 or CCL5 resulted in a rapid decrease of cell-surface CCR3 which was replicated using CCR3 transfectants. Progressive truncation of the CCR3 C terminus by 15 amino acids produced three constructs, Delta340, Delta325 and Delta310. Delta340 and Delta325 were able to bind CCL11 with affinities similar to wild-type CCR3. Delta340 transfectants exhibited enhanced migration and reduced receptor down-regulation in response to CCL11 and CCL13. Delta325 transfectants displayed chemotactic responses to CCL11 and CCL13 similar to wild-type CCR3, and had impaired down-regulation when stimulated with CCL13 but not CCL11. In contrast, neither the Delta325 nor Delta340 truncation affected chemotaxis or receptor down-regulation induced by CCL5. Delta310 transfectants bound CCL11 poorly and were biologically inactive. Inhibitors of p38 mitogen-activated protein kinase and PI3-kinase antagonized eosinophil shape change responses and chemotaxis of transfectants to CCL11 and CCL13. In contrast, shape change but not chemotaxis was sensitive to inhibition of the extracellular signal-regulated kinase kinase pathway suggesting differential regulation of the two responses. Thus, the CCR3 C terminus contains distinct domains responsible for the regulation of receptor desensitization and for coupling to chemotactic responses.

The regulation of systemic and local neutrophil activation is crucial to the clearance of infections and the successful resolution of inflammation without progress to tissue damage or disseminated inflammatory reactions. Using purified lipopolysaccharide (pLPS) and highly purified neutrophils, we have previously shown that Toll-like receptor 4 signaling is a potent neutrophil activator, but a poor stimulator of survival. In the presence of peripheral blood mononuclear cells (PBMCs), however, pLPS becomes a potent neutrophil survival factor. Interleukin (IL)-1beta has been identified as an important neutrophil activator and prosurvival cytokine, and is produced in abundance by LPS-stimulated PBMCs. We now show that IL-1beta fails to activate highly purified neutrophils or enhance their survival, but in the presence of PBMCs, IL-1beta induces neutrophil survival. We hypothesized that LPS-primed neutrophils might become responsive to IL-1beta, but were unable to demonstrate this. Moreover, IL-1ra failed to prevent pLPS + PBMC-dependent neutrophil survival. In studies of IL-1R1(-/-) mice, we found that LPS was still able to mediate neutrophil survival, and neutrophil survival was enhanced by the addition of monocytic cells. Thus an important paradigm of neutrophil regulation needs to be viewed in the context of a cellular network in which actions of IL-1beta on neutrophils are indirect and mediated by other cells.

Granulocyte apoptosis has been proposed as a fundamental, injury-limiting granulocyte-clearance mechanism. As such, inhibition of this process may prevent the resolution of inflammation. Our previous studies have shown that TNFalpha (tumour necrosis factor-alpha) has a bi-modal influence on the rate of constitutive neutrophil apoptosis in vitro, causing early acceleration and late inhibition of this process. The pro-apoptotic effect is uniquely TNFR1 (TNF receptor 1) and TNFR2-dependent and the latter survival process is mediated via phosphoinositide 3-kinase and NF-kappaB (nuclear factor-kappaB) activation. In the present study, we show that, in contrast with GM-CSF (granulocyte/macrophage colony-stimulating factor), the delayed addition (i.e. at 6 h) of TNFalpha increases its survival effect despite substantial loss of neutrophil TNFR1 and TNFR2 at that time. This paradox was resolved using PBMC (peripheral blood mononuclear cell)-deplete and 5% PBMC-replete neutrophil cultures, where the enhanced survival effect observed after delayed TNFalpha addition was shown to be PBMC-dependent. TNFR2-blocking antibodies had no effect on the late survival effect of TNFalpha, implying a TNFR1-dependent process. Finally, I-kappaBalpha (inhibitory kappaB-alpha) and NF-kappaB time-course studies demonstrated that the survival effects of both GM-CSF and TNFalpha could be explained by maintenance of functional NF-kappaB., (Copyright 2004 Biochemical Society)

Interactions between proinflammatory and cell maturation signals, and the pathways that regulate leukocyte migration, are of fundamental importance in controlling trafficking and recruitment of leukocytes during the processes of innate and adaptive immunity. We have investigated the molecular mechanisms by which selective Toll-like receptor (TLR)2 and TLR4 agonists regulate expression of CCR1 and CCR2 on primary human monocytes and THP-1 cells, a human monocytic cell line. We found that activation of either TLR2 (by Pam(3)CysSerLys(4)) or TLR4 (by purified LPS) resulted in down-modulation of both CCR1 and CCR2. Further investigation of TLR-induced down-modulation of CCR1 revealed differences in the signaling pathways activated, and chemokines generated, via the two TLR agonists. TLR2 activation caused slower induction of the NF-kappa B and mitogen-activated protein kinase signaling pathways and yet a much enhanced and prolonged macrophage-inflammatory protein 1 alpha (CC chemokine ligand 3) protein production, when compared with TLR4 stimulation. Enhanced macrophage-inflammatory protein 1 alpha production may contribute to the prolonged down-regulation of CCR1 cell surface expression observed in response to the TLR2 agonist, as preventing chemokine generation with the protein synthesis inhibitor cycloheximide, or CCR1 signaling with the receptor antagonist UCB35625, abolished TLR2- and TLR4-induced CCR1 down-modulation. This result suggests an autocrine pathway, whereby TLR activation can induce chemokine production, which then leads to homologous down-regulation of the cognate receptors. This work provides further insights into the mechanisms that regulate leukocyte recruitment and trafficking during TLR-induced inflammatory responses.

Chemokine receptors play an important role in the migration of leukocytes to sites of allergic inflammation in humans. In this study, we have identified increased expression of the chemokine receptor CXCR1 on CD4+ T lymphocytes derived from patients with atopic disease compared with normal donors. Enhanced expression of CXCR1 by atopic donors was identified on freshly isolated peripheral blood cells and on expanded cell populations derived from nasal mucosal biopsies and from the periphery. Identification of CXCR1 expression on CD4 cells in the nasal mucosa was confirmed by double immunofluorescence. In addition, expression of CXCR1 was dramatically decreased in patients undergoing successful treatment of allergic rhinitis by specific immunotherapy. CXCR1 provided a functional receptor capable of regulating T cells in the context of allergic disease, since expression of CXC chemokine ligand 8 was up-regulated at the site of allergic inflammation and freshly isolated CXCR1+CD4+ cells from atopic donors showed an enhanced functional response to this ligand. CXCR1 expression on CD4+ T cells was increased in vitro in response to the pro-Th2 cytokine IL-4. Phenotypic analysis reveals that IFN-gamma expression was lower in the CXCR1+CD4+ cells. The identification of CXCR1 as a marker of allergic rhinitis reveals a possible target for therapeutic intervention in atopic disease.

The CC chemokine receptor 4 (CCR4) shows selectivity for the recruitment of memory T cell subsets, including those of the T helper cell type 2 (Th2) phenotype. In humans, CCR4+ T cells are recruited to the asthmatic lung in response to allergen challenge; however, the contribution of this pathway to allergic disease remains uncertain. We therefore investigated the role of CCR4 in allergic airways inflammation in the guinea pig. Blockade of CCR4 with a specific antibody resulted in only minor changes in numbers of CCR4+ Th cells in the bronchoalveolar lavage fluid of allergen-challenged guinea pigs and failed to inhibit the generation of eotaxin/CC chemokine ligand (CCL)11 or macrophage-derived chemokine/CCL22 or the recruitment of inflammatory leukocytes to the lung. These data suggest that although CCR4 was originally proposed as a marker of Th2 status, antigen-specific Th2 cells are recruited to the lung predominantly by other pathways. This study casts doubts on the validity of CCR4 as a therapeutic target in the treatment of asthma.

We previously showed in a small group of donors that eosinophils from a subgroup of individuals responded equipotently to CC chemokine ligand (CCL)11/eotaxin and CCL3/macrophage-inflammatory protein-1alpha in assays of eosinophil shape change (CCL3/macrophage-inflammatory protein-1alpha-highly responsive (MHR) donors). In this study, we investigated the functional role of CCL3 in eosinophil responses in 73 donors. MHR donors, identified by their eosinophil shape change responses, represented approximately 19% of the donor pool. Eosinophils from these donors showed increased eosinophil CCR1 expression and also underwent CCL3-mediated chemotaxis and up-regulation of CD11b. All MHR donors gave a history of atopy-associated diseases. In a further study, we prospectively recruited 110 subjects, subdivided into nonatopics or atopics, and investigated expression of CCR1 and CCR3 on eosinophils, basophils, monocytes, and neutrophils. Eosinophil CCR1 expression was non-normally distributed in atopics, although higher CCR1 expression levels were not predictive of a diagnosis of atopy or atopic disease. We identified the CCR1 promoter and investigated its function. We found a minimal promoter within 177 bp of the transcription start site, and an upstream enhancer region that facilitated expression in leukocyte cell lines. Collectively, these data demonstrate that MHR individuals form an important subgroup that, when associated with a diagnosis of allergic disease, may require tailored therapy to modulate eosinophil recruitment. Identification of a functional CCR1 promoter will facilitate the study of possible genetic determinants underlying this potentially important clinical phenotype.

Neutrophil responses to commercial LPS, a dual Toll-like receptor (TLR)2 and TLR4 activator, are regulated by TLR expression, but are amplified by contaminating monocytes in routine cell preparations. Therefore, we investigated the individual roles of TLR2 and TLR4 in highly purified, monocyte-depleted neutrophil preparations, using selective ligands (TLR2, Pam(3)CysSerLys(4) and Staphylococcus aureus peptidoglycan; TLR4, purified LPS). Activation of either TLR2 or TLR4 caused changes in adhesion molecule expression, respiratory burst (alone, and synergistically with fMLP), and IL-8 generation, which was, in part, dependent upon p38 mitogen-activated protein kinase signaling. Neutrophils also responded to Pam(3)CysSerLys(4) and purified LPS with down-regulation of the chemokine receptor CXCR2 and, to a lesser extent, down-regulation of CXCR1. TLR4 was the principal regulator of neutrophil survival, and TLR2 signals showed relatively less efficacy in preventing constitutive apoptosis over short time courses. TLR4-mediated neutrophil survival depended upon signaling via NF-kappa B and mitogen-activated protein kinase cascades. Prolonged neutrophil survival required both TLR4 activation and the presence of monocytes. TLR4 activation of monocytes was associated with the release of neutrophil survival factors, which was not evident with TLR2 activation, and TLR2 activation in monocyte/neutrophil cocultures did not prevent late neutrophil apoptosis. Thus, TLRs are important regulators of neutrophil activation and survival, with distinct and separate roles for TLR2 and TLR4 in neutrophil responses. TLR4 signaling presents itself as a pharmacological target that may allow therapeutic modulation of neutrophil survival by direct and indirect mechanisms at sites of inflammation.

PGD(2), a major mast cell mediator, is a potent eosinophil chemoattractant and is thought to be involved in eosinophil recruitment to sites of allergic inflammation. In plasma, PGD(2) is rapidly transformed into its major metabolite delta(12)-PGJ(2), the effect of which on eosinophil migration has not yet been characterized. In this study we found that delta(12)-PGJ(2) was a highly effective chemoattractant and inducer of respiratory burst in human eosinophils, with the same efficacy as PGD(2), PGJ(2), or 15-deoxy-delta(12,14)-PGJ(2). Moreover, pretreatment of eosinophils with delta(12)-PGJ(2) markedly enhanced the chemotactic response to eotaxin, and in this respect delta(12)-PGJ(2) was more effective than PGD(2). delta(12)-PGJ(2)-induced facilitation of eosinophil migration toward eotaxin was not altered by specific inhibitors of intracellular signaling pathways relevant to the chemotactic response, phosphatidylinositol 3-kinase (LY-294002), mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (U-0126), or p38 mitogen-activated protein kinase (SB-202190). Desensitization studies using calcium flux suggested that delta(12)-PGJ(2) signaled through the same receptor, CRTH2, as PGD(2). Finally, delta(12)-PGJ(2) was able to mobilize mature eosinophils from the bone marrow of the guinea pig isolated perfused hind limb. Given that delta(12)-PGJ(2) is present in the systemic circulation at relevant levels, a role for this PGD(2) metabolite in eosinophil release from the bone marrow and in driving eosinophil recruitment to sites of inflammation appears conceivable.

Eotaxin and eotaxin-2, acting through CCR3, are potent eosinophil chemoattractants both in vitro and in animal models. In this study we examined the capacity of eotaxin and eotaxin-2 to recruit eosinophils and other inflammatory cells in vivo in human atopic and nonatopic skin. Skin biopsies taken after intradermal injection of eotaxin and eotaxin-2 were examined by immunohistochemistry. Allergen- and diluent-challenged sites were used as positive and negative controls. Eotaxin and eotaxin-2 produced a dose- and time-dependent local eosinophilia of comparable intensity in both atopic and nonatopic individuals. This was associated with an acute wheal and flare response at the site of injection and development of a cutaneous late phase reaction in a proportion of subjects. There was an accompanying decrease in mast cell numbers. Both chemokines also induced the accumulation of basophils and an unexpected early infiltration of neutrophils. Macrophages were prominent at the 24-h point. Although there was surface CCR3 expression on neutrophils in whole blood, we were unable to demonstrate any functional neutrophil responses to eotaxin in vitro. Thus, intradermal injection of eotaxin and eotaxin-2 in humans induced infiltration of eosinophils and other inflammatory cells as well as changes consistent with CC chemokine-induced mast cell degranulation.

We investigated the actions of a panel of nonsteroidal anti-inflammatory drugs on eosinophils, basophils, neutrophils, and monocytes. Indomethacin alone was a potent and selective inducer of eosinophil and basophil shape change. In eosinophils, indomethacin induced chemotaxis, CD11b up-regulation, respiratory burst, and L-selectin shedding but did not cause up-regulation of CD63 expression. Pretreatment of eosinophils with indomethacin also enhanced subsequent eosinophil shape change induced by eotaxin, although treatment with higher concentrations of indomethacin resulted in a decrease in the expression of the major eosinophil chemokine receptor, CCR3. Indomethacin activities and cell selectivity closely resembled those of prostaglandin D(2) (PGD(2)). Eosinophil shape change in response to eotaxin was inhibited by pertussis toxin, but indomethacin- and PGD(2)-induced shape change responses were not. Treatment of eosinophils with specific inhibitors of phospholipase C (U-73122), phosphatidylinositol 3-kinase (LY-294002), and p38 mitogen-activated protein kinase (SB-202190) revealed roles for these pathways in indomethacin signaling. Indomethacin and its analogues may therefore provide a structural basis from which selective PGD(2) receptor small molecule antagonists may be designed and which may have utility in the treatment of allergic inflammatory disease.

The chemokine eotaxin (CCL11) is a key player in the trafficking of eosinophils to normal tissues and in the tissue eosinophilia associated with human allergic disease. We have recently cloned equine eotaxin and here we report the production of rEq eotaxin, with and without a C-terminal fusion peptide, in a novel expression system utilising stably transfected insect cells. rEq eotaxin induced equine eosinophil migration and superoxide production in vitro. A shape change in human eosinophils that could be blocked by 7B11, a monoclonal antibody against human CCR3, was also observed. Biological activity was not dependent on an intact eotaxin C-terminus. These results suggest that equine eotaxin, like its human ortholog, may play a role in eosinophil accumulation and activation in the horse.

We studied the regulation of CCR4 expression in peripheral blood and in human models of cutaneous and pulmonary allergen challenge. CCR4 expression was detectable on freshly isolated CD4+ lymphocytes and in CD4+ and CD8+ T cell lines derived from blood of atopic donors. Numbers of CCR4+ cells were up-regulated in T cell lines expanded in the presence of IL-4. CCR4 mRNA was absent at baseline in normal subjects in lung and skin, but present at baseline in the lung of some atopics. Baseline expression of CCR4 mRNA and protein was higher in lung vs. skin, but allergen-induced increases in CCR4 mRNA+ cells were observed in both organs. CCR4 protein+ cells were present at higher levels after allergen challenge in atopics compared to normal subjects. CCR4 may be important in the recruitment of T lymphocytes at sites of allergic inflammation, in a non-organ-specific manner.

CC-chemokine receptor 3 (CCR3)-stimulating chemokines are likely to have important in vivo roles in the regulation of eosinophil, basophil, and potentially helper T cell type 2 and mast cell recruitment. We have developed techniques to investigate the actions of eotaxin and other chemokines on multiple leukocyte populations in whole blood, without cell purification steps that might alter leukocyte responsiveness. We have shown that the potency of eotaxin in whole blood is limited by Duffy antigen binding, which may modulate the actions of this chemokine in vivo. We have also investigated the efficacy and potency of a new panel of small molecule antagonists of CCR3 on responses of eosinophils, neutrophils, basophils, and monocytes to chemokines, using whole blood assays of shape change, chemokine receptor internalization, and CD11b upregulation. These small molecule antagonists cause selective and potent inhibition of CCR3 on eosinophils and basophils, are bioavailable in blood, and are prototypic antagonists potentially of benefit in the treatment of human allergic disease. Such whole blood methods may also be employed in the investigation of other small molecule chemokine receptor antagonists.

Leukocyte responsiveness to LPS is dependent upon CD14 and receptors of the Toll-like receptor (TLR) family. Neutrophils respond to LPS, but conflicting data exist regarding LPS responses of eosinophils and basophils, and expression of TLRs at the protein level in these granulocyte lineages has not been fully described. We examined the expression of TLR2, TLR4, and CD14 and found that monocytes expressed relatively high levels of cell surface TLR2, TLR4, and CD14, while neutrophils also expressed all three molecules, but at low levels. In contrast, basophils expressed TLR2 and TLR4 but not CD14, while eosinophils expressed none of these proteins. Tested in a range of functional assays including L-selectin shedding, CD11b up-regulation, IL-8 mRNA generation, and cell survival, neutrophils responded to LPS, but eosinophils and basophils did not. In contrast to previous data, we found, using monocyte depletion by negative magnetic selection, that neutrophil responses to LPS were heavily dependent upon the presence of a very low level of monocytes, and neutrophil survival induced by LPS at 22 h was monocyte dependent. We conclude that LPS has little role in the regulation of peripheral blood eosinophil and basophil function, and that, even in neutrophils, monocytes orchestrate many previously observed leukocyte LPS response patterns.

Background: Self-administered adrenaline syringes may be prescribed for patients at risk of life-threatening episodes of angio-oedema or anaphylaxis., Objectives: To determine whether patients are able to use these syringes appropriately and adequately., Methods: Twenty-nine consecutive patients who had been prescribed self-administered adrenaline syringes for severe angio-oedema were recruited. All completed a questionnaire (unsupervised), and were asked to demonstrate how to use a dummy syringe., Results: Three of 29 (10%) patients had been prescribed syringes in the absence of severe angio-oedema or collapse. Seventeen of 29 (59%) patients had been prescribed two syringes, and 21 of 29 (72%) kept a syringe with them at all times. Twenty of 28 (71%) patients had had the use of a syringe demonstrated to them with the initial prescription, but two of 29 (7%) had never been shown how to use it. Only six of 26 (23%) patients had been told to telephone for an ambulance after using a syringe. Only seven of 29 (24%) patients would use a syringe for an episode of collapse, whereas eight of 28 (29%) would use one for an episode of lip swelling. Nine of 21 (43%) patients had not been warned about adverse effects, although 13 of 20 (65%) given adrenaline had had at least one adverse effect. Of the 25 patients asked to demonstrate their use of a syringe, only 14 (56%) were able to perform all steps correctly, and three (12%) were unable to perform any of the steps. Despite this, all 29 patients felt confident about giving themselves an injection, and most felt more secure having been prescribed syringes., Conclusions: As self-administered adrenaline syringes are prescribed for life-threatening events, it is vital that they are given to appropriate patients with adequate written instructions and proper demonstration at the time of the initial prescription. As a result of this study we have developed a more detailed patient information leaflet, and all patients are shown how to use a syringe for a second time when attending the clinic for follow-up.

Th2 lymphocytes play a central role in the control and maintenance of allergic inflammation. The chemokine receptor CCR4 is preferentially expressed on the surface of Th2 lymphocytes polarised in vitro. However, CCR4 is found on the surface of a significant proportion of circulating memory T lymphocytes, some of which are capable of producing the Th1-associated cytokine interferon gamma. To investigate the function of CCR4 on guinea pig (gp) T lymphocytes, we identified the open-reading frame of gpCCR4, which encodes a 361-amino acid protein with 88 and 81% amino acid identity to human and murine CCR4 sequences, respectively. Cells transfected with gpCCR4 migrated toward the human and murine orthologues of the CCR4 ligands, macrophage-derived chemokine and thymus and activation-regulated chemokine. Surface expression of CCR4, using an anti-human CCR4 monoclonal antibody, 10E4, was detected on approximately 12% of guinea pig peripheral blood T helper cells, and CCR4(+) guinea pig thymocytes were detected in low numbers. However, CCR4(+) T helper cells constituted approximately 9% of the T lymphocyte population within the normal guinea pig lung and 52% of the guinea pig bronchoalveolar lavage fluid, which is consistent with a role for CCR4 in T lymphocyte development and trafficking through normal tissues. Subsequent analysis of chimeric chemokine receptors indicated that 10E4, a functional inhibitor of gpCCR4 responses, recognized the amino terminus of CCR4.

Selective leukocyte trafficking and recruitment is primarily regulated by a specific family of small proteins called "chemokines". This extended family shepherds and guides leukocytes through their lives, facilitating their development, regulating their interactions with other leukocyte types, and guiding their recruitment to sites of inflammation. Through the actions of chemokines, allergen sensitization is regulated in atopic asthma, through the controlled migration of dendritic cells, T- and B-lymphocytes, mast cells and basophils. Subsequently, atopic inflammation is driven by chemokine-directed recruitment of eosinophils, basophils and lymphocytes. Diseases from cancer to chronic obstructive pulmonary disease to interstitial fibrosis are all potential targets for chemokine receptor antagonism. Innate immunity (the early pattern-recognition responses to stimuli such as lipopolysaccharide, viral proteins and bacterial DNA) needs to bridge the gap to specific immunity and antibody production and immunological memory. Again, chemokines are likely to be fundamental mediators of these responses. Chemokines are fundamental regulators of leukocyte homeostasis and inflammation, and their antagonism by small molecule chemokine receptor antagonists may be of enormous importance in the future treatment of human respiratory disease.

Neutrophils have been implicated in the pathogenesis of many inflammatory lung diseases, including the acute respiratory distress syndrome, chronic obstructive pulmonary disease and asthma. The CXC chemokine interleukin (IL)-8, is a potent neutrophil recruiting and activating factor and the detection of IL-8 in clinical samples from patients with these diseases has led clinicians to believe that antagonism of IL-8 may be a practicable therapeutic strategy for disease management. Work over the last decade has concentrated on both the molecular mechanisms by which IL-8 is produced in the inflammatory setting and also on the manner in which IL-8 activates the neutrophil. Expression of the IL-8 gene appears to be controlled by several components of the inflammatory milieu. Whilst lipopolysaccharide, IL-1beta and tumor necrosis factor-alpha are capable of augmenting IL-8 production, IL-10 is a potent inhibitor of IL-8 synthesis and appears to play an auto-regulatory role. Regulation of the IL-8 gene is under the control of nuclear factor kappaB which appears to be a primary target for corticosteroid-mediated repression of IL-8 production. IL-8 exerts is effects on neutrophils by binding with high affinity to two receptors on its cell surface, the chemokine receptors CXCR1 and CXCR2. These closely related receptors belong to the superfamily of G-protein coupled receptors, proteins that historically have proved amenable to antagonism by small molecules. The recent descriptions in the literature of highly potent small molecule antagonists of CXCR2 and their success in blocking in vivo trafficking of neutrophils suggest that antagonism of IL-8 at the receptor level is a viable therapeutic strategy. Clinical trials of such compounds will ultimately provide crucial information currently lacking and will define whether or not IL-8 blockade provides future therapy in pulmonary disease.

Despite sharing considerable homology with the members of the monocyte chemoattractant protein (MCP) family, the CC chemokine eotaxin (CCL11) has previously been reported to signal exclusively via the receptor CC chemokine receptor 3 (CCR3). Using the monocyte cell line THP-1, we investigated the relative abilities of eotaxin and MCPs 1-4 to induce CCR2 signaling, employing assays of directed cell migration and intracellular calcium flux. Surprisingly, 1 microm concentrations of eotaxin were able to recruit THP-1 cells in chemotaxis assays, and this migration was sensitive to antagonism of CCR2 but not CCR3. Radiolabeled eotaxin binding assays performed on transfectants bearing CCR2b or CCR3 confirmed eotaxin binding to CCR2 with a K(d) of 7.50 +/- 3.30 nm, compared with a K(d) of 1.68 +/- 0.91 nm at CCR3. In addition, whereas 1 microm concentrations of eotaxin were able to recruit CCR2b transfectants, substimulatory concentrations of eotaxin inhibited MCP-1-induced chemotaxis of CCR2b transfectants and also inhibited MCP-1-induced intracellular calcium flux of THP-1 cells. Collectively, these findings suggest that eotaxin is a partial agonist of the CCR2b receptor. A greater understanding of the interaction of CCR2 with all of its ligands, both full and partial agonists, may aid the rational design of specific antagonists that hold great promise as future therapeutic treatments for a variety of inflammatory disorders.

Mucosal environments such as that of the nose are points of first contact between the human organism and its environment. At these sites the immune system must be regulated to differentiate between and respond appropriately to pathogens and harmless contaminants. T cell-driven immune responses broadly fall into Th1- or Th2-type phenotypes, with increasing evidence that the recruitment of these T lymphocyte subsets is mediated by selective expression of specific chemokine receptors. We have investigated the immunology of the normal nasal mucosa. We show that nasal T cell lines from normal individuals, expanded by culture in IL-2, show reduced expression of the Th2-type cytokines IL-4 and IL-5 compared with lines derived from the blood of the same subjects. These T cells also show reduced expression of the Th2-selective chemokine receptor, CCR3, but similar levels of CCR4 compared with the blood-derived lines. This apparent suppression of Th2 cytokine and CCR3 expression by nasal T cells was reversed by addition of IL-4 to the culture medium. These data are consistent with the presence of a nasal mucosal microenvironment that suppresses Th2 responses and may represent a protective measure against atopic allergic disease in humans and a favoring of Th1 responses to infectious agents. In contrast, T cell expression of CCR1 was higher in the nose than in the blood regardless of the culture medium cytokine environment in keeping with a role for this receptor in tissue homing or lymphocyte activation.

During the Phase I/II assessment of new therapies with the potential to suppress eosinophil and neutrophil inflammation, there is a need to assess the peripheral blood pharmacokinetic (PK) and pharmacodynamic (PD) profiles of the drug. This has relevance in respiratory disease since drugs that target eosinophillic inflammation are in development for asthma; whereas neutrophil-directed therapies are being introduced for treatment of chronic obstructive airways disease (COPD). Pharmacokinetic evaluation is required to determine the concentration of drug substance (and possibly metabolites) in peripheral blood at intervals following single or repeated dosing. Pharmacodynamic assessment is also required since many drug substances have a duration of action which is prolonged beyond the time when drug substance is detectable in the blood (see Fig. 1). Fig. 1. Whole blood pharmacodynamics. In preclinical studies, animal or human blood is treated with test agents. In clinical studies, human subjects are treated with drug and blood removed for analysis. GAFS, gated autofluorescence forward scatter; PK, pharmacokinetics; PD, pharmacoldynamics.

To investigate human basophil responses to chemokines, we have developed a sensitive assay that uses flow cytometry to measure leukocyte shape change as a marker of cell responsiveness. PBMC were isolated from the blood of volunteers. Basophils were identified as a single population of cells that stained positive for IL-3Ralpha (CDw123) and negative for HLA-DR, and their increase in forward scatter (as a result of cell shape change) in response to chemokines was measured. Shape change responses of basophils to chemokines were highly reproducible, with a rank order of potency: monocyte chemoattractant protein (MCP) 4 (peak at <1 nM) >/= eotaxin-2 = eotaxin-3 >/= eotaxin > MCP-1 = MCP-3 > macrophage-inflammatory protein-1alpha > RANTES = MCP-2 = IL-8. The CCR4-selective ligand macrophage-derived chemokine did not elicit a response at concentrations up to 10 nM. Blocking mAbs to CCR2 and CCR3 demonstrated that responses to higher concentrations (>10 nM) of MCP-1 were mediated by CCR3 rather than CCR2, whereas MCP-4 exhibited a biphasic response consistent with sequential activation of CCR3 at lower concentrations and CCR2 at 10 nM MCP-4 and above. In contrast, responses to MCP-3 were blocked only in the presence of both mAbs, but not after pretreatment with either anti-CCR2 or anti-CCR3 mAb alone. These patterns of receptor usage were different from those seen for eosinophils and monocytes. We suggest that cooperation between CCRs might be a mechanism for preferential recruitment of basophils, as occurs in tissue hypersensitivity responses in vivo.

Eotaxin is a potent eosinophil chemoattractant that acts selectively through CCR3, which is expressed on eosinophils, basophils, mast cells, and Th2-type T cells. This arm of the immune system is believed to have evolved to control helminthic parasites. We hypothesized that helminths may employ mechanisms to inhibit eosinophil recruitment, to prolong worm survival in the host. We observed that the excretory/secretory products of the hookworm Necator americanus inhibited eosinophil recruitment in vivo in response to eotaxin, but not leukotriene B(4), a phenomenon that could be prevented by the addition of protease inhibitors. Using Western blotting, N. americanus supernatant was shown to cause rapid proteolysis of eotaxin, but not IL-8 or eotaxin-2. N. americanus homogenate was fractionated by gel filtration chromatography, and a FACS-based bioassay measured the ability of each fraction to inhibit the activity of a variety of chemokines. This resulted in two peaks of eotaxin-degrading activity, corresponding to approximately 15 and 50 kDa molecular mass. This activity was specific for eotaxin, as responses to other agonists tested were unaffected. Proteolysis of eotaxin was prevented by EDTA and phenanthroline, indicating that metalloprotease activity was involved. Production of enzymes inactivating eotaxin may be a strategy employed by helminths to prevent recruitment and activation of eosinophils at the site of infection. As such this represents a novel mechanism of regulation of chemokine function in vivo. The existence of CCR3 ligands other than eotaxin (e.g., eotaxin-2) may reflect the evolution of host counter measures to parasite defense systems.

We describe a small molecule chemokine receptor antagonist, UCB35625 (the trans-isomer J113863 published by Banyu Pharmaceutical Co., patent WO98/04554), which is a potent, selective inhibitor of CCR1 and CCR3. Nanomolar concentrations of UCB35625 were sufficient to inhibit eosinophil shape change responses to MIP-1alpha, MCP-4, and eotaxin, while greater concentrations could inhibit the chemokine-induced internalization of both CCR1 and CCR3. UCB35625 also inhibited the CCR3-mediated entry of the human immunodeficiency virus-1 primary isolate 89.6 into the glial cell line, NP-2 (IC(50) = 57 nm). Chemotaxis of transfected cells expressing either CCR1 or CCR3 was inhibited by nanomolar concentrations of the compound (IC(50) values of CCR1-MIP-1alpha = 9.6 nm, CCR3-eotaxin = 93.7 nm). However, competitive ligand binding assays on the same transfectants revealed that considerably larger concentrations of UCB35625 were needed for effective ligand displacement than were needed for the inhibition of receptor function. Thus, it appears that the compound may interact with a region present in both receptors that inhibits the conformational change necessary to initiate intracellular signaling. By virtue of its potency at the two major eosinophil chemokine receptors, UCB35625 is a prototypic therapy for the treatment of eosinophil-mediated inflammatory disorders, such as asthma and as an inhibitor of CCR3-mediated human immunodeficiency virus-1 entry.

To investigate eosinophil stimulation by chemokines we developed a sensitive assay of leukocyte shape change, the gated autofluorescence/forward scatter assay. Leukocyte shape change responses are mediated through rearrangements of the cellular cytoskeleton in a dynamic process typically resulting in a polarized cell and are essential to the processes of leukocyte migration from the microcirculation into sites of inflammation. We examined the actions of the chemokines eotaxin, eotaxin-2, monocyte chemoattractant protein-1 (MCP-1), MCP-3, MCP-4, RANTES, macrophage inflammatory protein-1alpha (MIP-1alpha), and IL-8 on leukocytes in mixed cell suspensions and focused on the responses of eosinophils to C-C chemokines. Those chemokines acting on CCR3 induced a rapid shape change in eosinophils from all donors; of these, eotaxin and eotaxin-2 were the most potent. Responses to MCP-4 were qualitatively different, showing marked reversal of shape change responses with agonist concentration and duration of treatment. In contrast, MIP-1alpha induced a potent response in eosinophils from a small and previously undescribed subgroup of donors via a non-CCR3 pathway likely to be CCR1 mediated. Incubation of leukocytes at 37 degrees C for 90 min in the absence of extracellular calcium up-regulated responses to MCP-4 and MIP-1alpha in the majority of donors, and there was a small increase in responses to eotaxin. MIP-1alpha responsiveness in vivo may therefore be a function of both CCR1 expression levels and the regulated efficiency of coupling to intracellular signaling pathways. The observed up-regulation of MIP-1alpha signaling via non-CCR3 pathways may play a role in eosinophil recruitment in inflammatory states such as occurs in the asthmatic lung.

Certain C-C chemokines, signaling via the eotaxin receptor C-C chemokine receptor-3 (CCR3), are thought to be central mediators of eosinophil accumulation in allergic inflammation. To investigate the role of CCR3 in vivo, we cloned the guinea pig eotaxin receptor (guinea pig CCR3) from a genomic DNA library. We isolated a single-exon open reading frame coding for a 358-amino acid chemokine receptor protein with 67 and 69% homology to human and murine CCR3, respectively. When expressed in stable transfectants, this receptor bound 125I-labeled guinea pig eotaxin, 125I-labeled human monocyte chemotactic protein-3, and 125I-labeled human RANTES. In chemotaxis assays, guinea pig CCR3 transfectants responded only to guinea pig eotaxin, with a maximal effect at 100 nM. mAbs were raised that bound selectively to both guinea pig CCR3 transfectants and guinea pig eosinophils. One of these mAbs, 2A8, blocked both ligand binding to transfectants and their chemotaxis in response to eotaxin. The Ab also inhibited chemotaxis and the elevation of cytosolic calcium in guinea pig eosinophils in response to eotaxin. F(ab')2 fragments of 2A8 were prepared that retained the ability to inhibit eosinophil calcium responses to eotaxin. Pretreatment of (111)In-labeled eosinophils in vitro with F(ab')2 2A8 selectively inhibited their accumulation in response to eotaxin in vivo. These data demonstrate that functional blockade of eosinophil chemokine receptors can be achieved in vivo and provide further support for the development of novel anti-inflammatory drugs targeting eosinophil recruitment through chemokine receptor antagonism.

Human neutrophils undergo rapid homologous receptor desensitization following repeated stimulation with chemoattractants such as IL-8, C5a, and FMLP. It has also been demonstrated that cross-desensitization among these chemoattractant receptors occurs. We investigated the mechanisms underlying the cross-desensitization of responses to IL-8 induced by pretreatment with FMLP or C5a. In [125I]-labeled IL-8 binding studies we found that the cross-desensitization induced by FMLP or C5a was associated with a subsequent reduction in IL-8 binding to neutrophils. There was no recovery of [125I]-labeled IL-8 binding on removal of the C5a or FMLP pretreatment. FACS analysis using mAbs specific for the two IL-8R subtypes showed differential regulation of IL-8R A and IL-8R B cell surface expression after chemoattractant pretreatment. Homologous desensitization by IL-8 resulted in internalization of IL-8R A and IL-8R B, but only IL-8R A was completely re-expressed after removal of agonist. FMLP stimulation led to a substantial loss of IL-8R B from the cell surface, whereas C5a stimulation induced only a partial loss. In both cases there was no re-expression of IL-8R B on removal of the chemoattractant stimulation. C5a and FMLP did not affect IL-8R A expression. Calcium mobilization studies using melanoma growth stimulatory activity and IL-8 suggest that a sustained loss of IL-8R B may play a part in maintaining FMLP-induced IL-8R cross-desensitization. Chemoattractant-induced cross-desensitization of neutrophils may be of importance in regulating neutrophil accumulation during the inflammatory response in vivo.

A patient in whom chylothorax was the presenting feature of sarcoidosis is reported. Mediastinal lymphadenopathy was shown by computed tomographic scanning. Obstruction of the thoracic duct by enlarged lymph nodes or fibrosis is the probable cause of chylothorax in this case. The association of chylothorax and sarcoidosis is extremely rare.

A 67 year old woman developed a severe adenoviral pneumonitis whilst receiving immunosuppressive therapy. She showed clinical and radiological evidence of a response to treatment with nebulised and intravenous ribavirin and intravenous pooled normal human immunoglobulin. To our knowledge this is the first time that such a therapeutic approach has been used in the treatment of a condition which normally carries a very high mortality.