Jose M. Lora

The murine CCR3 receptor regulates both the role of eosinophils and mast cells in allergen-induced airway inflammation and hyperresponsiveness.

CCR3 is a chemokine receptor initially thought specific to eosinophils but subsequently identified on TH2 cell subsets, basophils, mast cells, neural tissue, and some epithelia. Because of the prominent role of these cells in allergic disease, including asthma, we generated mice deficient in CCR3 to determine its contribution in a model of allergic airway disease. Here we show that CCR3 is important for the basal trafficking of eosinophils to the intestinal mucosa but not the lung. In contrast, CCR3 disruption significantly curtails eosinophil recruitment to the lung after allergen challenge, with the majority of the eosinophils being arrested in the subendothelial space. Further, a role for CCR3 in mast cell homing has been identified; after sensitization and allergen challenge, we find increased numbers of intraepithelial mast cells in the trachea of knockout mice. Physiologically, we find that the net result of these complex cell fates after sensitization and allergen challenge is a paradoxical increase in airway responsiveness to cholinergic stimulation. These data underscore a more complex role for CCR3 in allergic disease than was anticipated.

PYPAF7, a Novel PYRIN-containing Apaf1-like Protein That Regulates Activation of NF-κB and Caspase-1-dependent Cytokine Processing

PYRIN-containing Apaf1-like proteins (PYPAFs) are members of the nucleotide-binding site/leucine-rich repeat (NBS/LRR) family of signal transduction proteins. We report here that PYPAF7 is a novel PYPAF protein that activates inflammatory signaling pathways. The expression of PYPAF7 is highly restricted to immune cells, and its gene maps to chromosome 19q13.4, a locus that contains a cluster of genes encoding numerous PYPAF family members. Co-expression of PYPAF7 with ASC results in the recruitment of PYPAF7 to distinct cytoplasmic loci and a potent synergistic activation of NF-κB. To identify other proteins involved in PYPAF7 and ASC signaling pathways, we performed a mammalian two-hybrid screen and identified pro-caspase-1 as a binding partner of ASC. Co-expression of PYPAF7 and ASC results in the synergistic activation of caspase-1 and a corresponding increase in secretion of interleukin-1β. In addition, PYPAF1 induces caspase-1-dependent cytokine processing when co-expressed with ASC. These findings indicate that PYPAF family members participate in inflammatory signaling by regulating the activation of NF-κB and cytokine processing.

ICOS is critical for T helper cell–mediated lung mucosal inflammatory responses

We examined the requirement for and cooperation between CD28 and inducible costimulator (ICOS) in effective T helper (TH) cell responses in vivo. We found that both CD28 and ICOS were critical in determining the outcome of an immune response; cytolytic T lymphocyte–associated antigen 4–immunoglobulin (CTLA-4–Ig), ICOS-Ig and/or a neutralizing ICOS monoclonal antibody attenuated T cell expansion, TH2 cytokine production and eosinophilic inflammation. CD28-dependent signaling was essential during priming, whereas ICOS–B7RP-1 regulated TH effector responses, and the up-regulation of chemokine receptors that determine T cell migration. Our data suggests a scenario whereby both molecules regulate the outcome of the immune response but play separate key roles: CD28 primes T cells and ICOS regulates effector responses.

Activated human hepatic stellate cells express the renin-angiotensin system and synthesize angiotensin II.

BACKGROUND & AIMS The renin-angiotensin system plays an important role in hepatic fibrogenesis. In other organs, myofibroblasts accumulated in damaged tissues generate angiotensin II, which promotes inflammation and extracellular matrix synthesis. It is unknown whether myofibroblastic hepatic stellate cells, the main hepatic fibrogenic cell type, express the renin-angiotensin system and synthesize angiotensin II. The aim of this study was to investigate whether quiescent and activated human hepatic stellate cells contain the components of the renin-angiotensin system and synthesize angiotensin II. METHODS Hepatic stellate cells were freshly isolated from normal human livers (quiescent hepatic stellate cells) and from human cirrhotic livers (in vivo activated hepatic stellate cells). Culture-activated hepatic stellate cells were used after a second passage of quiescent hepatic stellate cells. Angiotensinogen, renin, and angiotensin-converting enzyme were assessed by quantitative polymerase chain reaction. Angiotensin II production was assessed by enzyme-linked immunosorbent assay and immunohistochemistry. RESULTS Quiescent hepatic stellate cells barely express the renin-angiotensin system components--angiotensinogen, renin, and angiotensin-converting enzyme--and do not secrete angiotensin II. In contrast, both in vivo activated hepatic stellate cells and culture-activated hepatic stellate cells highly express active renin and angiotensin-converting enzyme and secrete angiotensin II to the culture media. Mature angiotensin II protein is also detected in the cytoplasm of in vivo activated and culture-activated hepatic stellate cells. Growth factors (platelet-derived growth factor and epidermal growth factor) and vasoconstrictor substances (endothelin-1 and thrombin) stimulate angiotensin II synthesis, whereas transforming growth factor-beta and proinflammatory cytokines have no effect. Vasodilator substances markedly attenuate the effect of endothelin-1. CONCLUSIONS After activation, human hepatic stellate cells express the components of the renin-angiotensin system and synthesize angiotensin II. These results suggest that locally generated angiotensin II could participate in tissue remodeling in the human liver.

PYPAF1, a PYRIN-containing Apaf1-like Protein That Assembles with ASC and Regulates Activation of NF-κB

The PYRIN domain is a recently identified protein-protein interaction domain that is found at the N terminus of several proteins thought to function in apoptotic and inflammatory signaling pathways. We report here that PYPAF1 (PYRIN-containing Apaf1-like protein 1) is a novel PYRIN-containing signaling protein that belongs to the nucleotide-binding site/leucine-rich repeat (NBS/LRR) family of signaling proteins. The expression of PYPAF1 is highly restricted to immune cells, and its gene maps to chromosome 1q44, a locus that is associated with the rare inflammatory diseases Muckle-Wells syndrome and familial cold urticaria. To identify downstream signaling partners of PYPAF1, we performed a mammalian two-hybrid screen and identified ASC as a PYRIN-containing protein that interacts selectively with the PYRIN domain of PYPAF1. When expressed in cells, ASC recruits PYPAF1 to distinct cytoplasmic loci and induces the activation of NF-κB. Furthermore, coexpression of PYPAF1 with ASC results in a potent synergistic activation of NF-κB. These findings suggest that PYPAF1 and ASC function as upstream activators of NF-κB signaling.

Functional screening of five PYPAF family members identifies PYPAF5 as a novel regulator of NF-κB and caspase-1

PYRIN‐containing Apaf‐1‐like proteins (PYPAFs) are a recently identified family of proteins thought to function in apoptotic and inflammatory signaling pathways. PYPAF1 and PYPAF7 proteins have been found to assemble with the PYRIN–CARD protein ASC and coordinate the activation of NF‐κB and pro‐caspase‐1. To determine if other PYPAF family members function in pro‐inflammatory signaling pathways, we screened five other PYPAF proteins (PYPAF2, PYPAF3, PYPAF4, PYPAF5 and PYPAF6) for their ability to activate NF‐κB and pro‐caspase‐1. Co‐expression of PYPAF5 with ASC results in a synergistic activation of NF‐κB and the recruitment of PYPAF5 to punctate structures in the cytoplasm. The expression of PYPAF5 is highly restricted to granulocytes and T‐cells, indicating a role for this protein in inflammatory signaling. In contrast, PYPAF2, PYPAF3, PYPAF4 and PYPAF6 failed to colocalize with ASC and activate NF‐κB. PYPAF5 also synergistically activated caspase‐1‐dependent cytokine processing when co‐expressed with ASC. These findings suggest that PYPAF5 functions in immune cells to coordinate the transduction of pro‐inflammatory signals to the activation of NF‐κB and pro‐caspase‐1.

Expression of the type 2 receptor for cysteinyl leukotrienes (CysLT2R) by human mast cells: Functional distinction from CysLT1R

Cysteinyl leukotrienes (cysLTs) mediate vascular leakage and bronchoconstriction through the smooth muscle-associated CysLT type 1 receptor (CysLT1R), one of at least two loosely homologous cysLT-binding G protein-coupled receptors. We previously reported that CysLT1R is expressed by cultured human mast cells (hMCs), and that priming these cells with IL-4 enhances their sensitivity to calcium flux and cytokine generation in response to cys-LTs and the nucleotide ligand, uridine diphosphate (UDP), without increasing their surface expression of CysLT1R. We now report that hMCs express the type 2 receptor for cysLTs (CysLT2R) as well, and that the amount of surface CysLT2R protein increases in response to priming with IL-4. The selective function of CysLT2R was evident based on uninhibited IL-8 secretion by IL-4-primed hMCs stimulated with cys-LTs or UDP in the presence of the selective CysLT1R antagonist MK571. MK571 did inhibit IL-5 generation, calcium flux, and phosphorylation of extracellular signal-regulated kinase. IL-8 secretion was inhibited by pertussis toxin and a selective p38 kinase inhibitor, SB203580. The CysLT2 response may permit the cys-LTs and nucleotides generated in infection and tissue injury to elicit IL-8 generation by hMCs, potentially leading to neutrophilic infiltration, a characteristic of aerosol challenge-induced late-phase responses and of sudden death associated with asthma.

Cysteinyl Leukotrienes Regulate Th2 Cell-Dependent Pulmonary Inflammation

The Th2 cell-dependent inflammatory response is a central component of asthma, and the ways in which it is regulated is a critical question. The cysteinyl leukotrienes (cys-LTs) are 5-lipoxygenase pathway products implicated in asthma, in particular, by their function as smooth muscle constrictors of airways and microvasculature. To elucidate additional roles for cys-LTs in the pathobiology of pulmonary inflammation, we used an OVA sensitization and challenge protocol with mice lacking leukotriene C4 synthase (LTC4S), the terminal enzyme for cys-LT generation. Ag-induced pulmonary inflammation, characterized by eosinophil infiltration, goblet cell hyperplasia with mucus hypersecretion, and accumulation and activation of intraepithelial mast cells was markedly reduced in LTC4Snull mice. Furthermore, Ag-specific IgE and IgG1 in serum, Th2 cell cytokine mRNA expression in the lung, and airway hyperresponsiveness to methacholine were significantly reduced in LTC4Snull mice compared with wild-type controls. Finally, the number of parabronchial lymph node cells from sensitized LTC4Snull mice and their capacity to generate Th2 cell cytokines ex vivo after restimulation with Ag were also significantly reduced. In contrast, delayed-type cutaneous hypersensitivity, a prototypic Th1 cell-dependent response, was intact in LTC4Snull mice. These findings provide direct evidence of a role for cys-LTs in regulating the initiation and/or amplification of Th2 cell-dependent pulmonary inflammation.

Differential expression of inflammatory chemokines by Th1- and Th2-cell promoting dendritic cells: A role for different mature dendritic cell populations in attracting appropriate effector cells to peripheral sites of inflammation

Protective immunity to pathogens depends on efficient immune responses adapted to the type of pathogen and the infected tissue. Dendritic cells (DC) play a pivotal role in directing the effector T cell response to either a protective T helper type 1 (Th1) or type 2 (Th2) phenotype. Human monocyte‐derived DC can be differentiated into Th1‐, Th2‐ or Th1/Th2‐promoting DC in vitro upon activation with microbial compounds or cytokines. Host defence is highly dependent on mobile leucocytes and cell trafficking is largely mediated by the interactions of chemokines with their specific receptors expressed on the surface of leucocytes. The production of chemokines by mature effector DC remains elusive. Here we assess the differential production of both inflammatory and homeostatic chemokines by monocyte‐derived mature Th1/Th2‐, Th1‐ or Th2‐promoting DC and its regulation in response to CD40 ligation, thereby mimicking local engagement with activated T cells. We show that mature Th1‐ and Th1/Th2‐, but not Th2‐promoting DC, selectively express elevated levels of the inflammatory chemokines CCL2/MCP‐1, CCL3/MIP‐1α, CCL4/MIP‐1β and CCL5/RANTES, as well as the homeostatic chemokine CCL19/MIP‐3β. CCL21/6Ckine is preferentially expressed by Th2‐promoting DC. Production of the Th1‐attracting chemokines, CXCL9/Mig, CXCL10/IP‐10 and CXCL11/I‐TAC, is restricted to Th1‐promoting DC. In contrast, expression of Th2‐associated chemokines does not strictly correlate with the Th2‐promoting DC phenotype, except for CCL22/MDC, which is preferentially expressed by Th2‐promoting DC. Because inflammatory chemokines and Th1‐associated chemokines are constitutively expressed by mature Th1‐promoting DC and CCL22/MDC is constitutively expressed by mature Th2‐promoting DC, we propose a novel role for mature DC present in inflamed peripheral tissues in orchestrating the immune response by recruiting appropriate leucocyte populations to the site of pathogen entry.

PK1/EG-VEGF induces monocyte differentiation and activation

Macrophages exist as sentinels in innate immune response and react by expressing proinflammatory cytokines and up‐regulating antigen‐presenting and costimulatory molecules. We report a novel function for prokineticin‐1 (PK1)/endocrine gland‐derived vascular endothelial growth factor. Screening of murine tissue sections and cells for specific binding site leads to the identification of macrophages as an in vivo cellular target for PK1. We demonstrate PK1 induces differentiation of murine and human bone marrow cells into the monocyte/macrophage lineage. Human peripheral blood monocytes respond to PK1 by morphological changes and down‐regulation of B7‐1, CD14, CC chemokine receptor 5, and CXC chemokine receptor 4. Monocytes treated with PK1 have elevated interleukin (IL)‐12 and tumor necrosis factor α and down‐regulated IL‐10 production in response to lipopolysaccharide. PK1 induces a distinct monocyte‐derived cell population, which is primed for release of proinflammatory cytokines that favor a T helper cell type 1 response.