Evaluation of the C5a anaphylatoxin and its receptor (CD88) in allergic lung disease

Allergic asthma is characterized by airflow obstruction, airway hyperresponsiveness (AHR) and chronic airway inflammation. We and others have reported that complement component C3 and the anaphylatoxin C3a receptor promote while C5 protects against the development of the biological and physiological hallmarks of allergic lung disease in mice. In this study, we assessed if the protective responses could be mediated by C5a, an activation-induced C5 cleavage product. Mice with ablation of the C5a receptor (C5aR) either by genetic deletion or by pharmacological blockade exhibited significantly exacerbated AHR compared to allergen-challenged wild-type (WT) mice. However, there were no significant differences in many of the other hallmarks of asthma such as airway infiltration by eosinophils or lymphocytes, pulmonary IL-4-producing cell numbers, goblet cell metaplasia, mucus secretion or total serum IgE levels. In contrast to elevated AHR, numbers of IL-5 and IL-13 producing pulmonary cells, and IL-5 and IL-13 protein levels, were significantly reduced in allergen-challenged C5aR-/- mice compared to allergen-challenged WT mice. Administration of a specific cysteinyl leukotriene receptor 1 (cysLT1R) antagonist before each allergen-challenge abolished AHR in C5aR-/- as well as in WT mice. Pretreatment with a C3aR antagonist dose-dependently reduced AHR in allergen-challenged WT and C5aR-/- mice. Additionally, allergen-induced upregulation of pulmonary C3aR expression was exaggerated in C5aR-/- mice compared to WT mice. In summary, deficiency or antagonism of C5aR in a mouse model of pulmonary allergy increased AHR, which was reversed or reduced by blockade of the cysLT1R and C3aR, respectively. In conclusion, this study suggests that C5a and C5aR mediate protection against AHR by suppressing cysLT and C3aR signaling pathways, which are known to promote AHR. This also supports important and opposing roles of complement components C3a/C3aR and C5a/C5aR in AHR. ^

Does exhaled nitric oxide predict asthma exacerbation in children?

Asthma is a chronic complex disorder of the respiratory tract that affects millions of people globally, a large percentage of which are children. Triggered by a host of factors such as allergens and changes in temperature, the pathophysiologic and clinical indices vary among patients and have contributed to difficulties in overall management of asthma. Shortly after exhaled nitric oxide (eNO) was discovered in higher concentrations in asthma patients, it was shown to be superior to other markers such as PEFR, FEV1 and sputum eosinophils in screening asthma patients. Studies have also noted promising results regarding the use of eNO to predict asthma exacerbation in adults while in children, asthma symptoms have been observed to be good predictors of asthma exacerbation. Currently however, the potential of eNO as a predictor of asthma exacerbation in children is yet to be examined. The objective of this study was to assess eNO potential to predict asthma exacerbation in children by examining the relationship between eNO and changes in pulmonary function, asthma symptoms and rescue medication use.^ The primary study "Air Toxics and Asthma in Children" (ATAC), recruited children aged 9 to 14 years with labile persistent asthma diagnosed at least one year earlier. The data obtained from 30 study participants, included exhaled nitric oxide concentration, PEFR, FEV1, asthma symptoms and frequency of emergency medication use.^ Descriptive statistics, Pearson's and Spearman's correlation tests were followed by a simple linear regression in which eNO was the independent (predictor) variable while FEV1, PEFR, asthma symptoms and frequency of emergency medication use were the dependent (outcome) variables.^ Results showed that eNO was associated with percent change in FEV1, day time wheeze, night time shortness of breath, but correlated only weakly with PEFR, amplitude percent of mean PEFR, FEV1, percent change in FEV1 and asthma symptoms.^ Further research is imperative to better define the role of eNO and understand intrinsic pathologic mechanisms towards asthma management in children.^

Lesion, and haematologic and serum characteristics of Weddell seals (Leptonychotes weddellii) sampled in McMurdo Sound, Antarctica

We examined and collected biomedical samples from Weddell seals (Leptonychotes weddellii) during studies of post-breeding-season foraging behaviour of adults and movements of weaned pups as a complement to ongoing studies on the ecology and population dynamics of the McMurdo seals (Stewart et al. 2000, 2003). Here we report on Weddell seal health assessments conducted during the 1996/97, 1997/98 and 1998/99 breeding seasons at the Delbridge Islands (77.68°S, 166.50°E), McMurdo Sound, Antarctica. Our objectives were to compile baseline biomedical data for Weddell seals in McMurdo Sound, and to identify infectious and non-infectious diseases affecting the population. Development of such a database, including information on normal background morbidity and mortality, is an important first step in evaluating natural versus anthropogenic impacts on population health (Geraci et al. 1999; Reddy et al. 2001). These data will be integral to international studies of southern ocean pinnipeds that seek to evaluate the influence of biotic and abiotic factors on the ecology of these apex predators.

Pig but not human interferon-γ initiates human cell-mediated rejection of pig tissue in vivo

Split-thickness pig skin was transplanted on severe combined immunodeficient mice so that pig dermal microvessels spontaneously inosculated with mouse microvessels and functioned to perfuse the grafts. Pig endothelial cells in the healed grafts constitutively expressed class I and class II major histocompatibility complex molecules. Major histocompatibility complex molecule expression could be further increased by intradermal injection of pig interferon-γ (IFN-γ) but not human IFN-γ or tumor necrosis factor. Grafts injected with pig IFN-γ also developed a sparse infiltrate of mouse neutrophils and eosinophils without evidence of injury. Introduction of human peripheral blood mononuclear cells into the animals by intraperitoneal inoculation resulted in sparse perivascular mononuclear cell infiltrates in the grafts confined to the pig dermis. Injection of pig skin grafts on mice that received human peripheral blood mononuclear cells with pig IFN-γ (but not human IFN-γ or heat-inactivated pig IFN-γ) induced human CD4+ and CD8+ T cells and macrophages to more extensivley infiltrate the pig skin grafts and injure pig dermal microvessels. These findings suggest that human T cell-mediated rejection of xenotransplanted pig organs may be prevented if cellular sources of pig interferon (e.g., passenger lymphocytes) are eliminated from the graft.

Severe reduction in leukocyte adhesion and monocyte extravasation in mice deficient in CC chemokine receptor 2

CC chemokine receptor 2 (CCR2) is a prominent receptor for the monocyte chemoattractant protein (MCP) group of CC chemokines. Mice generated by gene targeting to lack CCR2 exhibit normal leukocyte rolling but have a pronounced defect in MCP-1-induced leukocyte firm adhesion to microvascular endothelium and reduced leukocyte extravasation. Constitutive macrophage trafficking into the peritoneal cavity was not significantly different between CCR2-deficient and wild-type mice. However, after intraperitoneal thioglycollate injection, the number of peritoneal macrophages in CCR2-deficient mice did not rise above basal levels, whereas in wild-type mice the number of macrophages at 36 h was ≈3.5 times the basal level. The CCR2-deficient mice showed enhanced early accumulation and delayed clearance of neutrophils and eosinophils. However, by 5 days neutrophils and eosinophils in both CCR2-deficient and wild-type mice had returned to near basal levels, indicating that resolution of this inflammatory response can occur in the absence of macrophage influx and CCR2-mediated activation of the resident peritoneal macrophages. After intravenous injection with yeast β-glucan, wild-type mice formed numerous large, well-defined granulomas throughout the liver parenchyma, whereas CCR2-deficient mice had much fewer and smaller granulomas. These results demonstrate that CCR2 is a major regulator of induced macrophage trafficking in vivo.

Vaccination with irradiated autologous melanoma cells engineered to secrete human granulocyte–macrophage colony-stimulating factor generates potent antitumor immunity in patients with metastatic melanoma

We conducted a Phase I clinical trial investigating the biologic activity of vaccination with irradiated autologous melanoma cells engineered to secrete human granulocyte–macrophage colony-stimulating factor in patients with metastatic melanoma. Immunization sites were intensely infiltrated with T lymphocytes, dendritic cells, macrophages, and eosinophils in all 21 evaluable patients. Although metastatic lesions resected before vaccination were minimally infiltrated with cells of the immune system in all patients, metastatic lesions resected after vaccination were densely infiltrated with T lymphocytes and plasma cells and showed extensive tumor destruction (at least 80%), fibrosis, and edema in 11 of 16 patients examined. Antimelanoma cytotoxic T cell and antibody responses were associated with tumor destruction. These results demonstrate that vaccination with irradiated autologous melanoma cells engineered to secrete granulocyte–macrophage colony-stimulating factor stimulates potent antitumor immunity in humans with metastatic melanoma.

Impaired granulopoiesis, myelodysplasia, and early lethality in CCAAT/enhancer binding protein ɛ-deficient mice

Polymorphonuclear leukocytes are essential for host defense to infectious diseases. CCAAT/enhancer binding protein ɛ (C/EBPɛ) is preferentially expressed in granulocytes and lymphoid cells. Mice with a null mutation in C/EBPɛ develop normally and are fertile but fail to generate functional neutrophils and eosinophils. Opportunistic infections and tissue destruction lead to death by 3–5 months of age. Furthermore, end-stage mice develop myelodysplasia, characterized by proliferation of atypical granulocytes that efface the bone marrow and result in severe tissue destruction. Thus, C/EBPɛ is essential for terminal differentiation and functional maturation of committed granulocyte progenitor cells.

Recognition Signal for C-Mannosylation of Trp-7 in RNase 2 Consists of Sequence Trp-x-x-Trp

C2-α-Mannosyltryptophan was discovered in human RNase 2, an enzyme that occurs in eosinophils and is involved in host defense. It represents a novel way of attaching carbohydrate to a protein in addition to the well-known N- and O-glycosylations. The reaction is specific, as in RNase 2 Trp-7, but never Trp-10, which is modified. In this article, we address which structural features provide the specificity of the reaction. Expression of chimeras of RNase 2 and nonglycosylated RNase 4 and deletion mutants in HEK293 cells identified residues 1–13 to be sufficient for C-mannosylation. Site-directed mutagenesis revealed the sequence Trp-x-x-Trp, in which the first Trp becomes mannosylated, as the specificity determinant. The Trp residue at position +3 can be replaced by Phe, which reduces the efficiency of the reaction threefold. Interpretation of the data in the context of the three-dimensional structure of RNase 2 strongly suggests that the primary, rather than the tertiary, structure forms the determinant. The sequence motif occurs in 336 mammalian proteins currently present in protein databases. Two of these proteins were analyzed protein chemically, which showed partial C-glycosylation of recombinant human interleukin 12. The frequent occurrence of the protein recognition motif suggests that C-glycosides could be part of the structure of more proteins than assumed so far.

IL-4 and -5 prime human mast cells for different profiles of IgE-dependent cytokine production

Mast cells (MC) are stem cell factor-dependent tissue-based hematopoietic cells with substantial functional heterogeneity. Cord blood-derived human MC (hMC) express functional receptors for IL-5, and IL-5 mediates stem cell factor-dependent comitogenesis of hMC in vitro. Although IL-5 is not required for normal hMC development, we considered that it might prime hMC for their high-affinity Fc receptor for IgE (FcɛRI)-dependent generation of cytokines, as previously demonstrated for IL-4. Compared with hMC maintained in stem cell factor alone, hMC primed with IL-5 expressed 2- to 4-fold higher steady-state levels of TNF-α, IL-5, IL-13, macrophage inflammatory protein 1α, and granulocyte-macrophage colony-stimulating factor transcripts 2 h after FcɛRI crosslinking and secreted 2- to 5-fold greater quantities of the corresponding cytokines, except IL-13, at 6 h. Unlike IL-4, IL-5 priming did not enhance FcɛRI-dependent histamine release. Thus, IL-5 augments cytokine production by hMC by a mechanism distinct from that of IL-4 and with a different resultant profile of cytokine production. These observations suggest a potentially autocrine effect of IL-5 on hMC for amplification of allergic immune responses, in addition to its recognized paracrine effects on eosinophils, and implicate both IL-4 and IL-5 in the modulation of the hMC phenotype.

HCC-2, a human chemokine: Gene structure, expression pattern, and biological activity

Cloning and sequencing of the upstream region of the gene of the CC chemokine HCC-1 led to the discovery of an adjacent gene coding for a CC chemokine that was named “HCC-2.” The two genes are separated by 12-kbp and reside in a head-to-tail orientation on chromosome 17. At variance with the genes for HCC-1 and other human CC chemokines, which have a three-exon-two-intron structure, the HCC-2 gene consists of four exons and three introns. Expression of HCC-2 and HCC-1 as studied by Northern analysis revealed, in addition to the regular, monocistronic mRNAs, a common, bicistronic transcript. In contrast to HCC-1, which is expressed constitutively in numerous human tissues, HCC-2 is expressed only in the gut and the liver. HCC-2 shares significant sequence homology with CKβ8 and the murine chemokines C10, CCF18/MRP-2, and macrophage inflammatory protein 1γ, which all contain six instead of four conserved cysteines. The two additional cysteines of HCC-2 form a third disulfide bond, which anchors the COOH-terminal domain to the core of the molecule. Highly purified recombinant HCC-2 was tested on neutrophils, eosinophils, monocytes, and lymphocytes and was found to exhibit marked functional similarities to macrophage inflammatory protein 1α. It is a potent chemoattractant and inducer of enzyme release in monocytes and a moderately active attractant for eosinophils. Desensitization studies indicate that HCC-2 acts mainly via CC chemokine receptor CCR1.

Bromination of deoxycytidine by eosinophil peroxidase: A mechanism for mutagenesis by oxidative damage of nucleotide precursors

Oxidants generated by eosinophils during chronic inflammation may lead to mutagenesis in adjacent epithelial cells. Eosinophil peroxidase, a heme enzyme released by eosinophils, generates hypobromous acid that damages tissue in inflammatory conditions. We show that human eosinophils use eosinophil peroxidase to produce 5-bromodeoxycytidine. Flow cytometric, immunohistochemical, and mass spectrometric analyses all demonstrated that 5-bromodeoxycytidine generated by eosinophil peroxidase was taken up by cultured cells and incorporated into genomic DNA as 5-bromodeoxyuridine. Although previous studies have focused on oxidation of chromosomal DNA, our observations suggest another mechanism for oxidative damage of DNA. In this scenario, peroxidase-catalyzed halogenation of nucleotide precursors yields products that subsequently can be incorporated into DNA. Because the thymine analog 5-BrUra mispairs with guanine in DNA, generation of brominated pyrimidines by eosinophils might constitute a mechanism for cytotoxicity and mutagenesis at sites of inflammation.

Rational interleukin 2 therapy for HIV positive individuals: daily low doses enhance immune function without toxicity.

When administered in high doses to HIV positive (HIV+) individuals, interleukin 2 (IL-2) causes extreme toxicity and markedly increases plasma HIV levels. Integration of the information from the structure-activity relationships of the IL-2 receptor interaction, the cellular distribution of the different classes of IL-2 receptors, and the pharmacokinetics of IL-2 provides for the rationale that low IL-2 doses should circumvent toxicity. Therefore, to identify a nontoxic, but effective and safe IL-2 treatment regimen that does not stimulate viral replication, doses of IL-2 from 62,500 to 250,000 IU/m2/day were administered subcutaneously for 6 months to 16 HIV+ individuals with 200-500 CD4+ T cells/mm3. IL-2 was already detectable in the plasma of most HIV+ individuals even before therapy. Peak plasma IL-2 levels were near saturating for high affinity IL-2 receptors in 10 individuals who received the maximum nontoxic dose, which ranged from 187,500 to 250,000 IU/m2/day. During the 6 months of treatment at this dose range, plasma levels of proinflammatory cytokines remained undetectable, and plasma HIV RNA levels did not change significantly. However, delayed type hypersensitivity responses to common recall antigens were markedly augmented, and there were IL-2 dose-dependent increases in circulating Natural Killer cells, eosinophils, monocytes, and CD4+ T cells. Expanded clinical trials of low dose IL-2 are now warranted, especially in combination with effective antivirals to test for the prevention of immunodeficiency and the emergence of drug-resistant mutants and for the eradication of residual virions.

Phenotypic and physiologic characterization of transgenic mice expressing interleukin 4 in the lung: lymphocytic and eosinophilic inflammation without airway hyperreactivity.

To investigate the contribution of interleukin-4 (IL-4) to airway inflammation in vivo and to explore directly its relationship to airway reactivity, we created transgenic mice in which the murine cDNA for IL-4 was regulated by the rat Clara cell 10 protein promoter. Expression was detected only in the lung and not in thymus, heart, liver, spleen, kidney, or uterus. The expression of IL-4 elicited hypertrophy of epithelial cells of the trachea, bronchi, and bronchioles. Hypertrophy is due, at least in part, to the accumulation of mucus glycoprotein. Histologic examination of parenchyma revealed multinucleated macrophages and occasional islands of cells consisting largely of eosinophils or lymphocytes. Analysis of lung lavage fluid revealed the presence of a leukocytic infiltrate consisting of lymphocytes, neutrophils and eosinophils. Mice expressing IL-4 had greater baseline airway resistance but did not demonstrate hyperreactivity to methacholine. Thus, the expression of IL-4 selectively within the lung elicits an inflammatory response characterized by epithelial cell hypertrophy, and the accumulation of macrophages, lymphocytes, eosinophils, and neutrophils without resulting in an alteration in airway reactivity to inhaled methacholine.

Eosinophil recruitment into the respiratory epithelium following antigenic challenge in hyper-IgE mice is accompanied by interleukin 5-dependent bronchial hyperresponsiveness.

A murine model for antigen-induced bronchial hyperreactivity (BHR) and airway eosinophilia, two hallmarks of asthma, was developed using ovalbumin-immunized mice, which produce large amounts of IgE (named BP2, "Bons Producteurs 2," for High Line of Selection 2). A single intranasal ovalbumin challenge failed to modify the bronchial responses, despite the intense eosinophil recruitment into the bronchoalveolar lavage fluid and airways. When mice were challenged twice a day for 2 days or once a day for 10 days, BHR in response to i.v. 5-hydroxytryptamine or to inhaled methacholine was induced in BP2 mice but not in BALB/c mice. Histological examination showed that eosinophils reached the respiratory epithelium after multiple ovalbumin challenges in BP2 mice but remained in the bronchial submucosa in BALB/c mice. Total IgE titers in serum were augmented significantly with immunization in both strains, but much more so in BP2 mice. Interleukin 5 (IL-5) titers in serum and bronchoalveolar lavage fluid of BP2 mice were augmented by the antigenic provocation, and a specific anti-IL5 neutralizing antibody suppressed altogether airway eosinophilia and BHR, indicating a participation of IL-5 in its development. Our results indicate that the recruitment of eosinophils to the airways alone does not induce BHR in mice and that the selective effect on BP2 mice is related to their increased IgE titers associated with antigen-driven eosinophil migration to the epithelium, following formation and secretion of IL-5.

Murine eotaxin: an eosinophil chemoattractant inducible in endothelial cells and in interleukin 4-induced tumor suppression.

Guinea pig eotaxin is a recently described member of the Cys-Cys family of chemokines and is involved in a guinea pig model of asthma. To determine whether eotaxin is a distinctive member of this family and to understand its physiologic role, we have cloned the mouse eotaxin gene and determined its structure and aspects of its biologic function. The sequence relationship between the mouse and guinea pig genes indicates that eotaxin is indeed a distinct member of the chemokine family. Moreover, murine eotaxin maps to a region of mouse chromosome 11 that encodes other Cys-Cys chemokines. In addition, recombinant murine eotaxin protein has direct chemoattractant properties for eosinophils. The eotaxin gene is widely (but not ubiquitously) expressed in normal mice and is strongly induced in cultured endothelial cells in response to interferon gamma. Eotaxin is also induced locally in response to the transplantation of interleukin 4-secreting tumor cells, indicating that it likely contributes to the eosinophil recruitment and antitumor effect of interleukin 4. Such responses suggest that eotaxin may be involved in multiple inflammatory states.