Lakshmi Puttagunta

Cutting Edge: Human Eosinophils Regulate T Cell Subset Selection through Indoleamine 2,3-Dioxygenase

Allergy involves eosinophilia and Th2 polarization. Indoleamine 2,3-dioxygenase (IDO)-catalyzed conversion of tryptophan to kynurenines (KYN) regulates T cell function. We show that human eosinophils constitutively express IDO. Eosinophils treated with IFN-γ showed an 8-fold increase in IDO mRNA within 4 h; IL-3, IL-5, and GM-CSF had no effect on baseline IDO expression. IL-3 pretreatment of eosinophils reduced IFN-γ-induced IDO mRNA expression below baseline. Conversely, GM-CSF, but not IL-5, resulted in a 2-fold increase in IFN-γ-induced IDO. Treatment with IL-3, IL-5, GM-CSF, or IFN-γ alone expressed IDO enzymatic activity (the presence of KYN in supernatants 48 h postculture). CD28 cross-linking resulted in measurable KYN in culture supernatants, inhibitable by a neutralizing anti-IFN-γ. Coculture of eosinophils with an IFN-γ-producing T cell line, but not IL-4-producing T cell clone, led to apoptosis and inhibition of CD3 or CD3/CD28-induced proliferation. Eosinophils infiltrating asthmatic lung and associated lymphoid tissue exhibited intracellular IDO immunoreactivity. Eosinophils may, therefore, maintain Th2 bias through IDO.

Distribution of nitric oxide synthase in normal and cirrhotic human liver

Chronic liver disorders represent a serious health problem, considering that 300 million people worldwide are hepatitis B virus carriers, and 8,000–10,000 patients per year, in the U.S. alone, die as a result of liver failure caused by hepatitis C infection. Nitric oxide synthase (NOS) regulates hepatic vasculature; however, the patterns of expression and activity of NOS proteins in healthy and diseased human livers are unknown. Sections of diseased (n = 42) and control livers (n = 14) were collected during orthotopic liver transplants and partial hepatectomy. The diseased sections included alcoholic cirrhosis, viral hepatitis, cholestasis, acute necrosis, and uncommon pathologies including α1-anti-trypsin disorder. The endothelial NOS (eNOS), inducible NOS (iNOS), and neuronal NOS (nNOS) were studied by using the citrulline assay, Western immunoblot, immunohistochemistry, and in situ hybridization. The systemic generation of plasma NO metabolites was measured by HPLC. In control livers, Ca2+-dependent and –independent NOS activities were identified by Western analysis as eNOS and iNOS, respectively. The eNOS was uniformly distributed in the hepatocytes and also detected in the endothelium of hepatic arteries, terminal hepatic venules, sinusoids, and in biliary epithelium. The iNOS was detected in hepatocytes and localized mainly in the periportal zone of the liver acinus. This pattern of distribution of eNOS and iNOS in normal liver was confirmed by in situ hybridization. In diseased livers, there was a significant increase in Ca2+-independent NOS with the corresponding strong appearance of iNOS in the cirrhotic areas. The eNOS was translocated to hepatocyte nuclei. Thus, eNOS and iNOS proteins are differentially expressed in healthy human liver, and this expression is significantly altered in cirrhotic liver disorders.

Inhibition of Allergic Inflammation in the Airways Using Aerosolized Antisense to Syk Kinase

Activation of the protein tyrosine kinase Syk is an early event that follows cross-linking of FcγR and FcεR, leading to the release of biologically active molecules in inflammation. We reported previously that aerosolized Syk antisense oligodeoxynucleotides (ASO) depresses Syk expression in inflammatory cells, the release of mediators from alveolar macrophages, and pulmonary inflammation. To study the effect of Syk ASO in allergic inflammation and airway hyperresponsiveness, we used the Brown Norway rat model of OVA-induced allergic asthma. Syk ASO, delivered in a liposome, carrier/lipid complex by aerosol to rats, significantly inhibited the Ag-induced inflammatory cell infiltrate in the bronchoalveolar space, decreasing both neutrophilia and eosinophilia. The number of eosinophils in the lung parenchyma was also diminished. Syk ASO also depressed up-regulation of the expression of β2 integrins, α4 integrin, and ICAM-1 in bronchoalveolar lavage leukocytes and reversed the Ag-induced decrease in CD62L expression on neutrophils. Furthermore, the increase in TNF levels in bronchoalveolar lavage following Ag challenge was significantly inhibited. Syk ASO also suppressed Ag-mediated contraction of the trachea in a complementary model. Thus, aerosolized Syk ASO suppresses many of the central components of allergic asthma and inflammation and may provide a new therapeutic approach.

Voltage-gated potassium channels in human ductus arteriosus.

We studied tone in the human ductus arteriosus and show that the constriction to oxygen is due to inhibition of voltage-gated potassium channels and, in the acute phase, is independent of endothelin-1.

Spleen tyrosine kinase (Syk) as a novel target for allergic asthma and rhinitis

Allergic asthma and rhinitis are prevalent diseases in the modern world, both marked by inflammation of the airways. The spleen tyrosine kinase (Syk) plays a critical role in the regulation of such immune and inflammatory responses. Although Syk is best known as a key component of immunoreceptor signalling complexes in leukocytes, recent studies demonstrated Syk expression in cells outside the haematopoietic lineage. Moreover, in recent years, it has been established that Syk is involved in various signalling cascades including those originating from integrin and cytokine receptors. Thus, Syk likely has a much wider biological role than previously recognised. Specific inhibition of Syk using aerosolised antisense oligonucleotides in liposome complexes significantly decreased lung inflammatory responses in experimental asthma and acute lung injury models. In addition, pharmacological inhibitors of Syk have been recently developed with potential for use as therapeutics. However, in the development and the rational delivery of drugs targeting Syk, it is important to consider the multiple cell types that express this kinase and the potential effects of its inhibition on various physiological functions. This review focuses on the recent data and the emerging ideas about Syk as a therapeutic target.

Regulation of Basolateral Cl ) Channels in Airway Epithelial Cells: The Role of Nitric Oxide

The presence of basolateral Cl− channels in airway epithelium has been reported in several studies, but little is known about their role in the regulation of anion secretion. The purpose of this study was to characterize regulation of these channels by nitric oxide (NO) in Calu-3 cells. Transepithelial measurements revealed that NO donors activated a basolateral Cl− conductance sensitive to 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS) and anthracene-9-carboxylic acid. Apical membrane permeabilization studies confirmed the basolateral localization of NO-activated Cl− channels. Experiments using 8-bromo cyclic guanosine monophosphate (8Br-cGMP) and selective inhibitors of soluble guanylyl cyclase and inducible NO synthase (1H-[1, 2, 4] oxadiazolol-[4, 3-a] quinoxalin-1-one [ODQ] and 1400W [N-(3-Aminomethyl)benzyl)acetamidine], respectively) demonstrated that NO activated Cl− channels via a cGMP-dependent pathway. Anion replacement and 36Cl− flux studies showed that NO affected both Cl− and HCO3− secretion. Two different types of Cl− channels are known to be present in the basolateral membrane of epithelial cells: Zn2+-sensitive ClC-2 and DIDS-sensitive bestrophin channels. S-Nitrosoglutathione (GSNO) activated Cl− conductance in the presence of Zn2+ ions, indicating that ClC-2 channel function was not affected by GSNO. In contrast, DIDS completely inhibited GSNO-activated Cl− conductance. Bestrophin immunoprecipitation studies showed that under control conditions bestrophin channels were not phosphorylated but became phosphorylated after GSNO treatment. The presence of bestrophin in airway epithelia was confirmed using immunohistochemistry. We conclude that basolateral Cl− channels play a major role in the NO-dependent regulation of anion secretion in Calu-3 cells.

Expression of DP2 (CRTh2), a Prostaglandin D2 Receptor, in Human Mast Cells

PGD2 has long been implicated in allergic diseases. Recent cloning of a second PGD2 receptor, DP2 (also known as CRTh2), led to a greater understanding of the physiological and pathophysiological implications of PGD2. PGD2 signaling through DP1 and DP2 mediates different and often opposite effects in many cell types of the immune system. Although mast cells (MC) are the largest source of PGD2 in the body, there is little information about their potential expression of DP2 and its functional significance. In this study, we show that tissue MC in human nasal polyps express DP2 protein, and that human MC lines and primary cultured human MC express mRNA as well as protein of DP2. By immunohistochemistry, we detected that 34% of MC in human nasal polyps expressed DP2. In addition, flow cytometry showed that 87% of the LAD2 human MC line and 98% of primary cultured human MC contained intracellular DP2. However, we could not detect surface expression of DP2 on human MC by single cell analysis using imaging flow cytometry. Blocking of endogenous PGD2 production with aspirin did not induce surface expression of DP2 in human MC. Two DP2 selective agonists, DK-PGD2 and 15R-15-methyl PGD2 induced dose-dependent intracellular calcium mobilization that was abrogated by pertussis toxin, but not by three DP2 selective antagonists. MC mediator release including degranulation was not affected by DP2 selective agonists. Thus, human MC express DP2 intracellularly rather than on their surface, and the function of DP2 in human MC is different than in other immune cells such as Th2 cells, eosinophils and basophils where it is expressed on the cell surface and induces Th2 cytokine and/or granule associated mediator release. Further studies to elucidate the role of intracellular DP2 in human MC may expand our understanding of this molecule and provide novel therapeutic opportunities.

Differential expression of spleen tyrosine kinase Syk isoforms in tissues: effects of the microbial flora

Spleen tyrosine kinase (Syk) is expressed widely in hematopoietic and non-hematopoietic cells. The widespread distribution of Syk and its involvement in host defense and allergic reactions, prompted us analyze the influence of microbial exposure on Syk expression. We compared the distribution of Syk in various tissues of germ-free and conventional mice using immunohistochemistry, Western blot analysis and real time RT-PCR. Total Syk expression was similar between germ-free and conventional mice. Since it has been claimed that Syk isoforms are differentially expressed, we studied the distribution and abundance of Syk (L) and Syk (S) isoforms in tissues from these mice. In contrast to previous reports, we found broad tissue expression of Syk (S). Interestingly, in germ-free mice the amount of Syk (S) but not Syk L protein was selectively increased in lung and spleen. In summary, our study reveals new and broad tissue expression of both Syk isoforms and demonstrates that lack of microbial flora results in selectively increased expression of Syk (S) isoform in lung and spleen.

Detection of human papillomavirus type 16 in oropharyngeal squamous cell carcinoma using droplet digital polymerase chain reaction.

The incidence of oropharyngeal squamous cell carcinoma caused by oncogenic HPV (HPV‐OPSCC) is rising worldwide. HPV‐OPSCC is commonly diagnosed by RT‐qPCR of HPV‐16 E6 and E7 oncoproteins or by cyclin‐dependent kinase inhibitor 2A, multiple tumor suppressor 1 (p16) immunohistochemistry (IHC). Droplet digital PCR (ddPCR) has been recently reported as ultra‐sensitive and highly precise method of nucleic acid quantification for biomarker analysis. We aimed to validate this method for the detection of HPV‐16 E6 and E7 in HPV‐OPSCC.

Regulation of inflammation by Rac2 in immune complex-mediated acute lung injury

Acute lung injury (ALI) is an inflammatory disorder associated with recruitment and activation of neutrophils in lungs. Rac2, a member of the Rho GTPase subfamily, is an essential regulator of neutrophil degranulation, superoxide release, and chemotaxis. Here, we hypothesized that Rac2 is important in mediating lung injury. Using a model of IgG immune complex-mediated ALI, we showed that injury was attenuated in rac2(-/-) mice compared with wild-type (WT) mice undergoing ALI, with significant decreases in alveolar leukocyte numbers, vascular leakage, and the inflammatory mediators, myeloperoxidase (MPO) and matrix metalloproteinases (MMPs). Reduced injury in rac2(-/-) mice was not associated with diminished cytokine and chemokine production, since bronchoalveolar lavage (BAL) levels of IL-17, TNF, CCL3, CXCL1, and CXCL2 were similarly increased in WT and rac2(-/-) mice with ALI compared with sham-treated mice (no ALI). BAL levels of MMP-2 and MMP-9 were significantly decreased in the airways of rac2(-/-) mice with ALI. Immunohistochemical analysis revealed that MMP-2 and MMP-9 expression was evident in alveolar macrophages and interstitial neutrophils in WT ALI. In contrast, MMP-positive cells were less prominent in rac2(-/-) mice with ALI. Chimeric mice showed that Rac2-mediated lung injury was dependent on hematopoietic cells derived from bone marrow. We propose that lung injury in response to immune complex deposition is dependent on Rac2 in alveolar macrophages and neutrophils.