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The processing of pro-interleukin-1beta depends on activation of caspase-1. Controversy has arisen whether Toll-like receptor (TLR) ligands alone can activate caspase-1 for release of interleukin-1beta (IL-1beta). Here we demonstrate that human blood monocytes release processed IL-1beta after a one-time stimulation with either TLR2 or TLR4 ligands, resulting from constitutively activated caspase-1 and release of endogenous adenosine triphosphate. The constitutive activation of caspase-1 depends on the inflammasome components, apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), and NALP3, but in monocytes caspase-1 activation is uncoupled from pathogen-associated molecular pattern recognition. In contrast, macrophages are unable to process and release IL-1beta solely by TLR ligands and require a second adenosine triphosphate stimulation. We conclude that IL-1beta production is differentially regulated in monocytes and macrophages, and this reflects their separate functions in host defense and inflammation.

Severe acute respiratory syndrome (SARS) is caused by a novel coronavirus called SARS-CoV (1-3). The virus causes atypical pneumonia with diffuse alveolar damage with an overall mortality of Ϸ10% that ranges from 0% in children and 50% in persons over 65 (2). Coronaviruses bind to their glycoprotein receptors by the Ϸ200-kDa spike glycoprotein, S, on the viral envelope. Identification of virus receptors can provide insight into mechanisms of virus entry, tissue tropism, pathogenesis, and host range.Several types of receptors were previously identified for coronavirus S glycoproteins. The receptors for the murine coronavirus mouse hepatitis virus are murine carcinoembryonic antigen cell adhesion molecule 1 (CEACAM1) and related murine glycoproteins in the carcinoembryonic antigen family in the Ig superfamily (4). The receptors for human coronavirus 229E (HCoV-229E), transmissible gastroenteritis virus of swine, and feline coronavirus in genetic group 1 are aminopeptidase N (APN) glycoproteins (5-8).Angiotensin-converting enzyme 2 (ACE2) was found to be an efficient receptor for the S glycoprotein of SARS-CoV (9, 10). Because the Vero line of rhesus monkey kidney cells is highly susceptible to infection with SARS-CoV, Li et al. (9) used a codon-optimized soluble SARS-CoV spike glycoprotein (amino acids 12-672) fused to the Fc domain of human IgG1 to immunoprecipitate a putative receptor glycoprotein from Vero cell membranes and identified the simian ACE2 protein by mass spectrometry. They showed that expression of recombinant human ACE2 greatly enhanced the susceptibility of human 293T cells to infection by SARS-CoV. Wang et al. (10) independently demonstrated that human ACE2 is a receptor for SARS-CoV by transducing HeLa cells with a retrovirus library of cDNAs from Vero E6 cells and by using flow cytometry to select transduced cells that bound to purified soluble SARS-CoV S glycoprotein (amino acids 14-502) with a 6-histidine tag. They found that the simian cDNA in these cells, which encoded triosephosphate isomerase, enhanced expression of human ACE2 by inserting into the HeLa cell genome immediately upstream of the ACE2 ORF. Murine NIH 3T3 cells expressing recombinant human ACE2, but not those expressing recombinant triosephosphate isomerase, were susceptible to infection by HIV pseudovirus expressing SARS-CoV S protein.In this report, we describe the discovery of an additional receptor for SARS-CoV, CD209L (also called L-SIGN, DC-SIGNR, and DC-SIGN2) (11). Our strategy for identifying a SARS-CoV receptor was to transduce a human lung cDNA library carried by a retroviral vector into Chinese hamster ovary (CHO) cells and use flow cytometry to select transduced CHO cells that bound soluble, codon-optimized, c-myc-tagged SARSCoV S 590 glycoprotein (amino acids 1-590) expressed in 293T cells. The SARS-CoV S 590 -binding cells were then challenged with infectious SARS-CoV, and infection was demonstrated by detection of subgenomic viral RNA synthesis and immunofluorescence with antiviral antibody. The finding that ...

Biology of alveolar type II cells MASON RJ. Respirology 2006; 11 : S12-S15 Abstract: The purpose of this review is to highlight the many metabolic properties of alveolar type II cells, their production of surfactant, their role in innate immunity, and their importance in the repair process after lung injury. The review is based on the medical literature and results from our laboratory. Type II cells produce and secrete pulmonary surfactant and for that purpose they need to synthesize the lipids of surfactant. One of the regulators of lipogenesis is the transcription factor sterol regulatory element binding protein-1c . This is a key transcription factor regulating fatty acid synthesis. Type II cells also proliferate to restore the epithelium after lung injury, clear alveolar fluid by transporting sodium from the apical to the basolateral surface, and participate in the innate immune response to inhaled materials and organisms. The type II cell is, in many ways, the defender of the alveolus. However, the type II cells work in concert with the other cells in the gas exchange regions of the lung to keep the alveoli open and reduce inflammation due to irritants in the air we breathe. Key words: epithelial cells, phospholipids, surfactant.The alveolus must function for effortless, effective gas exchange. Moreover, this gas exchange unit must deal with a constant low level bombardment of particles, toxins and organisms from the environment. The interface with the environment consists of the surfactant surface film, a small amount of alveolar fluid, the alveolar epithelial cells, and alveolar macrophages. This brief review will focus on the role of the alveolar epithelial cells, especially type II cells, as defenders of the alveolar microenvironment (Fig. 1).The alveolar epithelium is composed of two main cell types, the alveolar type I cell and the alveolar type II cell. Alveolar type I cells are large flat cells through which gas exchange takes place. Type I cells cover about 95% of the alveolar surface, comprise about 8% of peripheral lung cells, and have a surface area of about 5000 µ m 2 per cell. Recent studies have indicated that type I cells have a unique pattern of gene expression and indicate that these cells will have defined metabolic functions.1 For example, type I cells have all the pumps and ion channels for transcellular sodium transport.2 What is not known is whether type I cells have the energetics to be a sodium absorbing epithelium. They appear to lack the large number of mitochondria characteristics of a transporting epithelium and, therefore, likely rely on anaerobic glycolysis to generate ATP. Nevertheless, although it is very difficult, these cells can be isolated and maintained in primary culture and with time we will learn more about their metabolic and physiologic functions. Type II cells cover about 5% of the alveolar surface area, comprise 15% of peripheral lung cells, and have an apical surface area of about 250 µ m 2 per cell.3 Type II cells have a distinct morphology with characteristic ...

Severe acute respiratory syndrome (SARS)-coronavirus (CoV) produces a devastating primary viral pneumonia with diffuse alveolar damage and a marked increase in circulating cytokines. One of the major cell types to be infected is the alveolar type II cell. However, the innate immune response of primary human alveolar epithelial cells infected with SARS-CoV has not been defined. Our objectives included developing a culture system permissive for SARS-CoV infection in primary human type II cells and defining their innate immune response. Culturing primary human alveolar type II cells at an airliquid interface (A/L) improved their differentiation and greatly increased their susceptibility to infection, allowing us to define their primary interferon and chemokine responses. Viral antigens were detected in the cytoplasm of infected type II cells, electron micrographs demonstrated secretory vesicles filled with virions, virus RNA concentrations increased with time, and infectious virions were released by exocytosis from the apical surface of polarized type II cells. A marked increase was evident in the mRNA concentrations of interferon-b and interferon-l (IL-29) and in a large number of proinflammatory cytokines and chemokines. A surprising finding involved the variability of expression of angiotensin-converting enzyme-2, the SARS-CoV receptor, in type II cells from different donors. In conclusion, the cultivation of alveolar type II cells at an air-liquid interface provides primary cultures in which to study the pulmonary innate immune responses to infection with SARS-CoV, and to explore possible therapeutic approaches to modulating these innate immune responses.Keywords: lung innate immune response; cytokine responses to SARS coronavirus; lung cell differentiation; air-liquid interface culturesSevere acute respiratory syndrome-associated coronavirus (SARSCoV) produces devastating viral pneumonia (1, 2). Pathologic changes are most prominent in the lungs, with disruptions of the epithelium in gas exchange areas and conducting airways (2-4). The epithelial cells of the alveoli and the conducting airways are the primary targets of SARS-CoV in the human lung, and they express the SARS receptor, angiotension-converting enzyme-2 (ACE2) (5-8). In autopsies of patients with SARS, coronavirus RNA and proteins have been detected in type II cells via immunocytochemisty and in situ hybridization (7,(9)(10)(11)(12)(13)(14). In the aged macaque model of SARS, in which the initial site of infection in the lung can be studied, virus infection was detected in both alveolar type I and type II cells (11,15). In human SARS autopsy specimens, the infection of alveolar macrophages was also suggested because they contained SARS antigens and formed multinucleated giant cells (2, 11). However, in vitro human alveolar macrophages, monocyte-derived dendritic cells, and monocytes are not readily susceptible to SARS-CoV, and these cell types show only very modest cytokine and interferon responses upon exposure to SARS-CoV (16-21).One of the major...

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Rationale: The pulmonary mononuclear phagocyte system is a critical host defense mechanism composed of macrophages, monocytes, monocyte-derived cells, and dendritic cells. However, our current characterization of these cells is limited because it is derived largely from animal studies and analysis of human mononuclear phagocytes from blood and small tissue resections around tumors.Objectives: Phenotypic and morphologic characterization of mononuclear phagocytes that potentially access inhaled antigens in human lungs.Methods: We acquired and analyzed pulmonary mononuclear phagocytes from fully intact nondiseased human lungs (including the major blood vessels and draining lymph nodes) obtained en bloc from 72 individual donors. Differential labeling of hematopoietic cells via intrabronchial and intravenous administration of antibodies within the same lobe was used to identify extravascular tissue-resident mononuclear phagocytes and exclude cells within the vascular lumen. Multiparameter flow cytometry was used to identify mononuclear phagocyte populations among cells labeled by each route of antibody delivery. Measurements and Main Results:We performed a phenotypic analysis of pulmonary mononuclear phagocytes isolated from whole nondiseased human lungs and lung-draining lymph nodes. Five pulmonary mononuclear phagocytes were observed, including macrophages, monocyte-derived cells, and dendritic cells that were phenotypically distinct from cell populations found in blood.Conclusions: Different mononuclear phagocytes, particularly dendritic cells, were labeled by intravascular and intrabronchial antibody delivery, countering the notion that tissue and blood mononuclear phagocytes are equivalent systems. Phenotypic descriptions of the mononuclear phagocytes in nondiseased lungs provide a precedent for comparative studies in diseased lungs and potential targets for therapeutics. Correspondence and requests for reprints should be addressed to Claudia V. Jakubzick, Ph.D., Department of Pediatrics and Immunology, National Jewish Health, 1400 Jackson Street, Denver, CO 80206. E-mail: jakubzickc@njhealth.org This article has an online supplement, which is accessible from this issue's table of contents at www.atsjournals.org The human respiratory tract has a branching structure that terminates in millions of alveoli, whose luminal surface covers an approximate area of 50 to 100 m 2 . In comparison to other barrier surfaces, such as the skin (2 m 2 ) and the gut (10 m 2 ), this surface area is massive, and therefore, comprises the body's largest interface with the ambient environment. Because of normal respiratory function, the average human exchanges 7,000 to 9,000 L of gas each day and inhales billions of particles, allergens, and microbes. Accordingly, the human lung constitutes a major site for the innate and adaptive immune responses. In this context, cells in the mononuclear phagocyte system (MPS), which consists of macrophages, monocytes, monocytederived cells, and dendritic cells (DCs), play critical roles. T...

Highly pathogenic influenza H5N1 virus continues to pose a threat to public health. Although the mechanisms underlying the pathogenesis of the H5N1 virus have not been fully defined, it has been suggested that cytokine dysregulation plays an important role. As the human respiratory epithelium is the primary target cell for influenza viruses, elucidating the viral tropism and innate immune responses of influenza H5N1 virus in the alveolar epithelium may help us to understand the pathogenesis of the severe pneumonia associated with H5N1 disease. Here we used primary cultures of differentiated human alveolar type II cells, alveolar type I-like cells, and alveolar macrophages isolated from the same individual to investigate viral replication competence and host innate immune responses to influenza H5N1 (A/HK/483/97) and H1N1 (A/HK/54/98) virus infection. The viral replication kinetics and cytokine and chemokine responses were compared by quantitative PCR (qPCR) and enzyme-linked immunosorbent assay (ELISA). We demonstrated that influenza H1N1 and H5N1 viruses replicated productively in type II cells and type I-like cells although with different kinetics. The H5N1virus replicated productively in alveolar macrophages, whereas the H1N1 virus led to an abortive infection. The H5N1 virus was a more potent inducer of proinflammatory cytokines and chemokines than the H1N1 virus in all cell types. However, higher levels of cytokine expression were observed for peripheral blood monocytederived macrophages than for alveolar macrophages in response to H5N1 virus infection. Our findings provide important insights into the viral tropisms and host responses of different cell types found in the lung and are relevant to an understanding of the pathogenesis of severe human influenza disease.