Inducible Nitric Oxide Synthase Immunoreactivity in the Granulomatous Intestinal Lesions of Naturally Occurring Bovine Johne's Disease

Abstract

Inducible nitric oxide synthase (iNOS) is important in the control of a number of intracellular pathogens, including mycobacteria, and is a marker of classic macrophage activation. In human granulomatous diseases such as leprosy, a spectrum of granulomatous lesions is described, ranging from the tuberculoid to lepromatous types. Tuberculoid granulomas are associated with enhanced iNOS production and improved clinical outcomes over the lepromatous types. The aim of this study is to determine whether an association exists between morphology of bovine Johne's disease granulomas and lesion macrophage effector functions. To accomplish this, we retrospectively evaluated 24 cases of bovine Johne's disease. In each case, we recorded the predominant granuloma morphology and evaluated iNOS immunoreactivity and bacterial burden by acid-fast stains and mycobacterial immunolabeling. The results of this study demonstrate that all cases had granulomas with features most similar to the lepromatous type. This morphology correlated with heavy bacterial burdens demonstrated by acid-fast staining and mycobacterial immunoreactivity. None of the cases had high expression of iNOS in mycobacterial-positive granulomas. When iNOS immunoreactivity was identified, it was usually located near the crypts and was distinct from the granulomatous foci. Inducible nitric oxide synthase (iNOS) plays a crucial role in host defense and is a major killing mechanism within macrophages. iNOS is one of three iso-forms of nitric oxide synthase that generates nitric oxide (NO) from L-arginine. iNOS expression is well described in macrophages and neutrophils; however, it has also been reported in several additional cell types including respiratory and digestive epithelia, cardiac myocytes, and endothelium. 10,16,34,41 Within seconds after formation, NO reacts with water and oxygen to form reactive intermediates including NO 2 , NO 3 , N 2 O 2 , and ON00. These reactive nitrogen intermedi-ates (RNI) are highly unstable and are directly toxic to many microorganisms. iNOS expression and RNI production are important for macrophage killing of a wide range of intracellular pathogens including His-toplasma capsulatum, Toxoplasma gondii, Listeria monocytogenes, Mycobacterium tuberculosis, and Ec-tromelia virus (reviewed in MacMicking et al. 30) iNOS expression is induced by several types of signals. Environmental factors such as hypoxia and bacterial components including lipopolysaccharide and mycobacterial lipoarabinomanins will lead to iNOS expression in macrophages. 23,37 In addition, iNOS is strongly induced by T-helper 1 (Th1)-type cytokines including interferon-(IFN), interleukin 2, and tumor necrosis factor-; therefore, iNOS expression is a marker of Th1-mediated macrophage activation. 15,20 Similar to other species of pathogenic mycobacteria, Mycobacterium avium subspecies paratuberculosis (M. a. ptb) is an intracellular pathogen of macrophages and monocytes. 46 The pathogenesis of M. a. ptb infection at the macrophage level is incompletely understood , wi...

Lesion Development and Immunohistochemical Changes in Granulomas from Cattle Experimentally Infected withMycobacterium bovis

“…In other mycobacterial diseases of cattle such as paratuberculosis, iNOS expression has been shown to be minimal or absent in granulomas with high numbers of acid-fast bacilli. 16 Similar to the present study, iNOS immunoreactivity in human granulomas, caused by mycobacterial and nonmycobacterial infectious agents, is greatest in smaller granulomas and minimal in large granulomas with extensive necrosis. 12 Interestingly, in the present study, iNOS expression was limited to cells within the granuloma and not present in surrounding tissue, suggesting that iNOS expression is dependent on conditions within the granuloma microenvironment.…”

“…5,13 Inhibition was abrogated through the use of arginine inhibitors that prevent NO synthesis. 5,13 Although experimental evidence exists that NO plays a role in controlling infection with members of the M. tuberculosis complex 5,13,28 and bovine macrophages have been shown to express iNOS in vitro 2,17,47 and in vivo, 14,16,19 the expression of iNOS in bovine tuberculosis appears to be distinct from that seen in the murine tuberculosis model. In contrast to those in mice, bovine macrophages require additional signals from bacterial products (e.g., lipopolysaccharide, lipoarabinomannan) in addition to IFN-c for maximal NO production.…”

Helicobacter bilis Colonization Enhances Susceptibility to Typhlocolitis Following an Inflammatory Trigger

“…After staining, sections were washed and counterstained with hematoxylin. A previously described semiquantitative approach was used to score the frequency of positive cells [25]. Immunoreactivity was scored as follows: 0 = no immunoreactive cells, 1 = rare immunoreactive cells, 2 = scattered immunoreactive cells, 3 = clusters of immunoreactive cells, 4 = nearly diffuse staining.…”

LiveMycobacterium aviumsubsp.paratuberculosisand a Killed-Bacterium Vaccine Induce Distinct Subcutaneous Granulomas, with Unique Cellular and Cytokine Profiles

“…H&E-stained granulomatous lesions were individually scored using a scoring system adapted from previous publications (17,21,55). Briefly, scores of 0 to 3 were assigned for individual parameters to reflect the degree and organization of that particular parameter within the lesion.…”