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. 1999 Jan;154(1):145-52.
doi: 10.1016/S0002-9440(10)65261-3.

Expression of inducible nitric oxide synthase in human granulomas and histiocytic reactions

F Facchetti  1 , W Vermi, S Fiorentini, M Chilosi, A Caruso, M Duse, L D Notarangelo, R Badolato
Affiliations

Expression of inducible nitric oxide synthase in human granulomas and histiocytic reactions

F Facchetti et al. Am J Pathol. 1999 Jan.

Abstract

Inducible nitric oxide synthase (iNOS) is required in immune response against infections and is involved in granuloma formation in animals; in murine macrophages, iNOS is induced by lipopolysaccharide and interferon-gamma. In contrast, the role of iNOS in human immune response against infections is still questioned, and its expression in granulomas is poorly investigated. Using Western blotting and immunohistochemistry, we investigated iNOS expression in human lymph nodes with nonspecific reactions and in tissues containing granulomas caused by mycobacteria, Toxoplasma, Cryptococcus neoformans, Leishmania, Bartonella, noninfectious granulomas (sarcoidosis, foreign body), and other hystiocitic reactions (Kikuchi's disease, Omenn syndrome). iNOS was undetectable in nonspecific reactive lymphadenitis, foreign-body granulomas, and Omenn syndrome, whereas it was strongly expressed in infectious granulomas, sarcoidosis, and Kikuchi's diseases. Immunohistochemistry demonstrated that iNOS was selectively expressed by the epithelioid and multinucleated giant cells within the granulomas. Use of an anti-nitrotyrosine antibody, recognizing nitrosilated amino acid residues derived from nitric oxide production, revealed a consistent positivity within the cells expressing iNOS, thus suggesting that iNOS is functionally active. Detection of cytokines by reverse transcriptase-polymerase chain reaction demonstrated that tissues that were positive for iNOS, also expressed the Thl-type cytokine interferon-gamma mRNA, but not the Th2-type cytokine interleukin-4. Taken together, these results indicate that iNOS is involved in different human immune reactions characterized by histiocytic/granulomatous inflammation and associated with Th1-type cytokine secretion.

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Figures

Figure 1.
Figure 1.
Expression of iNOS by Western blot analysis in a reactive lymph node (a) and in lymph nodes with mycobacterial granuloma (b and d), Bartonella/cat scratch granuloma (c), Kikuchi’s disease (e), and cryptococcal granuloma (f). The same filter was stripped and reblotted with an anti-β-actin antibody to allow comparability of the samples. Results are representative of four independent experiments.
Figure 2.
Figure 2.
Expression of iNOS and nitrotyrosine in infectious granulomas (d, C. neoformans lymphadenitis; e and f, Mycobacterium tuberculosis lymphadenitis; g to h, L. donovani splenic granuloma), and comparison with a reactive lymph node (a and b) and with a foreign-body granuloma (c). The numerous intra- and interfollicular CD68+ macrophages observed in the reactive lymph node parenchyma (a) and in the foreign body granuloma (c1) are totally negative for iNOS (b and c2). In contrast, the epithelioid and the multinucleated giant cells in infectious granulomas are strongly positive for iNOS (d, e, and g), and show granular cytoplasmic positivity for nitrotyrosine (f and g). In d and g, intracellular cryptococci and Leishmania bodies are indicated by arrows. Immunohistochemistry for CD68 (a and c1), iNOS (b, c2, d, e, and g), and nitrotyrosine (f and h), 3-amino-9-ethylcarbazole development, and hematoxylin counterstain. Magnification, ×60 (e), ×160 (c1 and c2), ×200 (a and b), ×300 (f), ×400 (d, g, and h).
Figure 3.
Figure 3.
Expression of iNOS in lymph nodes from patients with sarcoidosis (a), Kikuchi’s disease (b), and Omenn syndrome (d). Whereas iNOS is strongly expressed by the epithelioid and multinucleated giant cells within the sarcoid granuloma and by the aggregates of histiocytes in Kikuchi’s disease, no reactivity is observed by the numerous histiocytes in the lymph node from Omenn syndrome, which are labeled by CD68 (c). Eosinophils, which are typically numerous in Omenn syndrome, are recognized in (d) because of their endogenous peroxidase, which was not inhibited during immunostain. Immunohistochemistry for iNOS (a to c) and CD68 (c), 3-amino-9-ethylcarbazole development, and hematoxylin counterstain. Magnification, ×60 (c), ×160 (a, b, and d).
Figure 4.
Figure 4.
Detection of IFN-γ, IL-4, and β-actin mRNAs expression by RT-PCR. Total RNA was extracted from activated peripheral blood mononuclear cells (a), from tissue samples represented by a reactive lymph node (b), and lymph nodes with mycobacterial granuloma (c), Bartonella/cat scratch granuloma (d), Kikuchi’s disease (e), and sarcoidosis (f). Results are representative of two independent experiments.
Figure 5.
Figure 5.
Expression of NF-κB in a reactive lymph node (a) and in cryptococcus granuloma (b). In the reactive lymph node, NF-κB is mostly localized in the cytoplasm of lymphoid and nonlymphoid cells (a). Whereas in the cryptococcus granuloma, the multinucleated giant cells show evident nuclear translocation of NF-κB (b). In (b) two intracellular yeast cells stained by periodic acid-Schiff are also recognizable. Immunohistochemistry for NF-κB, 3–3′diaminobenzidine development, periodic acid-Schiff counterstain (b). Magnification, ×160 (a), ×400 (b).
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