High-Level Expression of NRAMP1 in Peripheral Blood Cells and Tuberculous Granulomas fromMycobacterium bovis-Infected Bovines

Abstract

ByWestern blotting, we demonstrate high-level expression of NRAMP1 proteins in peripheral blood cells and granulomas of Mycobacterium bovis-infected bovines. Immunohistochemistry of granulomatous lesions showed heavily labeled epithelioid macrophages and Langhans cells. These data suggest that M. bovis infection enhances NRAMP1 expression and that active tuberculosis can occur despite this response.Mycobacterium bovis is the main causative agent of bovine tuberculosis (TB), a disease of great concern in developing countries such as Mexico for its detrimental economic impact and its effect on human beings (7). To control bovine TB, most programs rely on tuberculin tests to identify infected cattle, which are then sacrificed. Tuberculin tests, however, are not entirely reliable, and false-negative and false-positive results are not uncommon (8,19,20,22). To fight this disease, it would be a great asset to develop resistant breeds (17), a goal at present unattainable, as little is known about genetic factors of bovine TB (18,28).During the last decade, the gene coding for the so-called natural resistance-associated macrophage protein (NRAMP1) has been the center of much attention. Nramp1 was first described for the mouse as a gene conferring resistance to Salmonella enterica serovar Typhimurium, Leishmania donovani, and M. bovis BCG. A single recessive point mutation in Nramp1 was shown previously to occur in BCG-susceptible mouse strains (11). In human beings, eight NRAMP1 alleles have been identified (13, 24), some of them apparently associated with susceptibility to TB (4, 14). In bovines, two Nramp1 alleles have been cloned (16), the predicted polypeptide being a transmembrane molecule with a conserved transporter motif and multiple phosphorylation sites (9).To gain information on the role of NRAMP1 in bovine TB, we investigated the expression of NRAMP1 in peripheral blood cells (PBC) and tuberculous granulomas of M. bovisinfected bovines. A polyclonal antiserum was kindly donated by Jennifer Blackwell (University of Cambridge Medical School) for this purpose. The antiserum was raised in rabbits against a recombinant polypeptide spanning 35 residues of the carboxyl end of mouse NRAMP1 (1). Tested in our laboratory, this antiserum reacted with a 65-kDa band on phorbol myristate acetate-activated cells of the murine monocyte line J774A (data not shown). It showed no reactivity with M. bovis proteins by Western blotting (data not shown). PBC were obtained from 20 animals from a herd with 50% TB prevalence; 10 cattle were purified protein derivative (PPD) positive, and 10 were PPD negative. For a control, PBC from five PPD-negative cattle belonging to a TB-free herd were employed. Tracheobronchial and mediastinal lymph nodes (LN) with TB granulomas were isolated at autopsy from five cattle with M. bovis infection confirmed by histology, Ziehl-Neelsen stains, and bacteriological cultures (data not shown). For the Western blot, cells and tissues were treated with RIPA buffer (50 mM Tris, 150 mM NaCl, 1% NP-40, 0.5% s...

Coexpression of NRAMP1, iNOS, and Nitrotyrosine in Bovine Tuberculosis

“…By Western blot, this antibody was shown to react with both the characteristic 65-kDa NRAMP1 band of J774 murine MØ activated with PMA and with a similar band in cattle PBMCs. 15 A monoclonal antiactin immunoglobulin G (IgG) antibody was purchased from Pierce (Rockford, IL). Polyclonal rabbit antibodies to iNOS and NT were obtained from Transduction Laboratories (Lexington, KY).…”

“…5,10 NRAMP1 is primarily expressed in MØs and tissues of the recticuloendothelial system, but its role in the resistance of or Vet Pathol 43: 709-717 (2006) susceptibility to mycobacterial infection remains undetermined. 3,4,35 In a recent study we demonstrated high expression of NRAMP1 in granulomas of M. bovis-infected cattle; 15 since granulomas are protective inflammatory reactions, 17,20,21 it was thought that NRAMP1 plays a defensive role against mycobacteria locally. This finding, in addition to recent studies showing a regulatory role for NRAMP1 in iNOS production, 2,18 prompted this study, which aims to analyze the expression of NRAMP1 and iNOS in M. bovis-infected cattle.…”

Relationship of Bovine SLC11A1 (Formerly NRAMP1) Polymorphisms to the Risk of Bovine Tuberculosis in Holstein Cattle

“…Naturally resistant animals had the highest phagocytosis index and showed greater microbial control after exposure to M. bovis stimuli, producing stronger pro-inflammatory responses compared with susceptible animals [23]. In seeking to understand the reasons for natural variation in resistance, a high expression of NRAMP1 in peripheral blood cells and tuberculous granulomas from M. bovisinfected cattle appears to support a functional association for the SLC11A1 gene with enhanced protection against mycobacteria [5,23]. In bovines, two SLC11A1 alleles have been cloned and mapped to chromosome 2q43, which corresponds to a region predicted to encode a transmembrane polypeptide molecule that contains a conserved transporter motif and multiple phosphorylation sites [6,[13][14][15].…”

“…The host immune response to M. bovis infection is complex; following initial exposure, the bacilli are phagocytosed by host macrophages via transporters such as natural resistance-associated macrophage protein 1 (NRAMP1), encoded by the solute carrier family 11 member 1 (SLC11A1) gene. This transporter -which functions as part of the innate immune responseplays a key role in inhibiting proliferation of Mycobacterium tuberculosis (M. tuberculosis), through its involvement in acidification of phagosomes [5]. The relationships between various polymorphisms in the human SLC11A1 gene and mycobacterial diseases have been explored, including INT4 (single nucleotide G>C change in intron 4, 469+14G/C, rs3731865), D543N (conservative single base G>A substitution at codon 543, resulting in a change to asparagine from aspartic acid), and 3'UTR TGTG (a deletion in the 3' untranslated region, 1729+55del) [6][7][8][9][10].…”

A microsatellite study of bovine solute carrier family 11 a1 (Slc11a1) gene diversity in Mexico in relation to bovine tuberculosis

“…Molecular genetics studies have focused on understanding host susceptibility and resistance factors such as innate, specific and regulatory immune response-related genes and their relationship to infectious pathogens. Allelic variants of several genes have been implicated in the genetic susceptibility to tuberculosis in some human populations, but little is known in cattle (Estrada-Chavez et al 2001).…”