Altered regulation of ELAVL1/HuR in HLA-B27-expressing U937 monocytic cells.

OBJECTIVE: To investigate the role of HLA-B27 expression in the regulation of RNA binding protein (RBP) Embryonic Lethal Abnormal Vision (ELAV) L1/Human antigen R (HuR) expression in Salmonella-infected or LPS-stimulated human monocytic cells, since HuR is a critical regulator of the post-transcriptional fate of many genes (e.g. TNFα) important in inflammatory response. METHODS: U937 monocytic cells were stably transfected with pSV2neo resistant vector (mock), wild type HLA-B27, or mutated HLA-B27 with amino acid substitutions in the B pocket. Cells were differentiated, infected with Salmonella enteritidis or stimulated with lipopolysaccharide. The expression levels of HuR protein and cleavage products (CP1 and CP2) were detected by Western blotting and flow cytometry. Specific inhibitors were used to study the role of PKR and p38 in HuR expression and generation of CPs. TNFα and IL-10 secretion after p38 and PKR inhibition were measured by ELISA. RESULTS: Full length HuR is overexpressed and HuR cleavage is disturbed in U937 monocytic cells expressing HLA-B27 heavy chains (HC). Increased full length HuR expression, disturbed cleavage and reduced dependence on PKR after infection correlate with the expression of glutamic acid 45 in the B pocket that is linked to the misfolding of HLA-B27. CONCLUSION: Results show that the expression of HLA-B27 HCs modulates the intracellular environment of U937 monocyte/macrophages by altering HuR regulation. This phenomenon is at least partly dependent on the misfolding feature of the B27 molecule. Since HuR is an important regulator of multiple genes involved in inflammatory response observations offer an explanation how HLA-B27 may modulate inflammatory response.

Phosphorylation of STAT-1 serine 727 is prolonged in HLA-B27-expressing human monocytic cells.

A tissue antigen, HLA-B27, is strongly associated with a group of rheumatic diseases called spondyloarthritides. Despite the intensive research, the exact role of HLA-B27 in the pathogenesis of these diseases is still unclear. Here we studied whether HLA-B27 modulates the phosphorylation of signal transducer and activator of transcription 1 (STAT-1) serine 727 residue and the localization of STAT-1 in Salmonella-infected human monocytic cells. In addition, we studied the role of signaling molecule double-stranded RNA activated protein kinase (PKR) in these modulatory effects. U937 human monocytic cell transfectants stably expressing wild type HLA-B27 or mutated HLA-B27 heavy chains with amino acid substitutions in the B pocket were prepared. The PMA-differentiated cells were infected with S. enteritidis. Western blotting was used to detect the phosphorylation of STAT-1, and to visualize the localization of STAT-1 in the cells confocal microscopy was used. Specific inhibitors were employed to study the role of PKR in STAT-1 phosphorylation. We discovered that the phosphorylation of STAT-1 serine 727 is prolonged in cells expressing misfolding forms of HLA-B27 after S. enteritidis infection, whereas in mock cells and in cells expressing mutated, non-misfolding HLA-B27 the phosphorylation of serine 727 is transient. Interestingly, STAT-1 serine 727 phosphorylation is partly dependent on PKR. In addition, more STAT-1 is localized in the nucleus of HLA-B27-expressing cells, even before an external trigger, when compared to mock cells. In conclusion, our results show that the phosphorylation of STAT-1 serine 727 residue is prolonged in HLA-B27-expressing monocyte-macrophage U937 cells after bacterial infection. This is of interest since the phosphorylation of serine 727 on STAT-1 is suggested to contribute to macrophage activation and promote inflammatory responses. Therefore, our results provide a mechanism which explains how the expression of an HLA-B27 molecule can impact the course of Salmonella infection and reactive arthritis.

HLA-B27 modulates intracellular growth of Salmonella pathogenicity island 2 mutants and production of cytokines in infected monocytic U937 cells.

BACKGROUND: Salmonella enterica serovar Enteritidis PT4 KS8822/88 replicates rapidly in HLA-B27-transfected human monocytic U937 cells. In this process, Salmonella pathogenicity island 2 (SPI-2) genes play a crucial role. Our previous study indicated that 118 Salmonella genes, including 8 SPI-2 genes were affected by HLA-B27 antigen during Salmonella infection of U937 cells. METHODS/PRINCIPAL FINDINGS: To further investigate Salmonella replication in HLA-B27-positive U937 monocytic cells, two SPI-2 genes, ssaS and sscA up-regulated most during Salmonella infection of HLA-B27-transfected U937 cells, were mutated by using one-step gene disruption method. Intracellular survival and replication of the mutants in the U937 cells was compared to that of the wild type strain. Surprisingly, the two mutated strains replicated significantly more than the wild type bacteria in HLA-B27-transfected cells. Secretion of tumor necrosis factor alpha (TNF-α) and interleukin 10 (IL-10) was significantly induced during the infection of HLA-B27-transfected U937 cells with the mutants. The results indicated that the certain SPI-2 genes in wild type bacteria suppress Salmonella intracellular growth and production of cytokines in infected HLA-B27-transfected cells. HLA-B27-associated modulation of Salmonella SPI-2 genes and cytokine production may have importance in the persistent infection of the bacteria and the pathogenesis of reactive arthritis. CONCLUSIONS: The study provides evidence that certain virulence factors of pathogens can reduce the intracellular growth in the host cells. We suggest that the limiting intracellular growth might be a strategy for persistence of bacteria in host cells, keeping a balance between pathogenic growth and pathogenesis.

Enhanced phosphorylation of STAT-1 is dependent on double-stranded RNA-dependent protein kinase signaling in HLA-B27-expressing U937 monocytic cells.

OBJECTIVE: To study the phosphorylation of STAT-1 in HLA-B27-transfected human monocytic cells and the role of the signaling molecules double-stranded RNA-dependent protein kinase (PKR) and p38 in STAT-1 phosphorylation. METHODS: U937 human monocytic cell transfectants stably expressing wild-type HLA-B27 or mutated HLA-B27 heavy chains with amino acid substitutions in the B pocket were prepared. Mock-transfected cells were prepared using the antibiotic resistance vectors (pSV2neo or RSV5neo) alone. Phorbol myristate acetate-differentiated cells were stimulated with lipopolysaccharide (LPS) or infected with Salmonella enteritidis. The phosphorylation and expression levels of STAT-1 protein were detected by Western blotting and flow cytometry. Specific inhibitors were added in cell culture to study the role of PKR and p38 in STAT-1 phosphorylation. RESULTS: STAT-1 was constitutively highly phosphorylated on the tyrosine 701 residue in HLA-B27-positive monocytic cells when compared to control cells, even prior to stimulation with LPS or bacteria. This phenotype was associated with the expression of HLA-B27 heavy chains that misfold. In addition, phosphorylation of STAT-1 was dependent on PKR. CONCLUSION: Our results show that STAT-1 tyrosine 701 is constitutively highly phosphorylated in the HLA-B27-expressing monocyte/macrophage cell line. Since phosphorylation of tyrosine 701 on STAT-1 is sufficient to induce interferon (IFN)-dependent genes, constitutive activity of this phosphorylation site may lead to the overexpression of IFN-dependent genes, as well as other STAT-1-dependent genes, in HLA-B27 monocyte/macrophages. Our results offer a mechanism by which B27 expression alone, without any external trigger, is potentially capable of inducing activation of STAT-1, a critical regulator of the inflammatory response.

Microarray analysis of response of Salmonella during infection of HLA-B27- transfected human macrophage-like U937 cells.

BACKGROUND: Human leukocyte antigen (HLA)-B27 is strongly associated with the development of reactive arthritis (ReA) in humans after salmonellosis. Human monocytic U937 cells transfected with HLA-B27 are less able to eliminate intracellular Salmonella enterica serovar Enteritidis than those transfected with control HLA antigens (e.g. HLA-A2). To investigate further the mechanisms by which HLA-B27-transfected cells allow increased replication of these bacteria, a DNA-based microarray was used for comparative genomic analysis of S. Enteritidis grown in HLA-B27- or HLA-A2-transfected cells. The microarray consisted of 5080 oligonucleotides from different serovars of Salmonella including S. Enteritidis PT4-specific genes. Bacterial RNA was isolated from the infected HLA-B27- or HLA-A2-transfected cells, reverse-transcribed to cDNA, and hybridized with the oligonucleotides on the microarrays. Some microarray results were confirmed by RT-PCR. RESULTS: When gene expression was compared between Salmonella grown in HLA-B27 cells and in HLA-A2 cells, 118 of the 4610 S. Enteritidis-related genes differed in expression at 8 h after infection, but no significant difference was detectable at 2 h after infection. These differentially expressed genes are mainly involved in Salmonella virulence, DNA replication, energy conversion and metabolism, and uptake and metabolism of nutrient substances, etc. The difference suggests HLA-B27-dependent modulation of Salmonella gene expression, resulting in increased Salmonella replication in HLA-B27-positive cells. Among the up-regulated genes were those located in Salmonella pathogenicity island (SPI)-2, which play a central role in intracellular survival and replication of Salmonella. CONCLUSIONS: This is the first report to show the regulation of Salmonella gene expression by HLA-B27 during infection of host cells. This regulation probably leads to increased Salmonella survival and replication in HLA-B27-positive cells. SPI-2 genes seem to contribute significantly to the increased replication.

Clever-1/Stabilin-1 regulates lymphocyte migration within lymphatics and leukocyte entrance to sites of inflammation.

Clever-1/Stabilin-1 is a scavenger receptor present on lymphatic and sinusoidal endothelium as well as on a subset of type II macrophages. It is also induced on vasculature at sites of inflammation. However, its in vivo function has remained practically unknown and this work addresses those unknown aspects. We demonstrate using in vivo models that Clever-1/Stabilin-1 mediates migration of T and B lymphocytes to the draining lymph nodes in vivo and identify the adhesive epitope of the Clever-1/Stabilin-1 molecule responsible for the interaction between lymphocytes and lymphatic endothelium. Moreover, we demonstrate that Ab blocking of Clever-1/Stabilin-1 efficiently inhibits peritonitis in mice by decreasing the entrance of granulocytes by 50%, while migration of monocytes and lymphocytes into the inflamed peritoneum is prevented almost completely. Despite efficient anti-inflammatory activity the Ab therapy does not dramatically dampen immune responses against the bacterial and foreign protein Ag tested and bacterial clearance. These results indicate that anti-Clever-1/Stabilin-1 treatment can target two different arms of the vasculature--traffic via lymphatics and inflamed blood vessels.

HLA-B27 and host-pathogen interaction.

HLA-B27 is a risk factor closely associated to spondyloarthropathies (SpA). One form of SpA is reactive arthritis (ReA), which develops as a complication after certain bacterial infections (e.g., Salmonellae, Yersiniae, Shigellae, Campylobacteriae and Chlamydiae). The development of infection-triggered complication is a complex train of events between the triggering bacteria and the host. Since most of the patients suffering from ReA are HLA-B27 positive, it has been proposed that HLA-B27 may modulate the interaction between ReA-triggering bacteria and host cell. Besides antigen presenting function, HLA-B27 displays other unusual properties that might be of importance in the development of ReA. These properties (homodimer formation and misfolding of HLA-B27 heavy chain in the endoplasmic reticulum (ER)) may trigger ER-stress signaling pathways in host cell, which in turn may modulate cell signaling in favor of ReA-triggering bacteria. Here we summarize the observations of HLA-B27 modulating the interaction between ReA-triggering bacteria and host cell and discuss potential mechanisms behind the interaction.

Identification of cytokines that might enhance the promoter activity of HLA-B27.

OBJECTIVE: Although the mechanism by which HLA-B27 induces ankylosing spondylitis is unclear, a minimum threshold of transcription is essential, a process controlled at the promoter. Our aim was to scan the effect of a panel of cytokines on the promoter of the HLA-B27 gene over serial timepoints. METHODS: The promoter region of B*2705 gene was cloned into a luciferase reporter, stably transfected into HeLa cells, and used to monitor the serial effect of 25 cytokines. Results of HLA-B27 promoter-reporter assays were compared to those of real-time polymerase chain reactions. RESULTS: After an initial delay, significant activation of the HLA-B27 promoter was observed with tumor necrosis factor-alpha (TNF-alpha), interferon-gamma (IFN-gamma), and IFN-beta. While early response of the HLA-B27 promoter was highest with TNF-alpha and IFN-gamma, ultimately the highest activity was observed with IFN-beta. CONCLUSION: The only promoter of HLA-B alleles studied in the past was that of HLA-B7, and always at only a single fixed timepoint of culture. This is the first study to show that activation of HLA-B27 promoter is a sequential event, and that TNF-alpha and IFN-beta are major participants at different timepoints.

Evidence that the p38 MAP kinase pathway is dysregulated in HLA-B27-expressing human monocytic cells: correlation with HLA-B27 misfolding.

OBJECTIVE: To investigate the cause of the enhanced intracellular replication of Salmonella enteritidis in HLA-B27-transfected U937 human monocytic cells and the contribution of HLA-B27 heavy chain (HC) misfolding. METHODS: U937 monocytic cell transfectants stably expressing pSV2neo resistant vector (mock), wild-type HLA-B27, or mutated HLA-B27 HCs with amino acid substitutions in the B pocket were differentiated, infected with S enteritidis, and treated with signaling pathway inhibitors or specific p38 small interfering RNA (siRNA). The numbers of living intracellular bacteria were determined with the colony-forming unit method. To visualize S enteritidis, the bacteria were transformed with green fluorescent protein, and studied by microscopy. RESULTS: Treatment with the p38 MAPK inhibitors or with p38 siRNA enhanced the replication of S enteritidis in U937 transfectants, whereas the other inhibitors had no effect. In mock-transfected cells and in cells expressing the mutated B27 HCs in which the misfolding had been corrected, p38 inhibitors impaired their ability to resist the replication of bacteria (mock, B27.A2B, B27.E45M, and B27.C67A). In contrast, the number of intracellular bacteria was not significantly increased in p38 inhibitor-treated cells expressing misfolded B27 HCs (B27g, B27cDNA, and B27.H9F). CONCLUSION: Our results show that p38 activity plays a crucial role in controlling intracellular S enteritidis in U937 cells. Enhanced replication of bacteria in B27-expressing cells requires that the HCs contain glutamic acid at position 45 and cysteine at position 67. Furthermore, in transfectants expressing misfolded B27 HCs, p38 inhibition had no significant effect on bacterial replication, suggesting that in these cells, the p38 pathway may not function properly.

Reactive arthritis or post-infectious arthritis?

The term 'reactive arthritis' was first used in 1969 to describe the development of sterile inflammatory arthritis as a sequel to remote infection, often in the gastrointestinal or urogenital tract. The demonstration of antigenic material (e.g. Salmonella and Yersinia lipopolysaccharide), DNA and RNA, and, in occasional cases, evidence of metabolically active Chlamydia spp. in the joints has blurred the boundary between reactive and post-infectious forms of arthritis. No validated and generally agreed diagnostic criteria exist, but the diagnosis of reactive arthritis is mainly clinical based on acute oligoarticular arthritis of larger joints that develops within 2-4 weeks of the preceding infection. In about 25% of patients, the infection can be asymptomatic. Diagnosis of the triggering infection is very helpful for the diagnosis of reactive arthritis. This is mainly achieved by isolating the triggering infection (stools, urogenital tract) by cultures (stool cultures for enteric microbes) or ligase reaction (Chlamydia trachomatis). However, after the onset of arthritis, this is less likely to be possible. Therefore, the diagnosis must rely on various serological tests to demonstrate evidence of previous infection, but, these serological tests are unfortunately not standardized. Treatment with antibiotics to cure Chlamydia infection is important, but the use of either short or prolonged courses of antibiotics in established arthritis has not been found to be effective for the cure of arthritis. The long-term outcome of reactive arthritis is usually good; however, about 25-50% of patients, depending on the triggering infections and possible new infections, subsequently develop acute arthritis. About 25% of patients proceed to chronic spondyloarthritis of varying activity.

A hypervariable N-terminal region of Yersinia LcrV determines Toll-like receptor 2-mediated IL-10 induction and mouse virulence.

The virulence antigen LcrV of Yersinia enterocolitica O:8 induces IL-10 in macrophages via Toll-like receptor 2 (TLR2). The TLR2-active region of LcrV is localized within its N-terminal amino acids (aa) 31-57. Sequencing of codons 25-92 of the lcrV gene from 59 strains of the three pathogenic Yersinia species revealed a hypervariable hotspot within aa 40-61. According to these sequence differences, seven LcrV groups were identified, with Y. pestis and Y. pseudotuberculosis represented in group I and the other six distributed within Y. enterocolitica. By testing LcrV sequence-derived synthetic oligopeptides of all seven LcrV groups in CD14/TLR2-transfected human embryonic kidney 293 cells, we found the highest TLR2 activity with a peptide derived from group IV comprising exclusively Y. enterocolitica O:8 strains. These findings were verified in murine peritoneal macrophages by using recombinant LcrV truncates representing aa 1-130 from different Yersinia spp. By systematically replacing charged aa residues by glutamine in synthetic oligopeptides, we show that the K42Q substitution leads to abrogation of TLR2 activity in both in vitro cell systems. This K42Q substitution was introduced in the lcrV gene from Y. enterocolitica O:8 WA-C(pYV), resulting in WA-C(pYVLcrV(K42Q)), which turned out to be less virulent for C57BL/6 mice than the parental strain. This difference in virulence was not observed in TLR2(-/-) or IL-10(-/-) mice, proving that LcrV contributes to virulence by TLR2-mediated IL-10 induction. LcrV is a defined bacterial virulence factor shown to target the TLR system for evasion of the host's immune response.

Aetiology and pathogenesis of reactive arthritis: role of non-antigen-presenting effects of HLA-B27.

Spondyloarthropathies are inflammatory diseases closely associated with human leukocyte antigen (HLA)-B27 by unknown mechanisms. One of these diseases is reactive arthritis (ReA), which is typically triggered by Gram-negative bacteria, which have lipopolysaccharide as an integral component of their outer membrane. Several findings in vivo and in vitro obtained from patients with ReA and from different model systems suggest that HLA-B27 modulates the interaction between ReA-triggering bacteria and immune cells by a mechanism unrelated to the antigen presentation function of HLA-B27. In this review we piece together a jigsaw puzzle from the new information obtained from the non-antigen-presenting effects of HLA-B27.

Enhanced intracellular replication of Salmonella enteritidis in HLA-B27-expressing human monocytic cells: dependency on glutamic acid at position 45 in the B pocket of HLA-B27.

OBJECTIVE: To reveal the cause of the impaired elimination of Salmonella enteritidis in HLA-B27-transfected human monocytic cells and to study whether the B pocket of HLA-B27 contributes to these modulatory effects. METHODS: Stable U937 cell transfectants expressing HLA-A2, B27, or different forms of B27 with amino acid substitutions in the B pocket were prepared. Mock-transfected cells were prepared using the antibiotic resistance vector (pSV2neo) alone. Cells were differentiated, infected with S enteritidis, and the number of live intracellular S enteritidis organisms was determined using the colony-forming unit method. To visualize intracellular S enteritidis, the bacteria were transformed with green fluorescent protein (GFP), and studied by confocal microscopy. RESULTS: Cells expressing wild-type HLA-B27 were more permissive of intracellular replication of S enteritidis compared with mock-transfected or A2-transfected controls. Cells expressing B27 with an altered B pocket composition having either 6 amino acid substitutions (B27.A2B; substitutions H9F, T24A, E45M, I66K, C67V, and K70H) or a single substitution (B27.E45M) were no longer permissive of S enteritidis replication. In contrast, cells expressing B27 with the single substitution of F for H at position 9 (B27.H9F) retained their permissiveness. Studies using GFP-transformed S enteritidis confirmed that the increase in the amount of intracellular bacteria in B27-expressing cells was due to replication of the bacteria. CONCLUSION: Our data indicate that HLA-B27 expression modulates the host-microbe interaction that results in an impaired capacity of monocytes to resist intracellular replication of S enteritidis. The phenotype is dependent on glutamic acid at position 45 in the B pocket and, thus, may be due to properties of the B27 heavy chain that are related to this residue. The ability of HLA-B27 to confer susceptibility to Salmonella-triggered reactive arthritis may occur, at least in part, through these modulatory effects.

Non-antigen presenting effects of HLA-B27.

Spondyloarthropathies (SpA) are a group of chronic rheumatic diseases, which show a strong asoociation with human leukocyte antigen (HLA)-B27. Although the association between HLA-B27 and the susceptibility to SpA was discovered thirty years ago, the exact mechanism by which HLA-B27 predisposes to disease development remains unclear. The classical role of MHC class I molecules is to present peptides for CD8+ T cells. Therefore, it has been proposed that the antigen presenting function of HLA-B27 is somehow altered in the patients developing SpA. However, despite extensive research, the attempts to create a comprehensive theory that would explain the role of HLA-B27 as an antigen presenting molecule in the development of SpA have been unsuccessful. Reactive arthritis (ReA) belongs to the group of SpA. It is a joint inflammation developing after certain bacterial infections e.g. Salmonella, Yersinia, and Chlamydia. Several unrelated observations indicate that HLA-B27 modulates the interaction between ReA-triggering bacteria and host cell. These findings suggest that HLA-B27 may possess functions, which are unrelated to antigen presentation. In this paper, we summarize these findings and discuss their potential impact in the development of SpA.

The role of infection in the pathogenesis of spondyloarthropathies with special reference to human leukocyte antigen-B27.

Spondyloarthropathies consist of many inflammatory diseases that are closely associated with human leukocyte antigen (HLA)-B27. One of these diseases is reactive arthritis (ReA), which is a joint inflammation that occurs after infections that are caused by certain gram-negative bacteria. The importance of these infections as causative agents of ReA has been clearly established. It is not clear, however, whether these infections contribute to the development of other forms of spondyloarthropathies. The exact mechanism by which HLA-B27 influences disease susceptibility in spondyloarthropathies remains to be determined. The role of HLA-B27 as an antigen-presenting molecule is certainly important in the pathogenesis of these diseases; however, recent data indicate that this molecule may exhibit other functions unrelated to antigen presentation, which may be important in the pathogenesis of ReA. In this paper, the authors summarize the current knowledge of the role of infection in the spondyloarthropathies.

HLA-B27 modulates nuclear factor kappaB activation in human monocytic cells exposed to lipopolysaccharide.

OBJECTIVE: To study whether HLA-B27 modifies some key factors controlling inflammatory responses on lipopolysaccharide (LPS) stimulation in human monocytic cells. METHODS: U937 human monocytic cells were stably transfected with either HLA-B27 genomic DNA, HLA-B27 complementary DNA, HLA-A2 genomic DNA, or with the resistant vector pSV2neo (mock) alone. The cells were stimulated with LPS. Electrophoretic mobility shift assay was performed to determine nuclear factor kappaB (NF-kappaB) and heat-shock factor 1 activities, Western blotting was performed to detect the expressions of inhibitory kappaBalpha (IkappaBalpha) and heat-shock proteins (HSPs), and enzyme-linked immunosorbent assay was performed to measure tumor necrosis factor alpha (TNFalpha) secretion. RESULTS: The expression of HLA-B27 modulated the response to LPS in U937 human monocytic cells. Stimulation with LPS led to faster degradation of IkappaBalpha regulatory proteins, accompanied by faster and prolonged activation of NF-kappaB in HLA-B27-expressing cells compared with HLA-A2 and mock transfectants. The secretion of TNFalpha upon LPS stimulation correlated well with the activation of NF-kappaB. No activation of the heat-shock response was observed. CONCLUSION: Our data indicate that HLA-B27 has effects on host responses to LPS that are unrelated to antigen presentation. Two crucial events in the development of arthritis, the activation of NF-kappaB and the secretion of TNFalpha, were found to be enhanced in HLA-B27-expressing cells upon LPS stimulation. Because LPS is known to be present in the inflamed joints of patients with reactive arthritis (ReA), the enhanced inflammatory response of HLA-B27-positive cells upon LPS stimulation offers an attractive explanation for the role of HLA-B27 in the development of ReA.

HLA-B27-transfected (Salmonella permissive) and HLA-A2-transfected (Salmonella nonpermissive) human monocytic U937 cells differ in their production of cytokines.

The cytokine secretion of the Salmonella-permissive, HLA-B27-positive U937 cells was examined, as it was previously shown that these cells kill Salmonella less efficiently than controls. Salmonella-permissive U937 cells showed upregulated production of interleukin 10 and to a lesser extent tumor necrosis factor alpha. HLA-B27-associated modulation of cytokine responses may have importance in the pathogenesis of reactive arthritis.

Elimination of Salmonella enterica serotype enteritidis in intestinal epithelial cells by mechanisms other than nitric oxide.

Production of nitric oxide (NO) by intestinal epithelial cells is induced after infection with Salmonella spp. or some other enteroinvasive bacteria. However, direct evidence of the role of NO in the elimination of intracellular pathogens in intestinal mucosa has not been established. This study investigated whether NO mediates killing of Salmonella enterica serovar Enteritidis in human intestinal epithelial cells by using parent Henle-407 cell line and a transfected cell line not capable of induced NO production (Henle-NO(def)). NO synthesis was studied as combined accumulation of nitrite and nitrate, as inducible nitric oxide synthase (iNOS) protein determined by Western blotting and as iNOS mRNA detected by reverse transcription (RT)-PCR. Although parent and Henle-NO(def) cells differed markedly in their ability to produce NO after infection, they eliminated S. Enteritidis equally, as determined by cfu counts. The presence of aminoguanidine, a selective iNOS inhibitor, during the infection blocked the production of NO but did not affect the elimination of the bacteria. These data suggest that NO does not have a direct role in the elimination of intracellular Salmonellae by human intestinal epithelial cells.