CXC chemokines Gro(alpha)/IL-8 and IP-10/MIG in Helicobacter pylori gastritis

Infection with Helicobacter pylori causes chronic active gastritis, which is characterized by neutrophils infiltrating the gastric epithelial layer and the underlying lamina propria as well as by T, B lymphocytes and macrophages accumulating in the lamina propria. In this study, the chemokine profile responsible for the recruitment of these inflammatory cells is investigated. Using both RNA/RNA in situ hybridization and immunohistochemistry, the expression of the neutrophil and/or lymphocyte-attractant CXC chemokines growth-related oncogene alpha (Gro(alpha)), IL-8, interferon-gamma (IFN-gamma)-inducible protein-10 (IP-10), monokine induced by IFN-gamma (MIG) and the CC chemokines macrophage inflammatory protein-1alpha (MIP-1alpha), -1beta, regulated on activation normal T cell expressed and secreted (RANTES) and monocyte chemoattractant protein-1 (MCP-1) is studied and microanatomically localized in the gastric mucosa. Macrophages in the lamina propria at sites with neutrophil infiltration and gastric epithelium infiltrated by neutrophils highly expressed the neutrophil-attractant chemokines Gro(alpha) and IL-8. Additionally, Gro(alpha) and IL-8 were expressed by neutrophils themselves localized within gastric epithelium, in the foveolar lumen and in the cellular debris overlying mucosal erosion. IP-10 and to a lower extent MIG, both selectively chemotactic for inflammatory T cells, were expressed by endothelial cells of gastric mucosal vessels and by mononuclear cells at sites with T cell infiltration. Expression of all other CC chemokines tested was significantly lower. These in vivo data indicate that a set of predominantly CXC chemokines modulates the inflammation in H. pylori gastritis. Gro(alpha) and IL-8 may play an important role in neutrophil trafficking from the mucosal vessel into the gastric epithelium, whereas IP-10 and MIG contribute to the recruitment of inflammatory T cells into the mucosa.     

The effect of X4 and R5 HIV-1 on C, C-C, and C-X-C chemokines during the early stages of infection in human PBMCs

To better define a mechanism underlying the increase in expression of certain proinflammatory chemokines during HIV-1 infection, we analyzed the effect of X4 HIV-1 infection on C, C-C, and C-X-C chemokine mRNA levels. We demonstrate that X4 HIV-1 infection augments the expression of RANTES, IP-10, MCP-1, and Ltn in peripheral blood mononuclear cells (PBMCs). R5 HIV-1 also induces an increase in both IP-10 and MCP-1 production. Binding of UV-inactivated HIV-1 elevates MCP-1, RANTES, MIP-1alpha, MIP-1beta, and IL-8 expression, but fails to alter the production of IP-10, suggesting that the induction of IP-10 is dependent on downstream events following viral internalization. Indeed, recombinant gp120 alone was able to stimulate an eightfold increase in MCP-1 expression, but was unable to induce any detectable increase in IP-10 protein. HIV-induced modulation of chemokine expression suggests a mechanism by which HIV-infected monocytes and T cells might recruit target cells to sites of active viral replication, thus potentially aiding in the spread of the virus.     

Helicobacter pylori Lipopolysaccharide Binds to CD14 and Stimulates Release of Interleukin-8, Epithelial Neutrophil-Activating Peptide 78, and Monocyte Chemotactic Protein 1 by Human Monocytes

Helicobacter pylori gastritis is characterized by leukocyte infiltration of the gastric mucosa. The aims of this study were to determine whether H. pylori-derived factors stimulate chemokine release from human monocytes and to ascertain whether H. pylori lipopolysaccharide (LPS) may be responsible for this effect. Human peripheral blood monocytes were exposed to an H. pylori water extract (HPE) or to purified H. pylori LPS. Levels of the chemokines interleukin-8 (IL-8), epithelial neutrophil-activating peptide 78 (ENA-78), and monocyte chemotactic protein 1 (MCP-1) were measured by enzyme-linked immunosorbent assay. The contribution of H. pylori LPS to monocyte activation was determined by using the LPS antagonist Rhodobacter sphaeroides lipid A (RSLA) and a blocking monoclonal antibody to CD14 (60bca). HPE increased monocyte secretion of IL-8, ENA-78, and MCP-1. Heat treatment of HPE did not reduce its ability to activate monocytes. Purified H. pylori LPS also stimulated monocyte chemokine production but was 1,000-fold less potent than Salmonella minnesota lipid A. RSLA blocked H. pylori LPS-induced monocyte IL-8 release in a dose-dependent fashion (maximal inhibition 82%, P < 0.001). RSLA also inhibited HPE-induced IL-8 release (by 93%, P < 0.001). The anti-CD14 monoclonal antibody 60bca substantially inhibited IL-8 release from HPE-stimulated monocytes (by 88%, P < 0.01), whereas the nonblocking anti-CD14 monoclonal antibody did not. These experiments with potent and specific LPS inhibitors indicate that the main monocyte-stimulating factor in HPE is LPS. H. pylori LPS, acting through CD14, stimulates human monocytes to release the neutrophil-activating chemokines IL-8 and ENA-78 and the monocyte-activating chemokine MCP-1. Despite its low relative potency, H. pylori LPS may play an important role in the pathogenesis of H. pylori gastritis.     

Primary human alveolar type II epithelial cell chemokine release: effects of cigarette smoke and neutrophil elastase

An early response to cigarette smoke is an influx of leukocytes into the lung. Alveolar epithelial type II (ATII) cells may contribute by releasing chemokines in response to cigarette smoke and neutrophil elastase (NE). Human ATII cells were purified from normal regions of lungs resected for carcinoma (n = 14). In vitro, these cells exhibited ATII cell characteristics: lamellar bodies, apical microvilli, tight junctions, and expressed surfactant apoprotein C. Basal ATII cell release of five chemokines ranked as follows: monocyte chemotactic protein (MCP)-1 > interleukin (IL)-8 > growth-related oncogene (GRO)-alpha > macrophage inflammatory protein (MIP)-1alpha > regulated on activation, normal T cell expressed and secreted (RANTES). MIP-1alpha and RANTES were often not detectable. After stimulation with a mixture of lipopolysaccharide/endotoxin (LPS), tumor necrosis factor-alpha, IL-1beta, and IFN-gamma, MCP-1 and IL-8 secretion rose 4-6-fold, whereas GRO-alpha rose 25-fold. NE stimulated IL-8 mRNA expression, and 10nM NE stimulated IL-8 secretion; however, 100 nM NE caused a decrease in extracellular IL-8, MCP-1, and GRO-alpha, attributed to proteolysis. Cigarette smoke extract (CSE) inhibited IL-8 mRNA expression and release of all chemokines. Glutathione protected against the effects of CSE, suggesting oxidative mechanisms. GRO-alpha, important in growth and repair, was sensitive to both stimulation, by LPS:cytokines, and inhibition, by CSE. Thus, contrary to the original hypothesis, high concentrations of NE and CSE resulted in reduced extracellular chemokine levels. We hypothesize that reduced ATII cell-derived chemokine levels compromise alveolar repair, contributing to cigarette smoke-induced alveolar damage and emphysema.     

Shiga Toxins Induce, Superinduce, and Stabilize a Variety of C-X-C Chemokine mRNAs in Intestinal Epithelial Cells, Resulting in Increased Chemokine Expression

Exposure of humans to Shiga toxins (Stxs) is a risk factor for hemolytic-uremic syndrome (HUS). Because Stx-producing Escherichia coli (STEC) is a noninvasive enteric pathogen, the extent to which Stxs can cross the host intestinal epithelium may affect the risk of developing HUS. We have previously shown that Stxs can induce and superinduce IL-8 mRNA and protein in intestinal epithelial cells (IECs) in vitro via a ribotoxic stress response. We used cytokine expression arrays to determine the effect of Stx1 on various C-X-C chemokine genes in IECs. We observed that Stx1 induces multiple C-X-C chemokines at the mRNA level, including interleukin-8 (IL-8), GRO-alpha, GRO-beta, GRO-gamma, and ENA-78. Like that of IL-8, GRO-alpha and ENA-78 mRNAs are both induced and superinduced by Stx1. Furthermore, Stx1 induces both IL-8 and GRO-alpha protein in a dose-response fashion, despite an overall inhibition in host cell protein synthesis. Stx1 treatment stabilizes both IL-8 and GRO-alpha mRNA. We conclude that Stxs are able to increase mRNA and protein levels of multiple C-X-C chemokines in IECs, with increased mRNA stability at least one mechanism involved. We hypothesize that ribotoxic stress is a pathway by which Stxs can alter host signal transduction in IECs, resulting in the production of multiple chemokine mRNAs, leading to increased expression of specific proteins. Taken together, these data suggest that exposing IECs to Stxs may stimulate a proinflammatory response, resulting in influx of acute inflammatory cells and thus contributing to the intestinal tissue damage seen in STEC infection.     

Chemokine expression during acute rejection of rat kidneys

During acute rejection of fully allogeneic rat renal allografts, few neutrophil granulocytes are detected, whereas an abundant infiltrate of macrophages and T lymphocytes becomes apparent. The mechanisms leading to this specific pattern of infiltration are not understood. We performed a sequential daily Northern blot analysis of the mRNA expression of the CC-chemokines MCP-1, MIP-1alpha and RANTES and of the CXC-chemokines GRO/KC and MIP-2 in rat renal isografts (LEW --> LEW, n = 1 per day) and allografts during acute rejection (DA --> LEW, n = 3 per day). MCP-1 gene expression strongly increased on days 3-4 after allotransplantation and returned to control levels on day 6. The expression of MIP-1alpha and RANTES continuously rose until day 3-4 and remained stable thereafter. Isografts displayed minor changes in CC-chemokine expression. In contrast to CC-chemokines, GRO/KC was expressed in low amounts during rejection and MIP-2 mRNA remained undetectable. In conclusion, the expression of the CC-chemokines MCP-1, MIP-1 and RANTES was clearly upregulated during rejection, whereas the mRNA of the CXC-chemokines MIP-2 and GRO/KC was not detected at all or remained at low levels. This pattern of chemokine gene expression is in good accordance with the predominant mononuclear leukocyte infiltrate in allografts.     

Control of chemokine production at the blood-retina barrier

Chemokine production at the blood-retina barrier probably plays a critical role in determining the influx of tissue-damaging cells from the circulation into the retina during inflammation. The blood-retina barrier comprises the retinal microvascular endothelium and the retinal pigment epithelium. Chemokine expression and production by human retinal microvascular endothelial cells (REC) have never been reported previously, so we examined the in vitro expression and production of monocyte chemoattractant protein-1 (MCP-1), regulated on activation of normal T-cell expressed and secreted (RANTES), macrophage inflammatory protein (MIP)-1alpha, MIP-1beta, interleukin (IL)-8, epithelial cell-derived neutrophil activating protein-78 (ENA-78) and growth related oncogene alpha (GROalpha) in these cells, both unstimulated and stimulated by cytokines likely to be present during the evolution of an inflammatory response. We compared this to expression and production of these chemokines in vitro in human retinal pigment epithelial cells (RPE). MCP-1 was expressed and produced constitutively by REC but all the chemokines were produced in greater amounts upon stimulation with the proinflammatory cytokines IL-1beta and tumour necrosis factor-alpha (TNF-alpha). MCP-1 and IL-8 were produced at much higher levels than the other chemokines tested. MIP-1alpha and MIP-1beta were present only at low levels, even after stimulation with IL-1beta and TNF-alpha. Cytokines with greater anti-inflammatory activity, such as IL-4, IL-10, IL-13, transforming growth factor-beta (TGF-beta) and IL-6, had little effect on chemokine production either by REC alone or after stimulation with IL-1beta and TNF-alpha. RPE, although a very different cell type, showed a similar pattern of expression and production of chemokines, indicating the site-specific nature of chemokine production. Chemokine production by REC and RPE is probably significant in selective leucocyte recruitment during the development of inflammation in the retina.     

Helicobacter pylori lipopolysaccharides preferentially induce CXC chemokine production in human monocytes

Helicobacter pylori infection can cause duodenal ulcers and may also induce gastric adenocarcinoma. The bacteria colonize the gastric mucosa and areas of gastric metaplasia in the duodenum for decades, resulting in active chronic inflammation in the infected areas. A characteristic feature of the infection is the ongoing recruitment of neutrophils to the infected sites. To evaluate the role of H. pylori lipopolysaccharides (LPS) in the recruitment of leukocytes to the gastric mucosa, we have examined the cytokine and chemokine production from human monocytes stimulated with LPS isolated from different H. pylori strains, as well as from several other gram-negative bacteria. Our results show that H. pylori LPS induce a large production of neutrophil-recruiting CXC chemokines (interleukin-8 and growth-related oncogene alpha) from purified human monocytes, to almost the same extent as Escherichia coli LPS. However, and in agreement with previous studies, H. pylori LPS was much less potent in inducing production of proinflammatory cytokines by purified human monocytes and was also a weak inducer of the CC chemokine RANTES. There was no difference between LPS preparations from different H. pylori strains in their ability to induce cytokines and chemokines. The preferential production of CXC chemokines after stimulation with H. pylori LPS indicates an important contribution of this molecule in maintaining neutrophil recruitment during the infection, irrespective of the infecting strain.     

Differential and additive effects of platelet-derived chemokines on monocyte arrest on inflamed endothelium under flow conditions

Platelet-derived chemokines, such as regulated on activation, normal T expressed and secreted (RANTES; CC chemokine ligand 5), platelet factor 4 [PF4; CXC chemokine ligand 4 (CXCL4)], and epithelial neutrophil-activating protein 78 (ENA-78; CXCL5), or precursors, such as beta-thromboglobulin, which can be processed to neutrophil-activating protein-2 (NAP-2; CXCL7), may play an important role in monocyte recruitment during atherogenesis. Platelets can deposit chemokines on inflamed endothelium; however, little is known about differential or additive effects of platelet chemokines on monocyte arrest. Here, we demonstrate that preincubation of activated human microvascular endothelial cells (HMVECs) with RANTES, PF4, or NAP-2 but not ENA-78 dose-dependently increased surface immobilization and subsequent monocyte arrest in flow. RANTES was the most potent and efficient arrest chemokine. Pretreatment of HMVECs with beta-thromboglobulin enhanced monocyte arrest in the presence of cathepsin G generating NAP-2. Combined pretreatment of HMVECs with RANTES and PF4 at suboptimal concentrations synergistically increased arrest, and preincubation with chondroitinase ABC abrogated RANTES- and PF4-induced monocyte arrest. This was associated with reduced expression of chondroitin sulfate, RANTES, and PF4 on the HMVEC surface. Perfusion of HMVECs with platelets known to deposit RANTES and PF4 on the endothelial surface enhanced monocyte arrest, which was inhibited by Met-RANTES, chondroitinase, or a blocking antibody to PF4 but not to ENA-78. The relevance of platelet-derived chemokines was confirmed in adhesion assays with activated whole blood, where Met-RANTES and to a lesser extent, antibodies to PF4 and NAP-2 inhibited arrest of CD14-positive monocytes. Thus, multiple platelet-derived chemokines and processable precursors, which can be presented by specific endothelial proteoglycans, may contribute and cooperate differentially to induce monocyte recruitment.     

TLR4 single-nucleotide polymorphisms alter mucosal cytokine and chemokine patterns in Mexican patients with Helicobacter pylori-associated gastroduodenal diseases

Helicobacter pylori is associated with peptic ulcer and gastric adenocarcinoma. Toll-like receptors (TLRs) participate in H. pylori recognition, and single-nucleotide polymorphisms (SNPs) in TLRs are associated with impaired immune response. We aimed to evaluate the association of TLR2/R753Q and TLR4/D299G/T399I SNPs with gastroduodenal diseases; and study the effect of SNPs on cytokine and chemokine expression in the gastric mucosa. Study included 450 Mexican patients with gastroduodenal diseases. SNPs in TLRs 2 and 4 genes were analyzed by allele-specific PCR. Cytokines and chemokines were assessed by qRT-PCR and immunoassay. TLR4/D299G/T399I polymorphisms were more frequent in duodenal ulcer and showed a trend in gastric cancer, when compared with non-atrophic gastritis. Patients with TLR4 polymorphisms expressed significantly lower levels of IL-1beta, IL-6, IL-8 and GRO-alpha; and higher levels of TNF-alpha, IL-10, MCP-1 and MIP-1alpha . SNPs in TLR4 gene had an association with severe H. pylori-associated disease and with modified pattern of inflammatory cytokines and chemokines in the gastric mucosa. These results suggest that TLR4 SNPs contributes importantly to the clinical outcome of H. pylori infection.     

Keratinocytes from patients with atopic dermatitis and psoriasis show a distinct chemokine production profile in response to T cell-derived cytokines

BACKGROUND: Atopic dermatitis (AD) and psoriasis are genetically determined inflammatory skin disorders. Keratinocytes actively participate in cutaneous inflammatory responses by elaborating various chemokines. OBJECTIVE: We investigated the capacity of IL-4, IFN-gamma, and TNF-alpha to modulate the expression of CCL and CXCL chemokines in cultured keratinocytes from patients and healthy individuals, as well as chemokine expression in situ. METHODS: Keratinocyte cultures were established from normal-looking skin of adult patients with AD or psoriasis vulgaris and from healthy subjects. Monocyte chemoattractant protein 1 (MCP-1)/CCL2, RANTES/CCL5, IL-8/CXCL8, and IFN-gamma-induced protein of 10 kd (IP-10)/CXCL10 production was evaluated at the mRNA and protein levels by using RNase protection assay and ELISA, respectively. The expression of the same chemokines was studied in chronic lesional skin by means of immunohistochemistry or in situ hybridization. RESULTS: Only IL-8 mRNA was detected in unstimulated ke-ratinocyte cultures. MCP-1 and IP-10 were potently induced by IFN-gamma, whereas IL-8 and RANTES were preferentially upregulated by TNF-alpha and, to a lesser extent, by IFN-gamma. IL-4 weakly induced IP-10, RANTES, and IL-8 but not MCP-1. Keratinocytes of patients with AD invariably responded with significantly earlier and higher RANTES expression. By contrast, keratinocytes of patients with psoriasis displayed much higher levels of both constitutive and induced IL-8 and a stronger induction of MCP-1 and IP-10. RANTES and MCP-1 mRNA(+) keratinocytes were detected in the basal layer of lesions of patients with AD and psoriasis. IP-10 and IL-8 were consistently upregulated in the epidermis of patients with psoriasis but not in lesions of patients with AD. CONCLUSIONS: Keratinocytes of patients with AD and psoriasis show an intrinsically abnormal and different chemokine production profile and may thus favor the recruitment of distinct leukocyte subsets into the skin.     

Expression of CC and CXC chemokines and chemokine receptors in human leprosy skin lesions

We have investigated the expression of chemokines and their receptors in leprosy skin lesions using immunohistochemistry. Skin biopsies from 25 leprosy patients across the leprosy spectrum, 11 patients undergoing type I reversal reactions and four normal donors were immunostained by ABC peroxidase method using antibodies against CC and CXC chemokines and their receptors. Using an in situ hybridization technique we have also studied the expression of monocyte chemoattractant protein 1 (MCP-1), RANTES and interleukin (IL)-8 chemokines mRNA in leprosy skin lesions. Chemokines and receptor expression was detected in all leprosy skin biopsies. Expression of CC chemokines MCP-1 (P < 0.01) and RANTES (P < 0.01) were elevated significantly in borderline tuberculoid leprosy in reversal reaction compared to non-reactional borderline tuberculoid leprosy, but there was no difference in the expression of IL-8 chemokine. Surprisingly, there was no significant difference in the expression of CC (CCR2 and CCR5) and CXC (CXCR2) chemokine receptors across the leprosy spectrum. Similarly, there was no significant difference in the expression of mRNA for MCP-1, regulated upon activation normal T cell expressed and secreted (RANTES) and IL-8 chemokines. Here, the presence of a neutrophil chemoattractant IL-8 in leprosy lesions, which do not contain neutrophils, suggests strongly a role of IL-8 as a monocyte and lymphocyte recruiter in leprosy lesions. These results suggest that the chemokines and their receptors, which are known to chemoattract T lymphocytes and macrophages, are involved in assembling the cellular infiltrate found in lesions across the leprosy spectrum.     

Profiling chemokines, cytokines and growth factors in human early pregnancy decidua by protein array

PROBLEM: In human decidua, a significant increase of leukocytes including CD56(++)CD16(-) uterine natural killer (uNK) cells and CD14(+) monocytes has been observed with the onset of pregnancy. The mechanisms required for the recruitment of those cells to the uterus are still under debate. Cytokines and chemokines have been suggested as key factors for the regulation of these complex cellular migration and interaction processes. METHOD OF STUDY: Investigation of the expression patterns of cytokines, chemokines and growth factors in human decidual tissue (7-8 weeks of gestation) was performed by protein array analysis. Isolated decidual leukocytes, i.e. CD56(++)CD16(-) uNK, CD14(+) monocytes as well as cytotrophoblast (CTB) and stromal cells were tested separately. RESULTS: This analysis revealed the production of monocyte attracting chemokines (GRO, MCP-1), angiogenetic substances (EGF, VEGF, Angiogenin) as well as granulocyte activating peptides (ENA-78, IL-1beta, RANTES, IL-8) by decidual tissue and its cell subsets. CONCLUSION: The observed pattern supports the role of decidua as a tissue which promotes angiogenesis, attracts monocytes and modulates the function of the latter.