Cytokines and pregnancy in rheumatic disease

Cytokines are important mediators involved in the successful outcome of pregnancy. The concept of pregnancy as biased toward a Th2 immune response states that Th1 type cytokines are associated with pregnancy failure and that Th2 cytokines are protective and counteract pregnancy-related disorders. Studies at the level of the maternal-fetal interface, in the maternal circulation and in cells of peripheral blood have shown that the Th2 concept of pregnancy is an oversimplification. Both Th1 and Th2 type cytokines play a role at different stages of pregnancy and are adapted to the localization and function of cells and tissues. The changes of local and systemic cytokine patterns during pregnancy correspond to neuroendocrine changes with hormones as powerful modulators of cytokine expression. Several autoimmune disorders show a modulation of disease activity during and after pregnancy. In rheumatic diseases with a predominance of a Th1 immune response, a shift to a Th2 type immune response during pregnancy has been regarded as beneficial. Studies of pregnant patients with rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE) have shown a cytokine expression similar to that found in healthy pregnant women. Significant differences were present only for a few cytokines and seemed related to the activity of the underlying disease. Interestingly, a gestational increase of cytokine inhibitors interleukin 1 receptor antagonist (IL-1ra) and soluble tumor necrosis factor receptor (sTNFR) in the circulation corresponded to low disease activity in RA. The influence of hormones and cytokines on autoimmune disease is an issue for further study.

Increased ENA-78 in the follicular fluid of patients with endometriosis

BACKGROUND: It is known that endometriosis is an inflammatory disease and those patients seem to have lower pregnancy rates. The aim of the study was to investigate the concentrations of chemokines and proinflammatory cytokines in the follicular fluid of patients with and without endometriosis. METHODS: Follicular aspiration, recovering follicular fluid during assisted reproductive treatment, follicular fluid storage and analysis of chemokines and proinflammatory cytokines were carried out. Tumor necrosis factor-alpha, interleukin-1 beta, interleukin-6, interleukin-8, interleukin-15, leukemia inhibitory factor, epithelial neutrophil-activating peptide 78, regulated upon activation, normal T-cell expressed and secreted, and growth-regulated oncogene-alpha were analyzed in the follicular fluid and compared between women with (n =47) and without endometriosis (n = 279). RESULTS: The above cytokines were detected in the follicular fluid samples. Epithelial neutrophil-activating peptide 78 levels were significantly higher in follicular fluid from endometriosis patients than from controls (p = 0.008). Increases (to twice the control level) were also observed for tumor necrosis factor-alpha and for interleukin-6. CONCLUSIONS: Increased follicular fluid levels of epithelial neutrophil-activating peptide 78, tumor necrosis factor-alpha and interleukin-6 indicate that these cytokines may influence oocyte quality and fecundability of women with endometriosis by deteriorating the microenvironment in the human follicle.

Predictors of inflammation in response to anthracycline-based chemotherapy for breast cancer

Although chemotherapy for breast cancer can increase inflammation, few studies have examined predictors of this phenomenon. This study examined potential contributions of demographics, disease characteristics, and treatment regimens to markers of inflammation in response to chemotherapy for breast cancer. Thirty-five women with stage I-III-A breast cancer (mean age 50 years) were studied prior to cycle 1 and prior to cycle 4 of anthracycline-based chemotherapy. Circulating levels of inflammatory markers with high relevance to breast cancer were examined, including C-reactive protein (CRP), tumor necrosis factor-alpha (TNF-alpha), Interleukin-1 receptor antagonist (IL1-RA), vascular endothelial growth factor (VEGF), soluble intercellular adhesion molecule-1 (sICAM-1), Interleukin- (IL-6), soluble P-selectin (sP-selectin), and von Willebrand factor (vWf). Chemotherapy was associated with elevations in VEGF (p < or = 0.01), sICAM-1 (p < or = 0.01), sP-selectin (p < or = 0.02) and vWf (p < or = 0.05). Multiple regression analysis controlling for age and body mass index (BMI) showed that higher post-chemotherapy levels of inflammation were consistently related to higher pre-chemotherapy levels of inflammation (ps < or =0.05) as well as to certain disease characteristics. Post-chemotherapy IL-6 levels were higher in patients who had larger tumors (p < or = 0.05) while post-chemotherapy VEGF levels were higher in patients who had smaller tumors (p < or = 0.05). Post-chemotherapy sP-selectin levels were highest in women who had received epirubicin, cytoxan, 5-fluorouracil chemotherapy (p < or = 0.01). These findings indicate that chemotherapy treatment can be associated with elevations in certain markers of inflammation, particularly markers of endothelial and platelet activation. Inflammation in response to chemotherapy is most significantly related to inflammation that existed prior to chemotherapy but also potentially to treatment regimen and to certain disease characteristics.

Expression and regulation of antimicrobial peptide rCRAMP after bacterial infection in primary rat meningeal cells

Bacterial meningitis is characterized by an inflammation of the meninges and continues to be an important cause of mortality and morbidity. Meningeal cells cover the cerebral surface and are involved in the first interaction between pathogens and the brain. Little is known about the role of meningeal cells and the expression of antimicrobial peptides in the innate immune system. In this study we characterized the expression, secretion and bactericidal properties of rat cathelin-related antimicrobial peptide (rCRAMP), a homologue of the human LL-37, in rat meningeal cells after incubation with different bacterial supernatants and the bacterial cell wall components lipopolysaccharide (LPS) and peptidoglycan (PGN). Using an agar diffusion test, we observed that supernatants from meningeal cells incubated with bacterial supernatants, LPS and PGN showed signs of antimicrobial activity. The inhibition of rCRAMP expression using siRNA reduced the antimicrobial activity of the cell culture supernatants. The expression of rCRAMP in rat meningeal cells involved various signal transduction pathways and was induced by the inflammatory cytokines interleukin-1, -6 and tumor necrosis factor alpha. In an experimental model of meningitis, infant rats were intracisternally infected with Streptococcus pneumoniae and rCRAMP was localized in meningeal cells using immunohistochemistry. These results suggest that cathelicidins produced by meningeal cells play an important part in the innate immune response against pathogens in CNS bacterial infections.

The potential association between gingival crevicular fluid inflammatory mediators and adverse pregnancy outcomes: a systematic review

OBJECTIVES The association between periodontal disease and adverse pregnancy outcomes (APO), primarily preterm birth (PTB), is still controversially discussed in the literature. Therefore, the aim of the present systematic review was to analyze the existing literature on the potential association between inflammatory mediators detected in gingival crevicular fluid (GCF) and APO. MATERIALS AND METHODS MEDLINE (PubMed) and EMBASE databases were searched for entries up to April 2012 and studies were selected by two independent reviewers. RESULTS The majority of the eight studies included confirmed a positive association between GCF mediators, such as interleukin-1β, prostaglandin E2, and tumor necrosis factor-alpha, and APO. Due to the heterogeneity and variability of the available studies, no meta-analysis could be performed. CONCLUSIONS A positive association between GCF inflammatory mediator levels and APO/PTB might be present but the results need to be considered with great caution because of the heterogeneity and variability among the studies. Further studies with an adequate number of patients allowing for an appropriate analysis are warranted to definitely confirm this association. CLINICAL RELEVANCE The present findings suggest that an association between GCF inflammatory mediator levels and APO might exist.

Matrix metalloproteinase inhibition lowers mortality and brain injury in experimental pneumococcal meningitis

Pneumococcal meningitis (PM) results in high mortality rates and long-lasting neurological deficits. Hippocampal apoptosis and cortical necrosis are histopathological correlates of neurofunctional sequelae in rodent models and are frequently observed in autopsy studies of patients who die of PM. In experimental PM, inhibition of matrix metalloproteinases (MMPs) and/or tumor necrosis factor (TNF)-converting enzyme (TACE) has been shown to reduce brain injury and the associated impairment of neurocognitive function. However, none of the compounds evaluated in these studies entered clinical development. Here, we evaluated two second-generation MMP and TACE inhibitors with higher selectivity and improved oral availability. Ro 32-3555 (Trocade, cipemastat) preferentially inhibits collagenases (MMP-1, -8, and -13) and gelatinase B (MMP-9), while Ro 32-7315 is an efficient inhibitor of TACE. PM was induced in infant rats by the intracisternal injection of live Streptococcus pneumoniae. Ro 32-3555 and Ro 32-7315 were injected intraperitoneally, starting at 3 h postinfection. Antibiotic (ceftriaxone) therapy was initiated at 18 h postinfection, and clinical parameters (weight, clinical score, mortality rate) were recorded. Myeloperoxidase activities, concentrations of cytokines and chemokines, concentrations of MMP-2 and MMP-9, and collagen concentrations were measured in the cerebrospinal fluid. Animals were sacrificed at 42 h postinfection, and their brains were assessed by histomorphometry for hippocampal apoptosis and cortical necrosis. Both compounds, while exhibiting disparate MMP and TACE inhibitory profiles, decreased hippocampal apoptosis and cortical injury. Ro 32-3555 reduced mortality rates and cerebrospinal fluid TNF, interleukin-1β (IL-1β) and collagen levels, while Ro 32-7315 reduced weight loss and cerebrospinal fluid TNF and IL-6 levels.

Differential effect of p47phox and gp91phox deficiency on the course of Pneumococcal Meningitis.

Bacterial meningitis is a severe inflammatory disease of the central nervous system and is characterized by massive infiltration of granulocytes into the cerebrospinal fluid (CSF). To assess the role of NADPH oxidase-derived reactive oxygen species (ROS) in pneumococcal meningitis, mice deficient in either the gp91 subunit (essential for functioning of the phagocyte enzyme) or the p47 subunit (essential for functioning of homologous enzymes in nonphagocytic cells) were intracisternally infected with live Streptococcus pneumoniae, and defined disease parameters were measured during the acute stage of infection. While none of the parameters measured (including CSF bacterial titers) were significantly different in gp91(-/-) and wild-type mice, the infection in p47(-/-) mice was associated with significantly increased inflammation of the subarachnoid and ventricular space, disruption of the blood-brain barrier, and the presence of interleukin-1 beta, tumor necrosis factor alpha, and matrix metalloproteinase 9 in the cortex. These changes were associated with approximately 10-fold-higher CSF bacterial titers in p47(-/-) mice than in wild-type mice (P < 0.001). In contrast to infection with live bacteria, the inflammatory response, including CSF leukocytosis, was significantly attenuated in p47(-/-) mice (but not gp91(-/-) mice) challenged with a fixed number of heat-inactivated pneumococci. Impairment of the host defense appeared to be responsible for the higher bacterial titers in p47(-/-) mice. Therefore, these results indicate that ROS generated by a gp91-independent NADPH oxidase(s) are important for establishing an adequate inflammatory response to pneumococcal CSF infection.

Effects of stimulus with proinflammatory mediators on nitric oxide production and matrix metalloproteinase activity in explants of cranial cruciate ligaments obtained from dogs

OBJECTIVE To evaluate the origin and degree of activity of nitric oxide (NO) and matrix metalloproteinase (MMP) in explants of cranial cruciate ligaments (CCLs) obtained from dogs and cultured with and without inflammatory activators. SAMPLE POPULATION Tissue specimens obtained from 7 healthy adult Beagles that were (mean +/- SD) 4.5 +/- 0.5 years old and weighed 12.5 +/- 0.8 kg. PROCEDURE The CCLs were harvested immediately after dogs were euthanatized, and specimens were submitted for explant culture. Cultures were stimulated by incubation with a combination of interleukin-1, tumor necrosis factor-alpha, and lipopolysaccharide, or they were not stimulated. Culture supernatants were examined for production of NO nitrite-nitrate metabolites (NOts) and activity of MMP Cultured specimens were evaluated by use of immunohistochemical analysis to detect activity of inducible NO synthase (iNOS). RESULTS All ligament explants produced measurable amounts of NOts. Stimulated cultures produced significantly more NOts after incubation for 24 and 48 hours, compared with nonstimulated cultures. Production of MMP in supernatants after incubation for 48 hours was significantly higher in stimulated cultures than in nonstimulated cultures. Cells with positive staining for iNOS were detected on all slides. Positively stained cells were predominantly chondroid metaplastic. There was a significant difference in intensity of cell staining between stimulated and non-stimulated cultures. CONCLUSIONS AND CLINICAL RELEVANCE Explant cultures of intact CCLs obtained from dogs produce iNOS-induced NO. Stimulation of chondroid metaplastic cells in CCL of dogs by use of inflammatory activators can increase production of iNOS, NOts, and MMP.

Generation and functional characterization of ovine bone marrow-derived macrophages.

A method for the culturing and propagation of ovine bone marrow-derived macrophages (BMM) in vitro is described. Bone marrow cells from sterna of freshly slaughtered sheep were cultured in hydrophobic (teflon foil) bags in the presence of high serum concentrations (20% autologous serum and 20% fetal calf serum). During an 18 day culture period in the absence of added conditioned medium, and without medium change, a strong enrichment of mononuclear phagocytes was achieved. Whereas the number of macrophages increased four to fivefold during this time, granulocytes, lymphoid cells, stem cells and undifferentiated progenitor cells were reduced to less than 3% of their numbers at Day 0. This resulted in BMM populations of 94 +/- 3% purity. These cells had morphological and histochemical characteristics of differentiated macrophages, and they performed functions similar to those of non-activated, unprimed human monocyte-derived macrophages. Thus, they avidly ingested erythrocytes coated with IgG of heterologous or homologous origin. They expressed a modest level of procoagulant activity, but upon triggering with lipopolysaccharide (LPS), a marked increase in cell-associated procoagulant activity was observed. LPS triggering promoted the secretion of interleukin-1, as evidenced by measurement of murine thymocyte costimulatory activity, and transforming growth factor-beta. Using the mouse L929 cell cytotoxicity assay as an indication of tumor necrosis factor (TNF) activity, no TNF activity was detected in the same supernatants, a result possibly due to species restriction. BMM generated low levels of O2- upon triggering with phorbol 12-myristate 13-acetate (PMA). On the other hand, no O2- production was observed upon stimulation with zymosan opsonized with ovine or human serum. Using luminol-enhanced chemiluminescence (CL) as a more sensitive indicator of an oxidative burst, both PMA or zymosan were able to trigger CL, but the response was subject to partial inhibition by sodium azide, an inhibitor of myeloperoxidase. This points to non-macrophage cells contributing also to the CL response, and is consistent with the view that unprimed BMM elicit a low oxidative burst upon triggering with strong inducers of a burst. Our functional characterization now allows us to apply priming and activation protocols and to relate their effect to functional alterations.

Culture of ovine bone marrow-derived macrophages and evidence for serum factors distinct from M-CSF contributing to their propagation in vitro.

An in vitro system allowing the culture of ovine bone marrow-derived macrophages (BMMs) is described. Bone marrow (BM) cells from the sternum of 4- to 9-month-old sheep were cultured in liquid suspension in hydrophobic bags with medium containing 20% autologous serum and 20% fetal calf serum (FCS). Cells with macrophage characteristics were positively selected and increased four- to five-fold between day (d) 0 and d18. Granulocytes and cells of lymphoid appearance including progenitor cells were negatively selected and were diminished 50-fold during this 18-d culture. The addition of macrophage colony-stimulating factor (M-CSF)-containing supernatants to liquid cultures did not significantly improve the yield of BMM in 18-d cultures. In contrast, cell survival at d6 and macrophage cell yield at d18 depended on the concentration and source of serum in the culture medium. FCS and 1:1 mixtures of FCS and autologous serum were superior to autologous serum alone. Analysis of growth requirements of ovine BMMs suggested that they are under more complex growth control than their murine counterparts. In an [3H]thymidine incorporation assay with BM cells collected at different times of culture, d3 or d4 BM cells responded to human recombinant M-CSF, human recombinant granulocyte-macrophage colony-stimulating factor (GM-CSF), bovine GM-CSF, murine M-CSF or murine M-CSF-containing supernatants, and bovine interleukin 1 beta (IL-1 beta) in decreasing order of magnitude. Likewise, pure murine BMM populations harvested at d6 responded to homologous GM-CSF, IL-3, and human or murine M-CSF. FCS did not stimulate the proliferation of murine BMMs (d6) and of ovine BM cells (d3 or d4). In contrast, ovine BM cells harvested at d12 responded to FCS by proliferation in a dose-dependent manner but failed to proliferate in the presence of human or murine M-CSF or M-CSF-containing supernatants of mouse and sheep fibroblasts containing mouse macrophage growth-promoting activity. Likewise, various cytokine-containing supernatants and recombinant cytokines (murine IL-3, murine and human GM-CSF, murine and bovine IL-1 beta) did not promote proliferation of ovine d12 BM cells to an extent greater than that achieved with 15% FCS alone. Thus, ovine BMM proliferation is under the control of at least two factors acting in sequence, M-CSF and an unidentified factor contained in FCS. The ovine BMM culture system may provide a model for the analysis of myelomonocytopoiesis in vitro.

IL1B and DEFB1 Polymorphisms Increase Susceptibility to Invasive Mold Infection After Solid Organ Transplantation.

BACKGROUND  Single nucleotide polymorphisms (SNPs) in immune genes have been associated with susceptibility to invasive mold infection (IMI) among hematopoietic stem cell (HSCT) but not solid organ transplant (SOT) recipients. METHODS  24 SNPs from systematically selected genes were genotyped among 1101 SOT recipients (715 kidneys, 190 liver, 102 lungs, 79 hearts, 15 other) from the Swiss Transplant Cohort Study. Association between SNPs and the endpoint were assessed by log-rank test and Cox regression models. Cytokine production upon Aspergillus stimulation was measured by ELISA in PBMCs from healthy volunteers and correlated with relevant genotypes. RESULTS  Mold colonization (N=45) and proven/probable IMI (N=26) were associated with polymorphisms in interleukin-1 beta (IL1B, rs16944; log-rank test, recessive mode, colonization P=0.001 and IMI P=0.00005), interleukin-1 receptor antagonist (IL1RN, rs419598; P=0.01 and P=0.02) and β-defensin-1 (DEFB1, rs1800972; P=0.001 and P=0.0002, respectively). The associations with IL1B and DEFB1 remained significant in a multivariate regression model (IL1B rs16944 P=0.002; DEFB1 rs1800972 P=0.01). Presence of two copies of the rare allele of rs16944 or rs419598 was associated with reduced Aspergillus-induced IL-1β and TNFα secretion by PBMCs. CONCLUSIONS  Functional polymorphisms in IL1B and DEFB1 influence susceptibility to mold infection in SOT recipients. This observation may contribute to individual risk stratification.

Regulation of NAMPT in human gingival fibroblasts and biopsies

Adipokines, such as nicotinamide phosphoribosyltransferase (NAMPT), are molecules, which are produced in adipose tissue. Recent studies suggest that NAMPT might also be produced in the tooth-supporting tissues, that is, periodontium, which also includes the gingiva. The aim of this study was to examine if and under what conditions NAMPT is produced in gingival fibroblasts and biopsies from healthy and inflamed gingiva. Gingival fibroblasts produced constitutively NAMPT, and this synthesis was significantly increased by interleukin-1β and the oral bacteria P. gingivalis and F. nucleatum. Inhibition of the MEK1/2 and NFκB pathways abrogated the stimulatory effects of F. nucleatum on NAMPT. Furthermore, the expression and protein levels of NAMPT were significantly enhanced in gingival biopsies from patients with periodontitis, a chronic inflammatory infectious disease of the periodontium, as compared to gingiva from periodontally healthy individuals. In summary, the present study provides original evidence that gingival fibroblasts produce NAMPT and that this synthesis is increased under inflammatory and infectious conditions. Local synthesis of NAMPT in the inflamed gingiva may contribute to the enhanced gingival and serum levels of NAMPT, as observed in periodontitis patients. Moreover, local production of NAMPT by gingival fibroblasts may represent a possible mechanism whereby periodontitis may impact on systemic diseases.

Cytokine induction in human mononuclear cells stimulated by IgG-coated culture surfaces and by IgG for infusion

The effect of IgG on cytokine production by human mononuclear cells (MNC) was studied. Tumor necrosis factor-alpha (TNF) was determined both by bioassay and by immunoassay. Interleukin-1 (IL1) was measured by a thymocyte costimulator assay, which was shown to be completely inhibitable by polyclonal anti-IL1. Precautions were taken to avoid inadvertent exposure of the studied cells to endotoxin. In a first model, TNF and IL1 production by adherent MNC in IgG-coated cluster plates were determined. IgG induced a strong TNF response, usually leveling off after 6 hr, and was comparable in kinetics and magnitude with an LPS-induced response. The thymocyte co-stimulatory activity response was relatively weak and peaked at 6 hr. In contrast, LPS-induced co-stimulatory activity production steadily increased over 24 hr. In a second model, MNC in suspension cultures containing autologous serum were exposed to IgG for intravenous use (IgG-IV). Cells exposed to IgG-IV produced higher amounts of cytokines than control counterparts and were primed for enhanced production of cytokines upon a second, unrelated stimulus. This implies that the effect of IgG-IV on suspended MNC resembles that of surface-adsorbed IgG and raises the possibility that cytokine release is an integral part of the mechanism of action of infused IgG. Evidence is presented suggesting that both surface IgG and IgG-IV act directly on monocytes, in a Fc-dependent manner.

CCL5/RANTES is a key chemoattractant released by degenerative intervertebral discs in organ culture.

Release of chemotactic factors in response to tissue damage has been described for different musculoskeletal tissues, including the intervertebral disc (IVD). This study investigated the chemoattractants that are released by induced degenerative IVDs and may be involved in recruiting mesenchymal stem cells (MSCs). Bovine caudal discs were cultured within a bioreactor and loaded under conditions that mimicked physiological or degenerative settings. Between days 4-6, medium was replaced by PBS, which was subsequently used for proteomic, ELISA and immunoprecipitation analyses of secreted chemokines and cytokines. A Boyden chamber assay was used to observe human MSC migration towards native and chemokine depleted media. Gene expression levels of chemokine receptors in human MSCs were analysed, and CCL5 was localised in bovine and human IVD by immunohistochemistry. Proteomic analysis revealed the presence of CCL5 and CXCL6 within conditioned media. Higher concentrations of CCL5 were found in the degenerative media, and a relationship was found between interleukin-1β and CCL5 concentration. Chemokine immunoprecipitation showed that MSCs had a significantly reduced chemotactic migration towards CCL5-immunoprecipitated and CCL5/CXCL6 co-immunoprecipitated media, whilst CXCL6 depletion did not change MSC chemotaxis. MSCs showed a significant increase in mRNA expression of the CCL5 receptors, CCR1 and CCR4, upon culture in degenerative media. Furthermore, CCL5 was identified in bovine and human disc tissue by immunohistochemistry. Hence, CCL5 may be a key chemoattractant that is produced and released by the intervertebral disc cells. Therefore, these factors could be used to enhance stem/progenitor cell mobilisation in regenerative therapies for early stages of disc degeneration.

HLA class II antigen expression in colorectal carcinoma tumors as a favorable prognostic marker.

The goal of this study was to determine the frequency of HLA class II antigen expression in colorectal carcinoma (CRC) tumors, its association with the clinical course of the disease, and the underlying mechanism(s). Two tissue microarrays constructed with 220 and 778 CRC tumors were stained with HLA-DR, DQ, and DP antigen-specific monoclonal antibody LGII-612.14, using the immunoperoxidase staining technique. The immunohistochemical staining results were correlated with the clinical course of the disease. The functional role of HLA class II antigens expressed on CRC cells was analyzed by investigating their in vitro interactions with immune cells. HLA class II antigens were expressed in about 25% of the 220 and 21% of the 778 tumors analyzed with an overall frequency of 23%. HLA class II antigens were detected in 19% of colorectal adenomas. Importantly, the percentage of stained cells and the staining intensity were significantly lower than those detected in CRC tumors. However, HLA class II antigen staining was weakly detected only in 5.4% of 37 normal mucosa tissues. HLA class II antigen expression was associated with a favorable clinical course of the disease. In vitro stimulation with interferon gamma (IFNγ) induced HLA class II antigen expression on two of the four CRC cell lines tested. HLA class II antigen expression on CRC cells triggered interleukin-1β (IL-1β) production by resting monocytes. HLA class II antigen expression in CRC tumors is a favorable prognostic marker. This association may reflect stimulation of IL-1β production by monocytes.