Development of a co-culture of keratinocytes and immune cells for in vitro investigation of cutaneous sulfur mustard toxicity.

Sulfur mustard (SM) is a chemical warfare agent causing skin blistering, ulceration and delayed wound healing. Inflammation and extrinsic apoptosis are known to have an important role in SM-induced cytotoxicity. As immune cells are involved in those processes, they may significantly modulate SM toxicity, but the extent of those effects is unknown. We adapted a co-culture model of immortalized keratinocytes (HaCaT) and immune cells (THP-1) and exposed this model to SM. Changes in necrosis, apoptosis and inflammation, depending on SM challenge, absence or presence and number of THP-1 cells were investigated. THP-1 were co-cultured for 24h prior to SM exposure in order to model SM effects on immune cells continuously present in the skin. Our results indicate that the presence of THP-1 strongly increased necrosis, apoptosis and inflammation. This effect was already significant when the ratio of THP-1 and HaCaT cells was similar to the ratio of Langerhans immune cells and keratinocytes in vivo. Any further increases in the number of THP-1 had only slight additional effects on SM-induced cytotoxicity. In order to assess the effects of immune cells migrating into skin areas damaged by SM, we added non-exposed THP-1 to SM-exposed HaCaT. Those THP-1 had only slight effects on SM-induced cytotoxicity. Notably, in HaCaT exposed to 300µM SM, necrosis and inflammation were slightly reduced by adding intact THP-1. This effect was dependent on the number of immune cells, steadily increasing with the number of unexposed THP-1 added. In summary, we have demonstrated that (a) the presented co-culture is a robust model to assess SM toxicity and can be used to test the efficacy of potential antidotes in vitro; (b) immune cells, damaged by SM strongly amplified cytotoxicity, (c) in contrast, unexposed THP-1 (simulating migration of immune cells into affected areas after exposure in vivo) had no pronounced adverse, but exhibited some protective effects. Thus, protecting immune cells from SM toxicity may help to reduce overall injury.

Functional polymorphisms in matrix metalloproteinases -1, -3, -9 and -12 in relation to cervical artery dissection.

BACKGROUND: Cervical artery dissection is a leading cause of cerebral ischemia in young adults. Morphological investigations have shown alterations in the extracellular matrix (ECM) of affected vessel walls. As matrix metalloproteinases (MMP) play a central role in the regulation of the ECM, an increased expression of these enzymes might lead to the endothelial damage in spontaneous cervical artery dissection (sCAD). Five different DNA polymorphisms in MMP-1, -3, -9 and -12 were tested for their frequency in patients with sCAD and compared with those of a control population. METHODS: Blood was sampled from 70 unrelated patients presenting consecutively in the department of neurology of the Aachen University Medical School with sCAD and from 87 control subjects living in the same area as the patients. The MMP polymorphisms were analyzed with hybridization probes using the LightCycler (Roche Diagnostics), by sequencing using the ABI 310 Genetic Analyzer (Applied Biosystems) and with the GeneScan program on a ABI 310 Genetic Analyzer. RESULTS: No statistically significant differences in the allelic distribution were found between sCAD patients and the controls. CONCLUSION: Alleles of these 5 functional polymorphisms of MMPs seem not to be associated with structural alterations in the blood vessel wall of sCAD patients. However, this does not exclude a pathogenetic role for MMPs in sCAD via secondary factors such as cytokines that are able to induce these enzymes in cervical blood vessel walls.

Cytokine response to endotoxin in individuals heterozygous for the Delta32 mutation of chemokine receptor CCR5.

Studies of mice with a targeted disruption of the CCR5 gene suggest that the CC chemokine receptor 5 (CCR5) is a determinant of the cytokine response to endotoxin. In humans, a naturally occurring mutation of the CCR5 gene is a 32-basepair (bp) deletion which precludes the translation of the gene into a functional transmembrane protein. To evaluate the cytokine phenotype of heterozygosity for the 32 deletion, we studied the endotoxin-stimulated release of tumor necrosis factor-alpha, Interleukin (IL)-6, IL-8, IL-10, and IL-12 in whole blood ex-vivo of healthy volunteers and patients undergoing elective cardiac bypass surgery. This operation represents a major surgical trauma associated with ischemia-reperfusion-injury and triggers a profound inflammatory response. In these patients, cytokine plasma concentrations were measured during and after cardiac surgery. No difference was found between the frequencies of the observed and expected CCR5 genotypes in the groups of individuals studied. Furthermore, no significant difference in ex-vivo or peri- and postoperative cytokine plasma concentrations was detected between CCR5 wild-type homozygotes and individuals carrying one defective CCR5 allele. Our results indicate that heterozygosity for the 32bp deletion of CCR5 is not associated with an altered cytokine response to endotoxin or to a major surgical trauma when compared with individuals homozygous for the wild-type allele.

Linkage disequilibrium between tumor necrosis factor (TNF)-alpha-308 G/A promoter and TNF-beta NcoI polymorphisms: Association with TNF-alpha response of granulocytes to endotoxin stimulation.

OBJECTIVE: Controversial data have been reported on the association between the tumor necrosis factor (TNF)-alpha-308 G[U279C]A promoter polymorphism or the TNF-alpha I polymorphism with TNF-alpha plasma concentrations. The purpose of this study was to evaluate whether there is a linkage disequilibrium between the two polymorphisms. Moreover, the influence of these polymorphisms on the TNF-alpha synthesis of activated granulocytes was studied. DESIGN: Analysis of TNF-alpha concentrations of human whole blood after endotoxin stimulation. SETTING: Medical research laboratory. PATIENTS: Healthy human volunteers. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Healthy human volunteers were genotyped for both TNF polymorphisms by means of polymerase chain reaction. TNF-alpha plasma concentrations were determined with chemiluminescence after incubation of whole blood with endotoxin. A strong (p <.0001) linkage disequilibrium was found for the TNF-beta I and the TNF-alpha-308 genetic polymorphisms. Almost all individuals homozygous for the TNF-B2 allele of the TNF-beta I polymorphism were also TNF-alpha-308 G homozygotes. Carriers of the TNF-alpha-308 genotype AG had a significantly higher TNF-alpha production capacity than G homozygotes. The TNF-beta I genotype TNF-B1/TNF-B2 was associated with significantly higher TNF-alpha concentrations than the genotype TNF-B2/TNF-B2. Individuals homozygous for the TNF-B2 and the TNF-alpha-308 G alleles had a significantly reduced TNF-alpha response compared with individuals heterozygous for both TNF polymorphisms. CONCLUSIONS: A linkage disequilibrium between the two TNF polymorphisms was found. This study revealed a significant association between genotype and phenotype for both TNF polymorphisms. Heterozygosity for both TNF polymorphisms is associated with an increased TNF-alpha response.

The cytokine synthesis by heterozygous carriers of the Toll-like receptor 4 Asp299Gly polymorphism does not differ from that of wild type homozygotes.

Previous studies have found that heterozygosity for the A896G mutation of the endotoxin receptor TLR4 confers susceptibility to Gram-negative infections and septic shock. To evaluate the underlying mechanisms, we studied the association of the TLR4 polymorphism with endotoxin-induced cytokine synthesis in human whole blood. Monocyte CD14 density and monocyte count were also determined. Healthy individuals were genotyped by means of a real-time polymerase chain reaction. Plasma concentrations of TNF-alpha, IL-6, and IL-8 were measured by chemiluminescence. No significant differences in cytokine synthesis were observed between heterozygous individuals and homozygous carriers of the wild type allele. Our study suggests that heterozygosity for this TLR4 mutation is not a major factor determining the cytokine response to endotoxin.

Tumor necrosis factor gene polymorphisms, leukocyte function, and sepsis susceptibility in blunt trauma patients.

The tumor necrosis factor alpha (TNF-alpha) -308 G/A and TNF-beta NcO1 polymorphisms have been described to be associated with an increased risk for sepsis in critically ill patients. Functional consequences associated with these polymorphisms remain unclear. We compared the genotype distribution of these TNF polymorphisms with susceptibility to severe sepsis and leukocyte function in blunt trauma patients (n = 70; mean injury severity score, 24 points [range, 4 to 57). Severe sepsis was defined according to the American College of Chest Physicians-Society of Critical Care Medicine consensus conference criteria. Genotyping for the NcO1 polymorphism (alleles TNFB1 and TNFB2) was performed by PCR and digestion of the products with NcO1, and that for the TNF-alpha -308 G/A polymorphism (alleles TNF1 and TNF2) was performed by real-time PCR. Leukocyte function was assessed by measurement of the production of endotoxin-induced cytokines (TNF-alpha, interleukin-6 [IL-6], and IL-8) in whole blood. TNF-alpha, IL-6, and IL-8 were determined by enzyme-linked immunosorbent assay. For the genotypes of the TNF-alpha -308 G/A polymorphism, differences in the frequency of development of severe sepsis were not detectable. Patients developing severe sepsis after trauma were significantly more likely to possess a homozygous genotype of the TNF-beta NcO1 polymorphism. Compared with heterozygotes, the odds ratio for the TNFB2/B2 genotype for the development of severe posttraumatic sepsis was 11 (P = 0.01), and that for the TNFB1/B1 genotype was 13 (P = 0.014). TNF-alpha -308:TNF-beta NcO1 haplotype analysis showed that the TNFB2:TNF2 haplotype is significantly negatively associated with development of severe sepsis. Patients homozygous for the TNFB1 or TNFB2 allele showed a persistently higher cytokine-producing capacity during at least 4 to 8 days after trauma than the heterozygotes. In patients homozygous for the TNF1 allele, a higher TNF-alpha- and IL-8-producing capacity was found only at day 1 after trauma. Although the TNF-beta NcO1 polymorphism appears to be less likely to be causative for development of severe sepsis after trauma, it is thus far the only genetic marker identified which can be used as a relevant risk estimate for severe sepsis in trauma patients immediately after the injury.

The -260 C-->T promoter polymorphism of the lipopolysaccharide receptor CD14 and severe sepsis in trauma patients.

OBJECTIVE: CD14, expressed on the cell surface of monocytes and hepatic Kupffer cells, interacts with Gram-positive and Gram-negative bacteria. Upon CD14 stimulation, these cells respond with the enhanced release of cytokines involved in the pathophysiology of sepsis. The purpose of this study was to evaluate whether the genotype distribution of the -260 C-->T promoter polymorphism of the CD14 gene is associated with the development of severe sepsis in trauma patients. PATIENTS AND PARTICIPANTS: Fifty-eight severely injured blunt trauma patients with an injury severity score of 16 or more and without pre-existing chronic diseases. MEASUREMENTS AND RESULTS: Genotyping for the single nucleotide exchange polymorphism of the CD14 gene was performed by means of a real-time polymerase chain reaction with fluorescence-labeled hybridization probes. Diagnosis of severe sepsis was based on the criteria of the ACCP/SCCM criteria. Fourteen out of the 58 patients (24.6%) developed a trauma-related severe sepsis. The overall allele frequency was 0.58 for the C allele and 0.42 for the T allele. The genotype distribution (TT 0.19, CT 0.47 and CC 0.35) did not differ significantly from a previously reported control group of healthy blood donors. There was no significant difference of the genotype distribution or allele frequency between trauma patients with severe sepsis and patients with an uncomplicated clinical course. CONCLUSIONS: This pilot study suggests that the CD14 -260 polymorphism is not associated with an increased risk of severe sepsis in trauma patients.

The CD14-260 C --> T promoter polymorphism co-segregates with the tumor necrosis factor-alpha (TNF-alpha)-308 G --> A polymorphism and is associated with the interleukin-1 beta (IL-1 beta) synthesis capacity of human leukocytes.

Genetic variations contribute to the interindividual variance in the cytokine response to endotoxin. The gene of tumor necrosis factor-alpha (TNF-alpha) carries a polymorphism at position -308 of the promoter, consisting of a G/A exchange. To further elucidate the inherited mechanisms influencing cytokine levels, healthy human blood donors were studied. Genotyping for the TNF-alpha -308 and the CD14 -260 C/T promoter polymorphisms was carried out by real-time polymerase chain reaction assay using specific fluorescence-labelled hybridisation probes. A human whole blood assay was used to study the leukocyte TNF-alpha and IL-1 beta synthesis capacity upon endotoxin stimulation. We found a linkage disequilibrium between the TNF-alpha -308 G/A and the CD14 -260 C/T polymorphisms (p = 0.043). The CD14 -260 polymorphism was associated with IL-1 beta levels (p = 0.033) and higher values were found in C homozygotes. No association was found between the CD14 -260 genotypes or the TNF-alpha -308 - CD14 -260 genotypes and the TNF-alpha response.

Can the interleukin-6 response to endotoxin be predicted? Studies of the influence of a promoter polymorphism of the interleukin-6 gene, gender, the density of the endotoxin receptor CD14, and inflammatory cytokines.

OBJECTIVES: To evaluate whether the -174 G/C promoter polymorphism of the interleukin-6 gene, gender, the monocyte density of the endotoxin receptor CD14, or the inflammatory cytokines tumor necrosis factor-alpha or interleukin-1beta influence the interleukin-6 response of whole blood to endotoxin. DESIGN: Analysis of interleukin-6 release from endotoxin-stimulated human whole blood. SETTING: Medical research laboratory. PATIENTS: Healthy human blood donors. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: The interleukin-6 -174 G/C and the tumor necrosis factor -308 G/A promoter polymorphisms were determined by real-time polymerase chain reaction assay by using specific fluorescence labeled hybridization probes. Monocyte CD14 expression was assessed by flow cytometry. After incubation of whole blood with endotoxin, plasma concentrations of interleukin-6, tumor necrosis factor-alpha, and interleukin-1beta were measured by means of chemiluminescence. The interleukin-6 concentrations were lower (p = .005) in individuals who were CG heterozygotes compared with individuals homozygous for the C or the G. The difference between C and G homozygotes was not significant (p = .67). The interleukin-6 response was enhanced in men compared with women (p = .015). There was no correlation between interleukin-6 concentrations and monocyte CD14 density. Interleukin-6 concentrations correlated with the concentrations of tumor necrosis factor-alpha (r = .59, p = .01) and interleukin-1beta (r = .47, p = .01). There was no linkage between the tumor necrosis factor -308 and the interleukin-6 -174 polymorphisms. CONCLUSIONS: The interleukin-6 response to endotoxin was influenced by gender and correlated with the concentrations of more proximal cytokine tumor necrosis factor-alpha and interleukin-1beta. The interleukin-6 -174 G/C promoter polymorphism can only partly predict the interleukin-6 response of human whole blood to endotoxin stimulation, and the results were different from previous reporter gene assays that reported higher interleukin-6 concentrations for the G allele. Tumor necrosis factor -308 G homozygotes produce the lowest tumor necrosis factor concentrations. The number of tumor necrosis factor -308 G homozygotes was not higher among interleukin-6 -174 heterozygotes, and thus this cannot account for their significantly smaller interleukin-6 production.

The interleukin-6 G(-174)C polymorphism and the ex vivo interleukin-6 response to endotoxin in severely injured blunt trauma patients.

The aim of this study was to evaluate whether the -174 G/C promoter polymorphism of the interleukin-6 (IL-6) gene is associated with the ex vivo, whole blood IL-6 response to endotoxin with the development of severe sepsis in severely injured, blunt trauma patients. Patients with a severe trauma and an injury severity score of 16 were included in the study. The IL-6 -174 G/C promoter polymorphism was determined by real-time polymerase chain reaction (PCR) assay using specific fluorescence-labelled hybridisation probes. Whole blood of the patients was stimulated with endotoxin and the IL-6 concentrations were measured by ELISA. There was no association between the IL-6 -174 genotypes and the ex vivo, stimulated IL-6 response: 25% of the patients developed severe sepsis later in the clinical course. These patients had higher IL-6 concentrations following whole blood stimulation on day 1 (p = 0.046) after the trauma than patients with uncomplicated post-traumatic recovery. The difference was even more significant on day 2 after the trauma (p = 0.02). High IL-6 responses in a whole blood stimulation assay with endotoxin on days 1 and 2 after a trauma are associated with severe post-traumatic sepsis. Genotyping for the IL-6 -174 G/C polymorphism does not allow early identification of trauma patients with a high, ex vivo IL-6 synthesis capacity.