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.     

Nutritional status and postoperative cytokine response in colorectal cancer patients

The present study was designed to investigate the relationship between pre-operative nutritional status and peri-operative regulation of the cytokine network, and to clarify its relation to clinical outcome in colorectal cancer patients. Protein-energy malnutrition was assessed using the creatinine height index. Peripheral venous blood samples were obtained peri-operatively, and the serum concentrations of interleukin (IL-)6, IL-1 receptor antagonist (ra), IL-6 soluble receptor (sR) C-reactive protein (CRP) and the percentage of peripheral neutrophils were determined. Excessive operative blood loss was associated with postoperative morbidity. Pre-operative malnutrition was associated with postoperative mortality when excessive bleeding occurred. Postoperative IL-6 response was exaggerated and postoperative IL-1ra response was suppressed in nutritionally depleted patients. The postoperative serum concentrations of IL-6sR in malnourished patients remained at the lowest levels when excessive bleeding occurred. In these patients, the percentage of peripheral neutrophils remained at high levels even after resolution of the postoperative cytokine storm. A marked activation of the pro-inflammatory cytokine network associated with a decreased antagonistic reaction and an increased consumption of IL-6sR became prominent in malnourished patients when they underwent intense surgical stress. These immunological disturbances may be relevant to neutrophil activation and subsequent clinical outcome.     

Plasma and urinary cytokine homeostasis and renal function during cardiac surgery without cardiopulmonary bypass

Cardiopulmonary bypass (CPB) significantly contributes to the plasma pro-inflammatory cytokine response at cardiac surgery. Complementary plasma and urinary anti-inflammatory cytokine responses have been described. The pro-inflammatory cytokines interleukin 8 (IL-8), tumour necrosis factor alpha (TNF-alpha) and interleukin 1beta (IL-1beta) have lower molecular weights than the anti-inflammatory cytokines interleukin 10 (IL-10), interleukin 1 receptor antagonist (IL-1ra) and TNF soluble receptor 2 (TNFsr2) and thus undergo glomerular filtration more readily. In vitro work suggests that proximal tubular cells are vulnerable to pro-inflammatory cytokine mediated injury. Accordingly, this study investigated the hypothesis that cardiac surgery without CPB would not have significant changes in plasma and urinary cytokines and proximal renal dysfunction. Eight patients undergoing coronary artery bypass grafting (CABG) without CPB were studied. Blood and urine samples were analysed for pro- and anti-inflammatory cytokines. Proximal tubular dysfunction was measured using urinary Nu-acetyl-beta-D-glucosaminidase (NAG)/creatinine and alpha(1)-microglobulin/creatinine ratios. Plasma IL-8, IL-10, IL-1ra and TNFsr2 were significantly elevated compared with baseline. Urinary IL-1ra and TNFsr2 were significantly elevated, as were urinary NAG/creatinine and alpha(1)-microglobulin/creatinine ratios. Two hours following revascularization, urinary IL-1ra correlated with urinary alpha(1)-microglobulin/creatinine ratios (P<0.05). As previously reported in CABG surgery with CPB, we now report that non-CPB cardiac surgery also has significant changes in plasma and urinary cytokine homeostasis and early proximal tubular injury. The correlation between urinary IL-1ra and alpha(1)-microglobulin/creatinine ratios is consistent with earlier suggestions of a mechanistic link between cytokine changes and proximal tubular dysfunction. The relative roles of CPB and non-CPB processes in producing inflammation still require definition.     

Interleukin-6 and perioperative thermoregulation and HPA-axis activation

Surgery is followed by an acute-phase response, including hypothalamo-pituitary-adrenal (HPA)-axis activation and fever. Considering its physiological properties and its behaviour in plasma after stress and surgery, the pro-inflammatory cytokine interleukin (IL)-6 is a putative candidate in eliciting these stress-related symptoms. However, evidence for this hypothesis is lacking. Rats subjected to individual psychological stress for 1h were injected intraperitoneally with saline or 3.33 microg per 100g rat neutralizing antibodies against rat IL-6. Thereafter, the single-housed rats were anaesthetized for 25 min, with or without undergoing a laparotomy. Intermittently, oesophageal temperatures were measured at defined time points. A parallel group of rats undergoing the same study protocol were decapitated, at time points when body temperatures differed, to obtain blood for measurement of plasma adrenocorticotropic hormone and corticosterone. Individual housing resulted in hyperthermia. Antibodies against IL-6 accelerated normalization of body temperature after individualizing stress, limited postoperative hyperthermia after laparotomy, but accentuated hyperthermia after anaesthesia alone. Antibody administration was not able to significantly influence the plasma hormone levels during any experiment. The present study indicates that IL-6 is a thermoregulatory factor during psychological, anaesthesiological and surgical stress, but the cytokine does not participate in HPA-axis activation until 6h after anaesthesia or surgery. A dose-finding study with antibodies against IL-6 ought to further identify the degree of contribution of IL-6 to perioperative thermoregulation.     

The diminished postoperative capacity of blood leukocytes to produce IL-6 is associated with high concentrations of IL-6 in the circulation

Surgical trauma is followed both by a transient increase of interleukin 6 (IL-6) concentrations in the serum and impaired function of circulating leukocytes. Perioperatively, we investigated the relationship of IL-6 concentrations in the serum with lipopolysaccharide (LPS)-stimulated cytokine production in the whole blood of patients undergoing elective major abdominal operations. In 50 patients, we found a transient increase of IL-6 concentrations in the serum. Six hours after skin incision, in vitro stimulated production of IL-6 and TNFalpha was diminished by 72% (P<0.05). A significant increase in cytokine production was observed three days postoperatively, however this was 63% below the preoperative values. Patients with high concentrations of circulating IL-6 showed a significantly lower stimulated IL-6 production than patients with low serum concentrations of IL-6. We conclude, that two opposing effects are associated with surgery: an activation leading to IL-6-release into the circulation, and a prolonged hyporesponsiveness of circulating leukocytes. These reactions are positively related.     

Inflammatory cytokines and soluble receptors after coronary artery bypass grafting

Much interest has been focused on the overexpression of proinflammatory cytokines, but studies on their soluble receptors are rare. For a comprehensive picture of cytokine activation in cardiac surgery, a combination of cytokines and the corresponding soluble receptor concentration should be determined. Blood samples were collected from the radial artery and coronary sinus perioperatively in ten patients undergoing coronary artery bypass grafting with cardiopulmonary bypass. TNF-alpha, IL-6, sTNFRI, sTNFRII, and sIL-6R levels in the plasma were determined. Systemic TNFRI, TNFRII and IL-6 increased significantly after reperfusion to the myocardium, while perioperative systemic sIL-6r levels were similar. Arterial and sinus levels of TNFRI, TNFRII and sIL-6r were similar before cardiopulmonary bypass. Five minutes after reperfusion to the myocardium, higher sinus TNFRI and TNFRII and lower sinus sIL-6R levels were observed as compared to the arterial levels. The myocardium release of sTNFRI (r=0.57, P=0.089) and sTNFRII (r=0.64, P=0.047) positively correlated with the change of cardiac index after cardiopulmonary bypass. Myocardium releases sTNFRI and sTNFRII after ischaemic-reperfusion injury, and this may be of benefit to cardiac performance. sIL-6R is constantly being produced in areas other than the myocardium, while sIL-6R levels are reduced by consumption in the myocardium after ischaemic-reperfusion injury.     

Increased systemic and adipose tissue cytokines in patients with HIV-associated lipodystrophy

The lipodystrophy syndrome (adipose tissue redistribution and metabolic abnormalities) observed with highly active antiretroviral therapy (HAART) during human immunodeficiency virus (HIV) infection may be related to increased proinflammatory cytokine activity. We measured acute cytokine (TNF-alpha, IL-6, leptin), glycerol, and lactate secretion from abdominal subcutaneous adipose tissue (SAT), and systemic cytokine levels, in HIV-infected subjects with and without lipodystrophy (HIVL+ and HIVL-, respectively) and healthy non-HIV controls. Lipodystrophy was confirmed and characterized as adipose tissue redistribution in HIVL+ compared with HIVL- and controls, by dual-energy X-ray absorptiometry and by whole body MRI. TNF-alpha secretion from abdominal SAT and circulating levels of IL-6, soluble TNF receptors I and II, and insulin were elevated in HIVL+ relative to HIVL- and/or controls, particularly in HIVL+ undergoing HAART. In the HIV-infected group as a whole, IL-6 secretion from abdominal SAT and serum IL-6 were positively associated with visceral fat and were negatively associated with the relative amount of lower limb adipose tissue (P < 0.01). Decreased leptin and increased lactate secretion from abdominal SAT were specifically associated with HAART. In conclusion, increased cytokine secretion from adipose tissue and increased systemic proinflammatory cytokine activity may play a significant role in the adipose tissue remodeling and/or the metabolic abnormalities associated with the HIV-lipodystrophy syndrome in patients undergoing HAART.     

Collecting And Measuring Wound Exudate Biochemical Mediators In Surgical Wounds

We describe a methodology by which we are able to collect and measure biochemical inflammatory and nociceptive mediators at the surgical wound site. Collecting site-specific biochemical markers is important to understand the relationship between levels in serum and surgical wound, determine any associations between mediator release, pain, analgesic use and other outcomes of interest, and evaluate the effect of systemic and peripheral drug administration on surgical wound biochemistry. This methodology has been applied to healthy women undergoing elective cesarean delivery with spinal anesthesia. We have measured wound exudate and serum mediators at the same time intervals as patient''s pain scores and analgesics consumption for up to 48 hours post-cesarean delivery. Using this methodology we have been able to detect various biochemical mediators including nerve growth factor (NGF), prostaglandin E2 (PG-E2) substance P, IL-1β, IL-2, IL-4, IL-6, IL-7, IL-8, IL-10, IL-12, IL-13, IL-17, TNFα, INFγ, G-CSF, GM-CSF, MCP-1 and MIP-1β. Studies applying this human surgical wound bioassay have found no correlations between wound and serum cytokine concentrations or their time-release profile (J Pain. 2008; 9(7):650-7).¹ We also documented the utility of the technique to identify drug-mediated changes in wound cytokine content (Anesth Analg 2010; 111:1452-9).²     

TH1/TH2 cytokine levels as an indicator for disease progression in human immunodeficiency virus type 1 infection and response to antiretroviral therapy

A recent theory stipulates that during the course of HIV infection, there is a shift in immune response from T-helper 1 to T-helper 2 responses, characterised by elevated secretions of relevant cytokines. Cytokine profiles of 15 asymptomatic (treatment na?ve) and 26 symptomatic (undergoing treatment) HIV-1 patients was determined to investigate the validity of this theory. HIV-1 RNA was quantified using the COBAS TaqMan HIV-1 test, CD4 T-cell counts with the FACSCalibur flow cytometer and IL-1, IL-4, IL-6, IL-10 and IFN-gamma cytokine levels by ELISA method. The asymptomatic group had significantly higher RNA levels (p-value; 0.000006) and lower CD4 T-cell counts than the symptomatic group indicating ongoing disease progression in the absence of antiretroviral treatment and a positive response to HIV treatment by the symptomatic group. IL-1, IL-4 and IFN-gamma were undetectable in most study subjects. IL-10 and IL-6 levels was relatively lower in the asymptomatic group (mean value; 206.352 pg/ml, 10.516 pg/ml) than the symptomatic group (mean value; 417.539, 18.387 pg/ml). Lower levels of proinflammatory cytokines (IL-1, IFN-gamma) in both study groups and elevated levels of anti-inflammatory cytokine IL-10, confirms that there is a shift in immune response as HIV infection progress to AIDS. In addition, the presence of a progressive trend of anti-inflammatory cytokine, IL-10 and proinflammatory cytokine, IL-6 in 12 symptomatic patients tested 3 months after antiretroviral therapy indicates an attempt by antiretrovirals to restore immune function.     

Interleukin-1 beta, interleukin-6 and TGF-beta in follicular tissue of impacted third molars

The clinical evaluation and management of impacted third molars remain challenging. The aim of the present study was to investigate possible associations between follicular tissue cytokines and radiographic manifestations of impacted third molar. The population included 72 patients who underwent surgical extraction of impacted third molars. All these patients underwent a preliminary panoramic radiograph. Levels of interleukin-1 beta (IL-1β), interleukin-6 (IL-6) and transforming growth factor beta (TGF-β) in tissue extracts were determined using ELISA. There were no significant differences between bony and tissue impaction as regards IL-1β, IL-6 and TGF-β levels. Moreover, the same results were obtained as far as the amount of pericoronal space and the presence or absence of a history of pericoronitis are concerned. These results suggest that radiographic findings or a history of pericoronitis are not associated with levels of expression of pro-inflammatory cytokines in patients undergoing surgical removal of impacted third molars. However, further studies are needed to address the possibility of variability during disease progression.     

Interleukin 8 production in whole blood assays: Is interleukin 10 responsible for the downregulation observed in sepsis?

Reduced cytokine production in ex vivo cultures has been regularly reported in patients suffering from sepsis syndrome. Using whole blood assays, we have now demonstrated that in sepsis patients, normal production of IL-8 was achieved with the higher concentration of lipopolysaccharide (LPS; 1 microg/ml) and with heat-killed streptococci, whereas the IL-8 production induced by lower LPS concentration (0.1 microg/ml) was significantly reduced as compared to healthy controls. In contrast, in patients undergoing cardiac surgery associated with cardio-pulmonary bypass, a group of patients with inflammation in the absence of infectious insult, none of the studied IL-8 productions were affected. Among the various anti-inflammatory cytokines known to regulate IL-8 production which we tested (i.e. IL-4, IL-10, IL-13, TGF-beta), IL-10 was the most active inhibitory cytokine in whole blood assays performed with blood samples from healthy subjects. However, its activity was not influenced by the amounts of LPS used. In addition, IL-10 also inhibited the heat-killed streptococci-induced IL-8 production and was the only cytokine to inhibit the release of IL-8 when TNF was added to LPS. It is worth noting that IL-13 which also inhibited the heat-killed streptococci-induced IL-8 production, failed to do so when the TNF production was analysed. Together, these data suggest that while circulating IL-10 in septic patients may be responsible for the hyporeactivity of circulating leukocytes, its presence is not sufficient to explain the observed dysregulation which occurs in septic patients.     

Effects of IL-10 and age on IL-6, IL-1beta, and TNF-alpha responses in mouse skeletal and cardiac muscle to an acute inflammatory insult.

Exaggerated proinflammatory cytokine responses can be observed with aging, and reduced levels of the anti-inflammatory cytokine IL-10 may contribute to these responses. IL-10 can reduce IL-6, IL-1beta, and TNF-alpha expression in nonmuscle tissues; however, no studies have examined the combined effects of IL-10 and age on cytokine responses in skeletal and cardiac muscle. These experiments tested the hypothesis that the absence of IL-10, in vivo, is associated with greater IL-6, TNF-alpha, and IL-1beta responses to an inflammatory challenge in skeletal and cardiac muscle and that aging exaggerates these responses. We compared IL-6, IL-1beta, and TNF-alpha mRNA and protein levels in skeletal and cardiac muscle of young (4 mo) and mature (10-11 mo) wild-type (IL-10(+/+)) and IL-10 deficient (IL-10(-/-)) mice following LPS. Skeletal and cardiac IL-6 mRNA and protein were elevated by LPS for IL-10(+/+) and IL-10(-/-) mice with greater responses in the IL-10(-/-) mice (P < 0.01). In skeletal muscle these effects were greater in mature than young mice (P < 0.01). IL-1beta mRNA and protein responses to LPS were greater in cardiac muscle of young but not mature IL-10(-/-) mice compared with IL-10(+/+) (P < 0.01). However, IL-1beta responses were greater in mature than young mice, but only in IL-10(+/+) groups (P < 0.05). The absence of IL-10 was associated with higher TNF-alpha protein levels in cardiac muscle (P < 0.05). The results provide the first in vivo evidence that the absence of IL-10 is associated with a greater IL-6 response to LPS in skeletal and cardiac muscles, and in skeletal muscle aging further exaggerates these responses.     

Tumour necrosis factor-alpha (TNFalpha) and interleukin-10 are crucial mediators in post-operative systemic inflammatory response and determine the occurrence of complications after major abdominal surgery

BACKGROUND: The course of serum cytokine levels in patients with postoperative systemic inflammatory response syndrome (SIRS) after major abdominal surgery remains currently unclear. METHODS: Blood was sampled pre- and post-operatively and on days 1 and 2 in 40 patients undergoing major abdominal surgery. Concentrations of tumour necrosis factor-alpha (TNFalpha), interleukin (IL) -6, IL-8, and IL-10 were measured by the LINCOplex assay; those of soluble triggering receptor expressed on myeloid cells-1 (sTREM-1) by an enzyme immunoassay. RESULTS: Compared to their pre-operative values, sTREM-1 was elevated on day 2; TNFalpha on day 1; IL-6 and IL-10 post-operatively and on days 1 and 2; and IL-8 post-operatively and on day 1. The duration of operation correlated with TNFalpha and IL-10 at all sampling times, and with IL-6 post-operatively. There were no differences in cytokine concentrations between patients who exhibited post-operative complications and those who did not. IL-10/TNFalpha below 30 was found in all patients with complications (100%) and in 20 patients without complications (64.5%, p: 0.043). CONCLUSIONS: SIRS following major surgery is characterised by complex alterations in cytokine concentrations. The balance between TNFalpha and IL-10 seems to determine the occurrence of post-operative complications.     

Urinary soluble IL-6 receptor levels after cardiopulmonary bypass

To investigate the possible role of soluble interleukin 6 receptor (sIL-6r) in the inflammatory response to cardiopulmonary bypass (CPB) with extracorporeal circulation (ECC), we examined the levels of sIL-6r in the urine of 50 patients undergoing coronary artery bypass surgery. The presence of sIL-6r in urine was confirmed in these patients, with levels rising from 6 pg/ml preoperatively to 19.5 pg/ml at 6 h and 41 pg/ml at 24 h after the start of cardiopulmonary bypass. Cardiopulmonary bypass leads to a rise in sIL-6r in urine that has not returned to normal after 24 h, suggesting a role for sIL-6r and IL-6 in the inflammatory response to such surgery. Determination of cytokine receptor presence in urine offers a non-invasive approach to the monitoring of the immune and inflammatory response to the stress of surgical and traumatic injury.     

The biological and clinical importance of the 'new generation' cytokines in rheumatic diseases

A better understanding of cytokine biology over the last two decades has allowed the successful development of cytokine inhibitors against tumour necrosis factor and interleukin (IL)-1 and IL-6. The introduction of these therapies should be considered a breakthrough in the management of several rheumatic diseases. However, many patients will exhibit no or only partial response to these therapies, thus emphasising the importance of exploring other therapeutic strategies. In this article, we review the most recent information on novel cytokines that are often members of previously described cytokine families such as the IL-1 superfamily (IL-18 and IL-33), the IL-12 superfamily (IL-27 and IL-35), the IL-2 superfamily (IL-15 and IL-21), and IL-17. Several data derived from experimental models and clinical samples indicate that some of these cytokines contribute to the pathophysiology of arthritis and other inflammatory diseases. Targeting of some of these cytokines has already been tested in clinical trials with interesting results.     

Plasma inflammatory cytokine response to surgical trauma in chronic depressed patients

We investigated inflammatory cytokine response in chronic depressed patients during abdominal surgery. Twenty-five major depressed patients (Group D) and twenty-five patients (Group C) as the control were studied. Plasma interleukin 6 (IL-6), interleukin 8 (IL-8) and tumour necrosis factor-alpha (TNF-alpha) concentrations were measured before and at 15 min after induction of anesthesia, the end of surgery, 24 h and 3 days after the operation. Plasma IL-6 concentrations in Group D at the end of the operation and 24 h after surgery were significantly lower than those of Group C. The plasma IL-6 concentration (87.1+/-55.3 pg/ml) of patients scoring more than 18 points in the Hamilton depression-rating score at the end of the operation was significantly higher than 57.5+/-76.7 pg/ml of patients scoring less than 18 points. Plasma IL-8 concentration (6.1+/-3.2 pg/ml) in Group D at the end of the operation was significantly lower than 8.7+/-4.2 pg/ml of Group C. We conclude that plasma IL-6 and IL-8 response to surgical trauma is inhibited in chronic depressed patients. The IL-6 response to surgical trauma is depending on the clinical state of depression.     

Peri- and post-operative course of cytokines and the metabolic activity of neutrophils in human liver transplantation.

Peri- and post-operative (up to day 7 after surgery) neutrophil chemiluminescence and the plasma concentrations of interleukin 6 (IL-6), IL-8, IL-10 and tumour necrosis factor alpha (TNF-alpha) were evaluated in the blood of patients undergoing liver transplantation. IL-6, IL-8 and IL-10 levels increased during early reperfusion and then returned to normal mostly within the first post-operative day. TNF-alpha was increased during the whole period observed. Spontaneous as well as activated neutrophil chemiluminescence was depressed in early reperfusion and remained low during the whole period followed. Samples collected during early reperfusion provided positive correlation for IL-6 vs IL-8 as well as for IL-6 and IL-8 vs chemiluminescence. The data were also evaluated with respect to the outcome of transplantation. Since IL-8, IL-10 and TNF-alpha levels increased significantly during the first post-operative week, mainly in a group of patients who developed serious complications within the first month after surgery, we proved a connection between peri- and early post-operative induction of cytokine release and the outcome of liver allograft transplantation.     

Plasma hepcidin correlates positively with interleukin-6 in patients undergoing pulmonary endarterectomy

Hepcidin, a recently discovered antimicrobial peptide synthesized in the liver, was identified to be the key mediator of iron metabolism and distribution. Despite our knowledge of hepcidin increased in recent years, there are only limited data on hepcidin regulation during systemic inflammatory response in human subjects. In a prospective study, the time course of plasma hepcidin was analyzed in relations to six inflammatory parameters - plasma cytokines and acute-phase proteins in patients undergoing uncomplicated pulmonary endarterectomy. Twenty-four patients (males, aged 52.6+/-10.2 years, treated with pulmonary endarterectomy in a deep hypothermic circulatory arrest) were enrolled into study. Hepcidin, interleukin (IL)-6, IL-8, tumor necrosis factor-alpha, C-reactive protein, alpha(1)-antitrypsin and ceruloplasmin arterial concentrations were measured before surgery and repeatedly within 120 h post-operatively. Hemodynamic parameters, hematocrit and markers of iron metabolism were followed up. In a postoperative period, hepcidin increased from preoperative level 8.9 ng/ml (6.2-10.7) (median and interquartile range) to maximum 16.4 ng/ml (14.1-18.7) measured 72 h after the end of surgery. Maximum post-operative concentrations of hepcidin correlated positively with maximum IL-6 levels. Both hepcidin and IL-6 maximum concentrations correlated positively with extracorporeal circulation time. In conclusions, the study demonstrated that plasma hepcidin is a positive acute-phase reactant in relation to an uncomplicated large cardiac surgery. Hepcidin increase was related to IL-6 concentrations and to the duration of surgical procedure. Our clinical findings are in conformity with recent experimental studies defining hepcidin as a type II acute-phase protein.