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Background: Excessive alcohol intake is associated with adverse immune response-related effects, however, acute and chronic abuse differently modulate monocyte activation. In this study, we have evaluated the phenotypic and functional changes of monocytes in acutely intoxicated healthy volunteers (HV).
Methods: Twenty-two HV consumed individually adjusted amounts of alcoholic beverages until reaching a blood alcohol level of 1‰ after 4h (T4). Peripheral blood was withdrawn before and 2h (T2), 4h (T4), 6h (T6), 24h (T24), and 48h (T48) after starting the experiment and stained for CD14, CD16 and TLR4. CD14brightCD16-, CD14brightCD16+ and CD14dimCD16+ monocyte subsets and their TLR4 expression were analyzed by flow cytometry. Inflammasome activation via caspase-1 in CD14+ monocytes was measured upon an ex vivo in vitro LPS stimulation. Systemic IL-1β and adhesion capacity of isolated CD14+ monocytes upon LPS stimulation were evaluated.
Results: The percentage of CD14+ monocyte did not change following alcohol intoxication, whereas CD14brightCD16- monocyte subset significantly increased at T2 and T24, CD14brightCD16+ at T2, T4 and T6 and CD14dimCD16+ at T4 and T6. The relative fraction of TLR4 expressing CD14+ monocytes as well as the density of TLR4 surface presentation increased at T2 and decreased at T48 significantly. TLR4+CD14+ monocytes were significantly enhanced in all subsets at T2. TLR4 expression significantly decreased in CD14brightCD16- at T48, in CD14brightCD16+ at T24 and T48, increased in CD14dimCD16+ at T2. IL-1β release upon LPS stimulation decreased at T48, correlating with TLR4 receptor expression. Alcohol downregulated inflammasome activation following ex vivo in vitro stimulation with LPS between T2 and T48 vs. T0. The adhesion capacity of CD14+ monocytes decreased from T2 with significance at T4, T6 and T48. Following LPS administration, a significant reduction of adhesion was observed at T4 and T6.
Conclusions: Alcohol intoxication immediately redistributes monocyte subsets toward the pro-inflammatory phenotype with their subsequent differentiation into the anti-inflammatory phenotype. This is paralleled by a significant functional depression, suggesting an alcohol-induced time-dependent hyporesponsiveness of monocytes to pathogenic triggers.
Blunt thoracic trauma (TxT) deteriorates clinical post-injury outcomes. Ongoing inflammatory changes promote the development of post-traumatic complications, frequently causing Acute Lung Injury (ALI). Club Cell Protein (CC)16, a pulmonary anti-inflammatory protein, correlates with lung damage following TxT. Whether CC16-neutralization influences the inflammatory course during ALI is elusive. Ninety-six male CL57BL/6N mice underwent a double hit model of TxT and cecal ligation puncture (CLP, 24 h post-TxT). Shams underwent surgical procedures. CC16 was neutralized by the intratracheal application of an anti-CC16-antibody, either after TxT (early) or following CLP (late). Euthanasia was performed at 6 or 24 h post-CLP. Systemic and pulmonary levels of IL-6, IL-1β, and CXCL5 were determined, the neutrophils were quantified in the bronchoalveolar lavage fluid, and histomorphological lung damage was assessed. ALI induced a significant systemic IL-6 increase among all groups, while the local inflammatory response was most prominent after 24 h in the double-hit groups as compared to the shams. Significantly increased neutrophilic infiltration upon double hit was paralleled with the enhanced lung damage in all groups as compared to the sham, after 6 and 24 h. Neutralization of CC16 did not change the systemic inflammation. However, early CC16-neutralization increased the neutrophilic infiltration and lung injury at 6 h post-CLP, while 24 h later, the lung injury was reduced. Late CC16-neutralization increased neutrophilic infiltration, 24 h post-CLP, and was concurrent with an enhanced lung injury. The data confirmed the anti-inflammatory potential of endogenous CC16 in the murine double-hit model of ALI.
Background: Polytraumatized patients undergo a strong immunological stress upon insult. Phagocytes (granulocytes and monocytes) play a substantial role in immunological defense against bacteria, fungi and yeast, and in the clearance of cellular debris after tissue injury. We have reported a reduced monocytes phagocytic activity early after porcine polytrauma before. However, it is unknown if both phagocyte types undergo those functional alterations, and if there is a pathogen-specific phagocytic behavior. We characterized the phagocytic activity and capacity of granulocytes and monocytes after polytrauma.
Methods: Eight pigs (Sus scrofa) underwent polytrauma consisting of lung contusion, liver laceration, tibial fracture and hemorrhagic shock with fluid resuscitation and fracture fixation with external fixator. Intensive care treatment including mechanical ventilation for 72 h followed. Phagocytic activity and capacity were investigated using an in vitro ex vivo whole blood stimulation phagocytosis assays before trauma, after surgery, 24, 48, and 72 h after trauma. Blood samples were stimulated with Phorbol-12-myristate-13-acetate and incubated with FITC-labeled E. coli, S. aureus or S. cerevisiae for phagocytosis assessment by flow cytometry.
Results: Early polytrauma-induced significant increase of granulocytes and monocytes declined to baseline values within 24 h. Percentage of E. coli-phagocytizing granulocytes significantly decreased after polytrauma and during further intensive care treatment, while their capacity significantly increased. Interestingly, both granulocytic phagocytic activity and capacity of S. aureus significantly decreased after trauma, although a recovery was observed after 24 h and yet was followed by another decrease. The percentage of S. cerevisiae-phagocytizing granulocytes significantly increased after 24 h, while their impaired capacity after surgery and 72 h later was detected. Monocytic E. coli-phagocytizing percentage did not change, while their capacity increased after 24–72 h. After a significant decrease in S. aureus-phagocytizing monocytes after surgery, a significant increase after 24 and 48 h was observed without capacity alterations. No significant changes in S. cerevisiae-phagocytizing monocytes occurred, but their capacity dropped 48 and 72 h.
Conclusion: Phagocytic activity and capacity of granulocytes and monocytes follow a different pattern and significantly change within 72 h after polytrauma. Both phagocytic activity and capacity show significantly different alterations depending on the pathogen strain, thus potentially indicating at certain and possibly more relevant infection causes after polytrauma.
Excessive neutrophil activation accompanied by delayed apoptotic cell death in inflammatory conditions causes progressive damage of cells and tissues, leading to life-threatening multiple organ dysfunction syndrome. Previous work suggested that circulating serum factors during inflammation are critically involved in the suppression of neutrophil cell death although the identity of these antiapoptotic mediators remained elusive. In this study, we identified the acute phase protein α-1 Antitrypsin (AAT) as a potent suppressor of staurosporine (STS)-induced apoptosis in human neutrophils through a mechanism implicating caspases-independent pathways. We show here that serum levels of AAT, potentially in part released by stimulated neutrophils, are markedly elevated in major trauma patients suffering from systemic inflammatory response syndrome (SIRS). Notably, AAT depletion from serum increased sensitivity of human neutrophils for STS-induced cell death. In fact, AAT was demonstrated to confer intrinsic apoptosis resistance by preventing PKC/Akt inactivation and subsequent proteasomal degradation of antiapoptotic Mcl-1 protein in response to STS treatment. Neither MAP kinase ERK1/2 nor caspases were found to be involved in AAT-triggered antiapoptotic pathways in neutrophils. In summary, these results establish a novel pivotal role of circulating AAT in mediating survival by antagonizing the proapoptotic action of the PKC inhibitor STS and should be considered for AAT augmentation therapies in future.
Objective: Trauma is the most common cause of death among young adults. Alcohol intoxication plays a significant role as a cause of accidents and as a potent immunomodulator of the post-traumatic response to tissue injury. Polytraumatized patients are frequently at risk to developing infectious complications, which may be aggravated by alcohol-induced immunosuppression. Systemic levels of integral proteins of the gastrointestinal tract such as syndecan-1 or intestinal fatty acid binding proteins (FABP-I) reflect the intestinal barrier function. The exact impact of acute alcohol intoxication on the barrier function and endotoxin bioactivity have not been clarified yet. Methods: 22 healthy volunteers received a precisely defined amount of alcohol (whiskey–cola) every 20 min over a period of 4 h to reach the calculated blood alcohol concentration (BAC) of 1‰. Blood samples were taken before alcohol drinking as a control, and after 2, 4, 6, 24 and 48 h after beginning with alcohol consumption. In addition, urine samples were collected. Intestinal permeability was determined by serum and urine values of FABP-I, syndecan-1, and soluble (s)CD14 as a marker for the endotoxin translocation via the intestinal barrier by ELISA. BAC was determined. Results: Systemic FABP-I was significantly reduced 2 h after the onset of alcohol drinking, and remained decreased after 4 h. However, at 6 h, FABP-I significantly elevated compared to previous measurements as well as to controls (p < 0.05). Systemic sCD14 was significantly elevated after 6, 24 and 48 h after the onset of alcohol consumption (p < 0.05). Systemic FABP-I at 2 h after drinking significantly correlated with the sCD14 concentration after 24 h indicating an enhanced systemic LPS bioactivity. Women showed significantly lower levels of syndecan-1 in serum and urine and urine for all time points until 6 h and lower FABP-I in the serum after 2 h. Conclusions: Even relative low amounts of alcohol affect the immune system of healthy volunteers, although these changes appear minor in women. A potential damage to the intestinal barrier and presumed enhanced systemic endotoxin bioactivity after acute alcohol consumption is proposed, which represents a continuous immunological challenge for the organism and should be considered for the following days after drinking.
Background: Severely injured patients experience substantial immunological stress in the aftermath of traumatic insult, which often results in systemic immune dysregulation. Regulatory T cells (Treg) play a key role in the suppression of the immune response and in the maintenance of immunological homeostasis. Little is known about their presence and dynamics in blood after trauma, and nothing is known about Treg in the porcine polytrauma model. Here, we assessed different subsets of Treg in trauma patients (TP) and compared those to either healthy volunteers (HV) or data from porcine polytrauma.
Methods: Peripheral blood was withdrawn from 20 TP with injury severity score (ISS) ≥16 at the admittance to the emergency department (ED), and subsequently on day 1 and at day 3. Ten HV were included as controls (ctrl). The porcine polytrauma model consisted of a femur fracture, liver laceration, lung contusion, and hemorrhagic shock resulting in an ISS of 27. After polytrauma, the animals underwent resuscitation and surgical fracture fixation. Blood samples were withdrawn before and immediately after trauma, 24 and 72 h later. Different subsets of Treg, CD4+CD25+, CD4+CD25+FoxP3+, CD4+CD25+CD127−, and CD4+CD25+CD127−FoxP3+ were characterized by flow cytometry.
Results: Absolute cell counts of leukocytes were significantly increasing after trauma, and again decreasing in the follow-up in human and porcine samples. The proportion of human Treg in the peripheral blood of TP admitted to the ED was lower when compared to HV. Their numbers did not recover until 72 h after trauma. Comparable data were found for all subsets. The situation in the porcine trauma model was comparable with the clinical data. In porcine peripheral blood before trauma, we could identify Treg with the typical immunophenotype (CD4+CD25+CD127−), which were virtually absent immediately after trauma. Similar to the human situation, most of these cells expressed FoxP3, as assessed by intracellular FACS stain.
Conclusion: Despite minor percental differences in the recovery of Treg populations after trauma, our findings show a comparable decrease of Treg early after polytrauma, and strengthen the immunological significance of the porcine polytrauma model. Furthermore, the Treg subpopulation CD4+CD25+CD127− was characterized in porcine samples.
Background: Tumor development remains one of the major obstacles following organ transplantation. Immunosuppressive drugs such as cyclosporine and tacrolimus directly contribute to enhanced malignancy, whereas the influence of the novel compound mycophenolate mofetil (MMF) on tumor cell dissemination has not been explored. We therefore investigated the adhesion capacity of colon, pancreas, prostate and kidney carcinoma cell lines to endothelium, as well as their beta1 integrin expression profile before and after MMF treatment. Methods: Tumor cell adhesion to endothelial cell monolayers was evaluated in the presence of 0.1 and 1 μM MMF and compared to unstimulated controls. beta1 integrin analysis included alpha1beta1 (CD49a), alpha2beta1 (CD49b), alpha3beta1 (CD49c), alpha4beta1 (CD49d), alpha5beta1 (CD49e), and alpha6beta1 (CD49f) receptors, and was carried out by reverse transcriptase-polymerase chain reaction, confocal microscopy and flow cytometry. Results: Adhesion of the colon carcinoma cell line HT-29 was strongly reduced in the presence of 0.1 μM MMF. This effect was accompanied by down-regulation of alpha3beta1 and alpha6beta1 surface expression and of alpha3beta1 and alpha6beta1 coding mRNA. Adhesion of the prostate tumor cell line DU-145 was blocked dose-dependently by MMF. In contrast to MMF's effects on HT-29 cells, MMF dose-dependently up-regulated alpha1beta1, alpha2beta1, alpha3beta1, and alpha5beta1 on DU-145 tumor cell membranes. Conclusion: We conclude that MMF possesses distinct anti-tumoral properties, particularly in colon and prostate carcinoma cells. Adhesion blockage of HT-29 cells was due to the loss of alpha3beta1 and alpha6beta1 surface expression, which might contribute to a reduced invasive behaviour of this tumor entity. The enhancement of integrin beta1 subtypes observed in DU-145 cells possibly causes re-differentiation towards a low-invasive phenotype.
Adhesion molecules of the integrin beta1 family are thought to be involved in the malignant progression renal cell carcinoma (RCC). Still, it is not clear how they contribute to this process. Since the hematogenous phase of tumour dissemination is the rate-limiting step in the metastatic process, we explored beta1 integrin alterations on several RCC cell lines (A498, Caki1, KTC26) before and after contacting vascular endothelium in a tumour-endothelium (HUVEC) co-culture assay. Notably, alpha2, alpha3 and alpha5 integrins became down-regulated immediately after the tumour cells attached to HUVEC, followed by re-expression shortly thereafter. Integrin down-regulation on RCC cells was caused by direct contact with endothelial cells, since the isolated endothelial membrane fragments but not the cell culture supernatant contributed to the observed effects. Integrin loss was accompanied by a reduced focal adhesion kinase (FAK) expression, FAK activity and diminished binding of tumour cells to matrix proteins. Furthermore, intracellular signalling proteins RCC cells were altered in the presence of HUVEC membrane fragments, in particular 14-3-3 epsilon, ERK2, PKCdelta, PKCepsilon and RACK1, which are involved in regulating tumour cell motility. We, therefore, speculate that contact of RCC cells with the vascular endothelium converts integrin-dependent adhesion to integrin-independent cell movement. The process of dynamic integrin regulation may be an important part in tumour cell migration strategy, switching the cells from being adhesive to becoming motile and invasive.
Objective: Trauma patients (TP) frequently develop an imbalanced immune response that often causes infectious postinjury complications. Monocytes show a diminished capability of both producing proinflammatory cytokines and antigen presentation after trauma. TLR2, TLR4, and TLR9 recognize pathogens and subsequently activate monocytes. While there are conflictive data about TLR2 and TLR4 expression after trauma, no studies about the expression of TLR2, TLR4, TLR9, and HLA-DR on monocytes from TP after their secondary ex vivo-in vitro “hit” have been reported.
Methods/Results: Ex vivo-in vitro lipopolysaccharide- (LPS-) stimulated blood from TP showed diminished interleukin- (IL-) 1β-release in TP for five postinjury days compared to healthy volunteers (HV). The recovery was observed at day 5. In parallel, monocytes from TP showed an impaired capability of TLR2, TLR4, and TLR9 expression after secondary stimulation compared to HV, while the measurement of unstimulated samples showed significant reduction of TLR4 and TLR9 at ED. Furthermore, HLA-DR decreased after trauma and was even more profound by stimulation of monocytes. Ratio of monocytes to leukocytes was significantly increased at days 6 and 7 after trauma compared to HV.
Conclusion: Impaired expression of TLRs and HLA-DR in acute inflammatory conditions may be responsible for the well-described monocyte paralysis after severe trauma.
Objective. Traumatic injury or severe surgery leads to a profound immune response with a diminished functionality of monocytes and subsequently their IL-1β release. IL-1β plays an important role in host immunity and protection against infections. Its biological activation via IL-1β-precursor processing requires the transcription of inflammasome components and their activation. Deregulated activity of NOD-like receptor inflammasomes (NLR) like NLRP3 that leads to the maturation of IL-1β has been described in various diseases. While the role of other inflammasomes has been studied in monocytes, nothing is known about NLRP3 inflammasome after a traumatic injury. Here, the role of the NLRP3 inflammasome in impaired monocyte functionality after a traumatic injury was analyzed.
Measurements and Main Results. Ex vivo-in vitro stimulation of isolated CD14+ monocytes with lipopolysaccharide (LPS) showed a significantly higher IL-1β secretion in healthy volunteers (HV) compared to trauma patients (TP) after admission. Reduced IL-1β secretion was paralleled by significantly lowered gene expression of NLRP3 in monocytes from TP compared to those of HV. Transfection of monocytes with NLRP3-encoding plasmid recovered the functionality of monocytes from TP regarding the IL-1β secretion.
Conclusions. This study demonstrates that CD14+ monocytes from TP are significantly diminished in their function and that the presence of NLRP3 components is necessary in recovering the ability of monocytes to produce active IL-1β. This recovery of the NLRP3 inflammasome in monocytes may imply a new target for treatment and therapy of immune suppression after severe injury.