Corneli W. van Aalst

Differences in Potency of CXC Chemokine Ligand 8-, CC Chemokine Ligand 11-, and C5a-Induced Modulation of Integrin Function on Human Eosinophils

The hypothesis was tested that different chemoattractants have different effects on the activity of integrins expressed by the human eosinophil. Three chemoattractants, CXCL8 (IL-8), CCL11 (eotaxin-1), and C5a were tested with respect to their ability to induce migration and the transition of eosinophils from a rolling interaction to a firm arrest on activated endothelial cells under flow conditions. CCL11 and C5a induced a firm arrest of eosinophils rolling on an endothelial surface, whereas CXCL8 induced only a transient arrest of the cells. The CXCL8- and CCL11-induced arrest was inhibited by simultaneously blocking α4 integrins (HP2/1) and β2 integrins (IB4). In contrast, the C5a-induced arrest was only inhibited by 30% under these conditions. The potency differences of C5a>CCL11>CXCL8 to induce firm adhesion under flow condition was also observed in migration assays and for the activation of the small GTPase Rap-1, which is an important signaling molecule in the inside-out regulation of integrins. Interestingly, only C5a was able to induce the high activation epitope of αMβ2 integrin recognized by MoAb CBRM1/5. The C5a-induced appearance of this epitope and Rap activation was controlled by phospholipase C (PLC), as was shown with the PLC inhibitor U73122. These data show that different chemoattractants are able to induce distinct activation states of integrins on eosinophils and that optimal chemotaxis is associated with the high activation epitope of the αMβ2 integrin. Furthermore, PLC plays an important role in the inside-out signaling and, thus, the activation status of integrins on eosinophils.

Homeostatic intracellular-free Ca2+ is permissive for Rap1-mediated constitutive activation of alpha4 integrins on eosinophils.

Although much progress has been made in understanding the molecular mechanisms underlying agonist-induced “inside-out” activation of integrins, little is known about how basal levels of integrin function are maintained. This is particularly important for nonactivated eosinophils, where intermediate activation of α4β1 integrin supports recruitment to endothelial cells under flow conditions. Depletion of intracellular Ca2+ and pharmacological inhibition of phospholipase C (but not other intracellular signaling molecules, including PI3K, ERK1/2, p38 MAPK, and tyrosine kinase activity) abrogated basal α4 integrin activity in nonactivated eosinophils. Basal α4 integrin activation was associated with activation of the small GTPase Rap1, a known regulator of agonist-induced integrin function. Basal Rap activation was dependent upon phospholipase C, but not intracellular Ca2+. However, depletion of intracellular Ca2+ in CD34+ hematopoietic progenitor cells abolished RapV12-mediated induction of α4 integrin activity. Thus, residual Rap activity or constitutively active Rap activity in Ca2+-depleted cells is not sufficient to induce α4 integrin activation. These data suggest that activation of functional α4 integrin activity in resting eosinophils is mediated by Rap1 provided that the intracellular-free Ca2+ is at a normal homeostatic concentration.

Physiological concentrations of leptin do not affect human neutrophils.

Leptin is an adipokine that is thought to be important in many inflammatory diseases, and is known to influence the function of several leukocyte types. However, no clear consensus is present regarding the responsiveness of neutrophils for this adipokine. In this study a 2D DIGE proteomics approach was used as an unbiased approach to identify leptin-induced effects on neutrophils. Additionally chemotaxis and survival experiments were performed to reproduce results from literature showing putative effects of leptin on these neutrophil responses. Leptin did not induce any significant changes in the proteome provided leptin was added at physiologically relevant concentrations (250 ng). Our leptin batches were biologically active as they induced proliferation in LeptinR expressing Ba/F3 cells. At high concentrations (25000 ng) leptin induced a change in neutrophil proteome. Seventeen differently regulated spots were identified of which twelve could be characterized by mass spectrometry. Two of these identified proteins, SerpinB1 and p40 phox, were chosen for further analysis but leptin-induced expression analyzed by western blot were highly variable. Additionally leptin also induced neutrophil survival at these high concentrations. No leptin-induced chemotaxis of human neutrophils was detected at any concentration. In conclusion, physiological concentrations of leptin do not affect neutrophils. High leptin concentrations induced survival and changes in the neutrophils proteome, but this was most likely mediated by an indirect effect. However, it cannot be ruled out that the effects were mediated by a yet not-identified leptin receptor on human neutrophils.

Proteomic profiling of peripheral blood neutrophils identifies two inflammatory phenotypes in stable COPD patients

BackgroundCOPD is a heterogeneous chronic inflammatory disease of the airways and it is well accepted that the GOLD classification does not fully represent the complex clinical manifestations of COPD and this classification therefore is not well suited for phenotyping of individual patients with COPD. Besides the chronic inflammation in the lung compartment, there is also a systemic inflammation present in COPD patients. This systemic inflammation is associated with elevated levels of cytokines in the peripheral blood, but the precise composition is unknown. Therefore, differences in phenotype of peripheral blood neutrophils in vivo could be used as a read out for the overall systemic inflammation in COPD.MethodOur aim was to utilize an unsupervised method to assess the proteomic profile of peripheral neutrophils of stable COPD patients and healthy age matched controls to find potential differences in these profiles as read-out of inflammatory phenotypes. We performed fluorescence two-dimensional difference gel electrophoresis with the lysates of peripheral neutrophils of controls and stable COPD patients.ResultsWe identified two groups of COPD patients based on the differentially regulated proteins and hierarchical clustering whereas there was no difference in lung function between these two COPD groups. The neutrophils from one of the COPD groups were less responsive to bacterial peptide N-formyl-methionyl-leucyl-phenylalanine (fMLF).ConclusionThis illustrates that systemic inflammatory signals do not necessarily correlate with the GOLD classification and that inflammatory phenotyping can significantly add in an improved diagnosis of single COPD patients.Trial registrationClinicaltrials.gov: NCT00807469 registered December 11th 2008

Preoperative protein profiles in cerebrospinal fluid in elderly hip fracture patients at risk for delirium: A proteomics and validation study

Background A neuroinflammatory response is suggested to play an important role in delirium, a common complication in older hospitalized patients. We examined whether hip fracture patients who develop postoperative delirium have a different proteome in cerebrospinal fluid (CSF) prior to surgery. Methods Patients (≥ 75 years) were admitted for hip fracture surgery. CSF was collected during spinal anaesthesia; proteins were separated using gel electrophoresis and identified with mass spectrometry. We compared the proteome of patients with and without postoperative delirium. Findings were validated in an independent, comparable cohort using immuno-assays. Results In the derivation cohort 53 patients were included, 35.8% developed postoperative delirium. We identified differences in levels of eight CSF proteins between patients with and without subsequent delirium: complement factor C3, contactin-1, fibulin-1 and I-beta-1,3-N-acetylglucosaminyltransferase were significantly lower in patients with postoperative delirium, while neural cell adhesion molecule-2, fibrinogen, zinc-α-2-glycoprotein and haptoglobin levels were significantly higher. In the validation cohort 21.2% of 52 patients developed postoperative delirium. Immuno-assays confirmed contactin-1 results although not statistically significant. Complement factor C3 was significantly higher in patients with postoperative delirium. Conclusion Our results show the complexity of pathophysiological mechanisms involved in delirium and emphasizes the need of independent validation of findings. General significance This study highlights the challenges and inconsistent findings in studies of delirium, a serious complication in older patients. We analysed proteins in CSF, the most proximal fluid to the brain. All patients were free from delirium at the time of sampling.

A comprehensive three-dimensional assay to assess neutrophil defense against bacteria

Neutrophil antibacterial capacity is measured in animal models and in vitro as an important indicator of neutrophil function. To be able to extrapolate their conclusions, in vitro experiments should mimic the in vivo situation. In vivo, antibacterial capacity depends on multiple steps of bacterial sensing, priming, chemotaxis, phagocytosis and intracellular killing. Therefore, we developed a simply executed assay that involves multiple steps in one assay. The neutrophils were incorporated into a three-dimensional matrix of fibrin fibers, in which they could freely migrate. The fibrin matrix provided a more physiological representation of tissue structure than a shaken suspension and extended ex vivo survival of neutrophils. Staphylococci endogenously producing GFP (Green Fluorescent Protein) provided a real-time quantification of the bacterial load without the need for lysing the fibrin matrix or counting of colony forming units on agar plates. The delay in bacterial outgrowth serves as a measure for the relative antibacterial capacity of the neutrophils. Additionally, neutrophil capacity could easily be measured high-throughput in a 96-wells format. In this new assay we study neutrophil behavior in a physiologically relevant setting and explore many functions of the neutrophil in a single test. The functional capacity of neutrophils from different in vitro treatments or different donors can directly be compared.

Postoperative delirium in elderly hip fracture patients: Preoperative CSF proteome suggests a neuroinflammatory response

After facial nerve axotomy in the mouse, a proportion of motorneurons located in the facial nucleus (FN) die over a period of several days and an immune response, characterized by microglial activation and T cell infiltration, occurs within the lesioned FN. Some studies using knock-out mice have described that, among T-cells, Th2 cell subset is responsible for promoting neuronal survival after facial nerve axotomy. The mechanisms underlying lymphocyte recruitment and activation within the central nervous system are not completely understood but a key role exerted by cytokine and chemokine expression may be expected. IL-6 is recognized as a pleiotropic and multifunctional cytokine with a key role in the control of the neuroinflammatory response. Previous studies in our group using the transgenic mice with astrocyte-targeted production of IL-6 (GFAP-IL6Tg) demonstrated a higher neuronal death associated with changes in the microglial activation pattern. In this context, the objective of the present study was to determine the specific lymphocyte subsets in the FN after nerve axotomy and the putative effects exerted by IL-6 production over the infiltration and activation of these lymphocyte subpopulations. To accomplish that, unilateral facial nerve resection was performed on GFAP-IL6Tg mice and their corresponding wild-type (WT) littermates. At 14 dpi and 21 dpi, the different subtypes of T helper cells infiltrated in the FN and the microglial expression of antigen-presenting molecules (MHC-II and costimulatory molecules) were analyzed using flow cytometry. Our results showed that, GFAP-IL6Tg animals presented higher number of CD3+ T-cells than WT at both 14 dpi and 21 dpi, with a remarkable increase of the pro-inflammatory Th1 cell subset at 21 dpi. Interestingly, CD11b+/CD45+ microglia/macrophages population expressed higher levels of MHC-II, CD80 and CD86 in GFAP-IL6Tg animals at 14 dpi. In conclusion, CNS-targeted IL-6 production leads to a higher infiltration of pro-inflammatory T helper cells and increased expression of antigen presenting molecules in microglia/macrophages correlating with higher neuronal death after facial nerve axotomy. Supported by the Spanish Ministry of Science and Innovation (BFU2011-27400) and an NHMRC project grant 632754.