Cytokines and chemokines contribute to the pathogenesis of acute disseminated encephalomyelitis (ADEM). Using a multiplex immunochemiluminescence ELISA, we measured 8 Th1/Th2 cytokines and 18 chemokines in the cerebrospinal fluid (CSF) and serum of 17 ADEM patients, 14 multiple sclerosis (MS) patients, and 7 healthy controls (HCs). Relative to HCs, ADEM patients had significantly high mean CSF concentrations of chemokines with attractant/activating properties towards neutrophils (CXCL1 and CXCL7), monocytes/T cells (CCL3 and CCL5), Th1 cells (CXCL10), and Th2 cells (CCL1, CCL22, and CCL17). Mean CSF concentrations of CXCL7, CCL1, CCL22, and CCL17 were higher in ADEM than in MS, whereas those of CCL11 were lower in MS than in ADEM and HCs. CSF pleocytosis correlated with CSF concentrations of CXCL1, CXCL10, CCL1, CCL17, and CCL22. Most of the functionally homologous chemokines correlated with each other. CSF Th1/Th2 cytokines were not detectable in most samples. Their mean concentrations did not differ in the three groups, and the same held for serum cytokines and chemokines. Our data suggest that the upregulation of chemokines active on neutrophils and Th2 cells differentiates ADEM from MS inflammation, and that both Th1 and Th2 chemokines might be produced in ADEM. Chemokines upregulated in ADEM could become CSF biomarkers after a posteriori evaluations in unselected case series.

A variety of chemokines has been shown to recruit human bone marrow-derived mesenchymal stem cells (MSC) and may be potential candidates for chemokine-based tissue regeneration approaches. The aim of our study was to determine whether the chemokine CXCL7 stimulates migration of human bone marrow-derived MSC and to analyze the effect of CXCL7 on the recruitment of MSC on the broad molecular level. Chemotaxis assays documented that high doses of CXCL7 significantly recruited MSC. Gene expression profiling using oligonucleotide microarrays showed that MSC treated with CXCL7 differentially expressed genes related to cell migration, cell adhesion and extracellular matrix remodeling. Pathway analysis showed that CXCL7 induced the expression of all chemokines binding the interleukin (IL) receptors A and B, CXCR1 and CXCR2, as well as the IL6 signal transducer (gp130) and its ligands IL6 and leukemia inhibitory factor (LIF). Induction of differentially expressed chemokines CXCL1-3, CXCL5, and CXCL6 as well as LIF and gp130 in MSC by CXCL7 was verified by real-time polymerase chain reaction. Immunoassay of cell culture supernatants confirmed elevated levels of the interleukins 6 and 8 in MSC upon treatment with CXCL7. Chemotaxis assays showed that interleukin 6 did not recruit MSC. In conclusion, CXCL7 significantly stimulates the migration of human MSC in vitro. Pathway analysis suggests that recruitment of human MSC by CXCL7 is supported by the induction of ligands of the interleukin 8 receptors, synergistically activating the respective signaling pathways.

Solid tumour and leukemic cells expressing chemokine receptors, metastasize to chemokine-secreting organs. Chemokines indirectly affect tumour development by attracting immunocompetent cells with pro- or anti-tumoral activities. Various membrane-associated and soluble proteases selectively cleave specific chemokines. Precursor plasma chemokines (CXCL7, CCL14) need to be proteolytically processed to obtain receptor affinity. Angiogenic CXC chemokines (CXCL1, CXCL8) have increased CXCR1/CXCR2 affinity after limited NH2-terminal processing, whereas truncated angiostatic chemokines (CXCL10) show lower CXCR3 affinity without loss of angiostatic potential. NH2-terminally cleaved monocyte chemotactic proteins (CCL2, CCL7, CCL8) have impaired capacity to attract tumour-associated macrophages and function as receptor antagonists for intact CC chemokines. Migration of Th1/CCR5+ and Th2/CCR4+ effector lymphocytes toward CCR5 (CCL5, CCL3L1) and CCR4 (CCL22) ligands is affected by cleavage. Although proteolytical processing of chemokines is well studied in vitro, the direct or indirect effects on tumour invasion and metastasis are only poorly evaluated.

The CXC chemokines platelet factor 4 (PF-4/CXCL4) and connective tissue-activating peptide III (CTAP-III) are released by activated human platelets in micromolar concentrations. So far, neutrophils have been recognized to cleave the precursor CTAP-III to form the active chemokine neutrophil-activating peptide 2 (NAP-2/CXCL7) through limited proteolysis by membrane-associated cathepsin G. Here we show for the first time that activated human skin mast cells (MCs) convert CTAP-III into biologically active NAP-2 through proteolytic cleavage by released chymase. A direct comparison on a cell number basis revealed that unstimulated MCs exceed the CTAP-III-processing potency of neutrophils about 30-fold, whereas MCs activated by IgE cross-linking exhibit even 1000-fold higher CTAP-III-processing capacity than fMLP-stimulated neutrophils. Intriguingly, PF-4 counteracted MC- as well as neutrophil-mediated NAP-2 generation at physiologically relevant concentrations. Addressing the underlying mechanism, we obtained evidence that PF-4 acts as an inhibitor of the CTAP-III-processing enzymes cathepsin G and chymase without becoming cleaved itself as a competitive substrate. Because cleavage of the CTAP-III-unrelated substrate substance P was also affected by PF-4, our results suggest a regulatory role for PF-4 not only in NAP-2 generation but also in neutrophil- and MC-mediated processing of other physiologically relevant inflammatory mediators.

Strategies for improved homing of mesenchymal stem cells (MSCs) to a place of injury are being sought and it has been shown that natural killer (NK) cells can stimulate MSC recruitment. Here, we studied the chemokines behind this recruitment. Assays were performed with bone marrow human MSCs and NK cells freshly isolated from healthy donor buffy coats. Supernatants from MSC-NK cell co-cultures can induce MSC recruitment but not to the same extent as when NK cells are present. Antibody arrays and ELISA assays confirmed that NK cells secrete RANTES (CCL5) and revealed that human NK cells secrete NAP-2 (CXCL7), a chemokine that can induce MSC migration. Inhibition with specific antagonists of CXCR2, a receptor that recognizes NAP-2, abolished NK cell-mediated MSC recruitment. This capacity of NK cells to produce chemokines that stimulate MSC recruitment points toward a role for this immune cell population in regulating tissue repair/regeneration.

Early pro-angiogenic cells (EPCs) have been shown to be involved in neovascularization, angiogenesis and re-endothelialization and cathepsin L inhibition blunted their pro-angiogenic effect. In the present study, we have analysed and mapped the proteome and secretome of human EPCs, utilizing a combination of difference in-gel electrophoresis (DIGE) and shotgun proteomics. A population of 206 protein spots were analysed, with 171 being identified in the cellular proteome of EPCs. 82 proteins were identified in their conditioned medium, including the alternative macrophage markers C-C motif chemokine 18 (CCL18) and the hemoglobin scavenger receptor CD163 as well as platelet factor 4 (CXCL4) and platelet basic protein (CXCL7) with "platelet alpha granule" being returned as the top category according to the Gene Ontology Annotation. Apart from cathepsin L, the cathepsin L inhibitor also attenuated the release of a wide range of other cathepsins and lysosomal proteins such as legumain, but stimulated the secretion of members of the S100 protein family. The data presented here are the most comprehensive characterization of protein expression and secretion in human EPCs to date and highlight the potential importance of cysteine proteases in the processing of platelet factors for their pro-angiogenic potential. This article is part of a special issue entitled, "Cardiovascular Stem Cells Revisited".

BACKGROUND

Rhinoviruses (RVs) are the major triggers of asthma exacerbations. We have shown previously that lower respiratory tract symptoms, airflow obstruction, and neutrophilic airway inflammation were increased in experimental RV-induced asthma exacerbations.

OBJECTIVE

We hypothesized that neutrophil-related CXC chemokines and antimicrobial peptides are increased and related to clinical, virologic, and pathologic outcomes in RV-induced exacerbations of asthma.

METHODS

Protein levels of antimicrobial peptides (SLPI, HNP 1-3, elafin, and LL-37) and neutrophil chemokines (CXCL1/GRO-α, CXCL2/GRO-β, CXCL5/ENA-78, CXCL6/GCP-2, CXCL7/NAP-2, and CXCL8/IL-8) were determined in bronchoalveolar lavage (BAL) fluid of 10 asthmatics and 15 normal controls taken before, at day four during and 6 weeks post-experimental infection.

RESULTS

BAL HNP 1-3 and Elafin were higher, CXCL7/NAP-2 was lower in asthmatics compared with controls at day 4 (P = 0.035, P = 0.048, and P = 0.025, respectively). BAL HNP 1-3 and CXCL8/IL-8 were increased during infection (P = 0.003 and P = 0.011, respectively). There was a trend to increased BAL neutrophils at day 4 compared with baseline (P = 0.076). BAL HNP 1-3 was positively correlated with BAL neutrophil numbers at day 4. There were no correlations between clinical parameters and HNP1-3 or IL-8 levels.

CONCLUSIONS

We propose that RV infection in asthma leads to increased release of CXCL8/IL-8, attracting neutrophils into the airways where they release HNP 1-3, which further enhances airway neutrophilia. Strategies to inhibit CXCL8/IL-8 may be useful in treatment of virus-induced asthma exacerbations.

Dendritic cells (DCs) are the most potent antigen-presenting cells and populate many tissues where they may participate in inflammatory reactions. The infiltration of polymorphonuclear leucocytes (PMNLs) into tissues is a prominent feature of inflammation. The mechanisms of PMNL recruitment depend on chemotactic factors and adhesion molecules expressed on endothelial cells. The aim of the present study was to determine whether DCs participate in the early recruitment of PMNLs. Dendritic cells derived from peripheral blood monocytes were used for this study. PMNLs incubated with culture supernatant (CS) from untreated or from tumour necrosis factor-alpha (TNF-alpha)-treated (1 hr, 100 U/ml, 37 degrees ) monocyte-derived DCs (moDCs) had increased surface expression of both CD11b and CD18. Moreover, both untreated and TNF-alpha-treated moDCs induced PMNL chemotaxis. By blocking CXCL8, CXCL5, CXCL7 and Pan GRO (CXCL1, CXCL2, CXCL3), we observed that CXCL8/interleukin-8 might be the chemokine that induced the PMNL chemotactic activity in the CS of untreated and TNF-alpha-treated moDC. Furthermore, we investigated the regulation of CXCL8 production in moDCs by adhesion molecule engagement. Our data demonstrated that CD31, CD18, CD29 and CD49d participated in the adhesion of immature moDCs to endothelium. Moreover, engagement of domains 1-3 of CD31, but not of CD29 or CD18, decreased the production of CXCL8 by immature but not mature moDCs (which display lower CD31 levels than immature moDCs). Overall, these results suggest that DCs not only trigger a specific immune response, but also the innate immune response by recruiting PMNLs. Furthermore, our results also suggest that CXCL8 production by immature DCs might be regulated by signalling through CD31 during their migration through the vascular endothelium.

Chemokines play crucial roles in combating microbial infection and initiating tissue repair by recruiting neutrophils in a timely and coordinated manner. In humans, no less than seven chemokines (CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, CXCL7, and CXCL8) and two receptors (CXCR1 and CXCR2) mediate neutrophil functions but in a context dependent manner. Neutrophil-activating chemokines reversibly exist as monomers and dimers, and their receptor binding triggers conformational changes that are coupled to G-protein and β-arrestin signaling pathways. G-protein signaling activates a variety of effectors including Ca2+ channels and phospholipase C. β-arrestin serves as a multifunctional adaptor and is coupled to several signaling hubs including MAP kinase and tyrosine kinase pathways. Both G-protein and β-arrestin signaling pathways play important non-overlapping roles in neutrophil trafficking and activation. Functional studies have established many similarities but distinct differences for a given chemokine and between chemokines at the level of monomer vs. dimer, CXCR1 vs. CXCR2 activation, and G-protein vs. β-arrestin pathways. We propose that two forms of the ligand binding two receptors and activating two signaling pathways enables fine-tuned neutrophil function compared to a single form, a single receptor, or a single pathway. We summarize the current knowledge on the molecular mechanisms by which chemokine monomers/dimers activate CXCR1/CXCR2 and how these interactions trigger G-protein/β-arrestin-coupled signaling pathways. We also discuss current challenges and knowledge gaps, and likely advances in the near future that will lead to a better understanding of the relationship between the chemokine-CXCR1/CXCR2-G-protein/β-arrestin axis and neutrophil function.

Chemokines secreted by DC are instrumental for DC to regulate their own migratory capacities and to recruit T lymphocytes during local tumour immune response. Using the recently developed chemokine protein arrays, we analyzed 38 chemokines associated with monocyte-derived DC (MoDC), including the CC family (CCL2, CCL3, CCL4, CCL17, CCL18, CCL22, CCL23, CCL24, CCL27) and the CXC family (CXCL3, CXCL5, CXCL7, CXCL8, CXCL16) chemokines. Our results indicate that MoDC largely inherit the chemokines constitutively expressed by monocytes, with a significant induction of CCL17, CCL22 and CCL23. Spent culture supernatant collected from MoDC exhibited chemotatic abilities to activate CD4(+), CD8(+), and CD25(+) Foxp3(+) regulatory T cells (Tregs). Selective knock down of CCL22 and CCL17 expression by siRNA decreased the ratios of CD4(+) to CD8(+), as well as the frequency of Tregs recruited by MoDC. Intratumoural injection of MoDC transfected with siCCL22 and siCCL17, significantly reduced the number of Tregs while increasing the number of infiltrating CD8(+) T cells in human tumour xenografts in athymic nude mice. This study demonstrates that chemokine expression of MoDC is complex and may change dynamically. Using siRNA to selectively knock down chemokines which are highly chemoattractive to Tregs may consequentially alter the lymphocyte populations recruited into the tumour microenvironment, therefore has the potency to provide insight into cellular interactions in cancer immunology. This may lead to a new strategy for DC vaccine development to improve cancer immunobiotherapy.

BACKGROUND

Clinical and translational research on lung cancer patients undergoing surgical treatment can provide valuable scientific data and unique opportunity to study tumor microenvironment. CXC chemokines, which are members of a big family of cytokines, are undoubtedly involved in tumor growth regulation and metastasizing pathways. For better understanding of CXC chemokine involvement in the process of carcinogenesis we have studied the cohort of early stage non-small cell lung cancer patients undergoing surgery with curative intent. Our aim was to assess CXC chemokine ligand (CXCL) levels in patient blood samples representing systemic circulation and tumor microenvironment; assess CXC chemokine receptor (CXCR) expression in tumor tissue; and measure tumor infiltrating immune cell subpopulations.

METHODS

A total of 54 patients with NSCLC had radical lung resection were enrolled in a single center prospective study and were followed-up annually for up to six years. During surgical procedure peripheral and tumor draining blood samples were taken. CXCL1, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11 and CXCL12 levels were determined by ELISA, and chemokine concentration gradient was calculated. Tumor infiltrating immune cells (T helper cells, T cytotoxic cells, macrophages, B cells, plasma cells) and expression of CXCR1, CXCR2, CXCR3 and CXCR4 in tumor tissue were assessed by immunohistochemistry.

RESULTS

Statistically significant decrease in chemokine concentration was found for CXCL4 (P=0.002) and CXCL5 (P=0.011), and statistically significant concentration increase was found for CXCL7 (P=0.001) in total cohort. We have found statistically significant CXC chemokine concentration change for majority of chemokines-CXCL1 (P=0.002), CXCL4 (P=0.001), CXCL5 (P=0.013), CXCL7 (P=0.036), CXCL8 (P=0.026), CXCL9 (P=0.034) and CXCL10 (P=0.032) in a group of patients who had good clinical result after surgery with no evidence of relapse, on the other hand patients with cancer recurrence including local and systemic cancer spread did not show any change of chemokine concentration in blood except for CXCL1 (P=0.041). We have also found that chemokine levels and gradients correlate with CXC receptor expression and number of tumor infiltrating immune cell subpopulations.

CONCLUSIONS

Assessment of tumor microcirculation is useful for evaluation of different types of circulating biomarkers and application of our method can be very wide, integrating thoracic surgeons into translational cancer research.

Infection of humans with Ehrlichia chaffeensis, the etiologic agent of human monocytic ehrlichiosis, can cause hepatitis of various levels of severity. When the three human isolates of E. chaffeensis, each belonging to a different genogroup, are inoculated into severe combined immunodeficiency mice, the order of severity of clinical signs and bacterial burden detected in the liver is as follows (from greatest to least severity and highest to lowest burden): strain Wakulla, followed by strain Liberty, followed by strain Arkansas. In this article, we used microarray analysis to define transcriptional profiles characteristic of the histopathological features in the mouse liver. Cytokine and chemokine profiles and their receptor profiles were strikingly different among the three strains of E. chaffeensis: gamma interferon, CCL5, CXCL1, CXCL2, CXCL7, CXCL9, interleukin 2 receptor gamma (IL2Rgamma), IL21R, CCR2, and CXCR6 were highly upregulated with strain Arkansas; and tumor necrosis factor (TNF), CCL2, CCL3, CCL5, CCL6, CCL12, CCL20, CXCL2, CXCL7, CXCL9, CXCL13, TNF receptor superfamily 9 (TNFRSF9), TNFRSF13beta, IL1R2, IL2Rgamma, IL20Rbeta, IL21R, CCR1, CCR2, and CXCR4 were highly upregulated with strain Wakulla. With strain Liberty, only CXCL13 was highly upregulated, and IL13Ralpha2 was downregulated. In livers infected with the Arkansas strain, monocytes/macrophages and NK cells were enriched in the granulomas and an increase in NK cell marker mRNAs was detected. Livers infected with the Wakulla strain displayed infiltration of significantly more neutrophils and an increase in neutrophil marker mRNAs. Genes commonly upregulated in liver tissue infected with the three strains are other host innate immune and inflammatory response genes, including those encoding several acute-phase proteins. Genes downregulated commonly are related to host physiologic functions. The results suggest that marked modulation of host cytokine and chemokine profiles by E. chaffeensis strains underlies the distinct host liver disease.

OBJECTIVE

Platelet factor 4 tetramers (CXCL4 chemokine) form complexes with β2glycoprotein I (β2GPI), recognized by anti-β2GPI antibodies leading to platelet activation in antiphospholipid syndrome (APS), either primary (PAPS) or secondary (SAPS). Increased plasma levels of CXCL4 may favor this process; therefore we measured plasma levels of CXCL4, a CXCL4 variant (CXCL4L1) and as controls, platelet-derived chemokines CXCL7 (NAP-2) and CCL5 (RANTES), in APS, and disease controls such as patients with systemic lupus erythematosus (SLE) coronary artery disease (CAD) and healthy donors (HDs).

METHODS

Plasma samples and platelets were isolated from patients with APS (n = 87), SLE (n = 29), CAD (n = 14) and 54 HDs. Plasma levels of CXCL4, CXCL4L1, CXCL7 and CCL5 as well as intracellular platelet CXCL4 and CXCL4L1 were measured using ELISA. Platelet CXCL4 and CXCL4L1 RNA levels were determined by RT-PCR.

RESULTS

CXCL4, CXCL7 (NAP-2) and CCL5 (RANTES) plasma levels were significantly higher in patients with APS compared to both control groups (SLE, CAD) and HDs. CXCL4L1 plasma levels were also significantly higher in APS than in SLE and HDs, but lower from that of CAD patients. Statistically significant concordance was detected between CXCL4 and CXCL7 (p < 0.0001) or CCL5 (p < 0.0001) plasma levels in patients with APS, either PAPS or SAPS. CXCL4L1 plasma levels were inversely correlated with CXCL4 (P = 0.0027), CXCL7 (p = 0.012) and CCL5 (p = 0.023) in PAPS and positively with CXCL4 (p = 0.0191), CCL5 (p < 0.0001) and CXCL7 (P < 0.0001), in SAPS. Levels of CXCL4, CXCL4L1, CXCL7 and CCL5 were divided in "high" (exceeding a level defined as the mean of HDs and 3 SD) and "low" (below this level); The "CXCL4L1 high" group was characterized by increased IgG aCL, (p = 0.0215), double antibody positivity (either aCL or anti-β2GPI plus LA), (p = 0.0277), triple antibody positivity (aCL plus anti-β2GPI plus LA), (p = 0.0073) and thrombocytopenia (p = 0.0061), as well as with at least 1 thrombotic event or the last 5 years (p = 0.0001), or more than 3 thrombotic events ever (p = 0.0151).

CONCLUSIONS

Chemokines associated with platelet activation and immune cell chemotaxis were found to be elevated in APS patients' plasma and may contribute to the pathogenesis of the syndrome. High CXCL4L1 plasma levels are associated with the clinical expression of APS and should be prospectively evaluated as a biomarker.

OBJECTIVE

To investigate the chemokine expression profiles in the aqueous humor of wet age-related macular degeneration (wet AMD) patients and to correlate their levels with clinical findings.

METHODS

Undiluted aqueous humor samples (100-200 μl) were obtained from 16 wet AMD eyes and 12 control eyes. Forty chemokines were measured using a multiplex method. A 6×6 mm area of the macular region centered on the fovea was examined using spectral domain optical coherence tomography (SD-OCT).

RESULTS

The detection rates were 50% or more for 15 chemokines. Compared with the control group, the aqueous humor in wet AMD patients showed a significantly higher expression of CXCL10 (p=0.004), CCL14 (p=0.002), CXCL16 (p=0.013), CXCL7 (p=0.033), and CCL22 (p=0.037), while growth-related oncogene (GRO) was significantly decreased in the wet AMD patients (p=0.001). When compared with treatment-naïve patients, the recurrent group had significant upregulation of CXCL10 (p=0.012) and CCL22 (p=0.002). CXCL16 was positively correlated with lesion size, and CCL22 was higher in patients whose OCT images showed intraretinal fluid (IRF) or hyperreflective foci (HF).

CONCLUSIONS

Elevated levels of inflammation-related chemokines, including CXCL10, CCL14, CXCL16, CXCL7, and CCL22, in the aqueous humor of AMD patients may suggest a pathogenic role for inflammation. CXCL10 and CCL22 were more elevated in eyes with recurrent wet AMD than in treatment-naïve eyes. CXCL16 was positively correlated with lesion size. The increase in CCL22 was correlated with the presence of IRF or HF. These data may be of interest in the search for biomarkers associated with wet AMD and may potentially indicate different treatment strategies.

Increased expression of lymphangiogenesis factors VEGF-C/D and heparanase has been correlated with the invasion of cancer. Furthermore, chemokines may modify matrix to facilitate metastasis, and they are associated with VEGF-C and heparanase. The chemokine CXCL7 binds heparin and the G-protein-linked receptor CXCR2. We investigated the effect of CXCR2 blockade on the expression of VEGF-C/D, heparanase, and on invasion. CXCL7 siRNA and a specific antagonist of CXCR2 (SB225002) were used to treat CXCL7 stably transfected MCF10AT cells. Matrigel invasion assays were performed. VEGF-C/D expression and secretion were determined by real-time PCR and ELISA assay, and heparanase activity was quantified by ELISA. SB225002 blocked VEGF-C/D expression and secretion (P < .01). CXCL7 siRNA knockdown decreased heparanase (P < .01). Both SB225002 and CXCL7 siRNA reduced the Matrigel invasion (P < .01). The MAP kinase signaling pathway was not involved. The CXCL7/CXCR2 axis is important for cell invasion and the expression of VEGF-C/D and heparanase, all linked to invasion.

BACKGROUND

Glomerular endothelium dysfunction, which plays a crucial role in the pathogenesis of early diabetic nephropathy, might be caused by circulating metabolic abnormalities. Platelet microparticles, extracellular vesicles released from activated platelets, have recently emerged as a novel regulator of vascular dysfunction.

METHODS

We studied the effects of platelet microparticles on glomerular endothelial injury in early diabetic nephropathy in rats with streptozotocin-induced diabetes and primary rat glomerular endothelial cells. Isolated platelet microparticles were measured by flow cytometry.

RESULTS

Plasma platelet microparticles were significantly increased in diabetic rats, an effect inhibited in aspirin-treated animals. In cultured glomerular endothelial cells, platelet microparticles induced production of reactive oxygen species, decreased nitric oxide levels, inhibited activities of endothelial nitric oxide synthase and SOD, increased permeability of the glomerular endothelium barrier, and reduced thickness of the endothelial surface layer. Conversely, inhibition of platelet microparticles in vivo by aspirin improved glomerular endothelial injury. Further analysis showed that platelet microparticles activated the mammalian target of rapamycin complex 1 (mTORC1) pathway in glomerular endothelial cells; inhibition of the mTORC1 pathway by rapamycin or raptor siRNA significantly protected against microparticle-induced glomerular endothelial injury in vivo and in vitro. Moreover, platelet microparticle-derived chemokine ligand 7 (CXCL7) contributed to glomerular endothelial injury, and antagonizing CXCL7 using CXCL7-neutralizing antibody or blocking CXCL7 receptors with a competitive inhibitor of CXCR1 and CXCR2 dramatically attenuated such injury.

CONCLUSIONS

These findings demonstrate a pathogenic role of platelet microparticles in glomerular endothelium dysfunction, and suggest a potential therapeutic target, CXCL7, for treatment of early diabetic nephropathy.

Chemokines encompass a large family of proteins that act as chemoattractants and are involved in many biological processes. In particular, chemokines guide the migration of leukocytes during normal and inflammatory conditions. Recent studies reveal that the heterophilic interactions between chemokines significantly affect their biological activity, possibly representing a novel regulatory mechanism of the chemokine activities. The co-localization of platelet-derived chemokines in vivo allows them to interact. Here, we used nano-spray ionization mass spectrometry to screen eleven different CXC and CC platelet-derived chemokines for possible interactions with the two most abundant chemokines present in platelets, CXCL4 and CXCL7. Results indicate that many screened chemokines, although not all of them, form heterodimers with CXCL4 and/or CXCL7. In particular, a strong heterodimerization was observed between CXCL12 and CXCL4 or CXCL7. Compared to other chemokines, the main structural difference of CXCL12 is in the orientation and packing of the C-terminal alpha-helix in relation to the beta-sheet. The analysis of one possible structure of the CXCL4/CXCL12 heterodimer, CXC-type structure, using molecular dynamics (MD) trajectory reveals that CXCL4 may undergo a conformational transition to alter the alpha helix orientation. In this new orientation, the alpha-helix of CXCL4 aligns in parallel with the CXCL12 alpha-helix, an energetically more favorable conformation. Further, we determined that CXCL4 and CXCL12 physically interact to form heterodimers by co-immunoprecipitations from human platelets. Overall, our results highlight that many platelet-derived chemokines are capable of heterophilic interactions and strongly support future studies of the biological impact of these interactions.

BACKGROUND

The platelet (PLT) storage lesion remains incompletely understood. To gain a greater insight into the PLT storage lesion, a proteomic analysis of supernatant from leukofiltered pooled buffy-coat PLT concentrates (PCs) was undertaken.

METHODS

PCs were prepared in PLT additive solution and stored according to standard blood bank procedures. Supernatant samples were collected throughout 7 days of storage. Maps of supernatant proteins were generated by two-dimensional (2D) gel electrophoresis and mass spectrometry. Cytokine antibody microarrays and enzyme-linked immunosorbent assay were used to investigate bioactive molecules.

RESULTS

The 2D gel maps of PC supernatant proteins displayed many features of plasma protein maps. Several storage-induced protein changes were identified including modifications to major plasma proteins. PLT-derived proteins were also identified, including tremlike transcript 1 and integrin-linked kinase, which may influence PLT-endothelium interactions. Cytokine antibody microarrays revealed a number of bioactive proteins that have not been previously associated with PCs produced for transfusion, such as brain-derived neurotrophic factor (BDNF). The concentration of PLT-derived cytokines including BDNF, CXCL7, epidermal growth factor, PLT-derived growth factor (PDGF), and CCL5 significantly increased during storage of PCs. Extended storage from Day 5 to Day 7 caused significantly increased levels of BDNF, PDGF, and CCL5 in PC supernatant.

CONCLUSIONS

Proteomic techniques provide valuable new insight into the effects of storage on PCs and the contribution of soluble proteins to the development of the PLT storage lesion and recipient responses to PLT transfusion.

Platelet-derived chemokine CXCL7 (also known as NAP-2) plays a crucial role in orchestrating neutrophil recruitment in response to vascular injury. CXCL7 exerts its function by activating the CXC chemokine receptor 2 (CXCR2) receptor and binding sulfated glycosaminoglycans (GAGs) that regulate receptor activity. CXCL7 exists as monomers, dimers, and tetramers, and previous studies have shown that the monomer dominates at lower and the tetramer at higher concentrations. These observations then raise the question: what, if any, is the role of the dimer? In this study, we make a compelling observation that the dimer is actually the favored form in the GAG-bound state. Further, we successfully characterized the structural basis of dimer binding to GAG heparin using solution nuclear magnetic resonance (NMR) spectroscopy. The chemical shift assignments were obtained by exploiting heparin binding-induced NMR spectral changes in the WT monomer and dimer and also using a disulfide-linked obligate dimer. We observe that the receptor interactions of the dimer are similar to the monomer and that heparin-bound dimer is occluded from receptor interactions. Cellular assays also show that the heparin-bound CXCL7 is impaired for CXCR2 activity. We conclude that the dimer-GAG interactions play an important role in neutrophil-platelet crosstalk, and that these interactions regulate gradient formation and the availability of the free monomer for CXCR2 activation and intrathrombus neutrophil migration to the injury site.

BACKGROUND

Central retinal vein occlusion (CRVO) is a common disease characterized by a disrupted retinal blood supply and a high risk of subsequent vision loss due to retinal edema and neovascular disease. This study was designed to assess the concentrations of selected signaling proteins in the vitreous and blood of patients with ischemic CRVO.

METHODS

Vitreous and blood samples were collected from patients undergoing surgery for ischemic CRVO (radial optic neurotomy (RON), n = 13), epiretinal gliosis or macular hole (control group, n = 13). Concentrations of 40 different proteins were determined by an ELISA-type antibody microarray.

RESULTS

Expression of proteins enriched in the vitreous (CCL2, IGFBP2, MMP10, HGF, TNFRSF11B (OPG)) was localized by immunohistochemistry in eyes of patients with severe ischemic CRVO followed by secondary glaucoma. Vitreal expression levels were higher in CRVO patients than in the control group (CRVO / control; p < 0.05) for ADIPOQ (13.6), ANGPT2 (20.5), CCL2 (MCP1) (3.2), HGF (4.7), IFNG (13.9), IGFBP1 (14.7), IGFBP2 (1.8), IGFBP3 (4.1), IGFBP4 (1.7), IL6 (10.8), LEP (3.4), MMP3 (4.3), MMP9 (3.6), MMP10 (5.4), PPBP (CXCL7 or NAP2) (11.8), TIMP4 (3.8), and VEGFA (85.3). In CRVO patients, vitreal levels of CCL2 (4.2), HGF (23.3), IGFBP2 (1.23), MMP10 (2.47), TNFRSF11B (2.96), and VEGFA (29.2) were higher than the blood levels (vitreous / blood, p < 0.05). Expression of CCL2, IGFBP2, MMP10, HGF, and TNFRSF11B was preferentially localized to the retina and the retinal pigment epithelium (RPE).

CONCLUSIONS

Proteins related to hypoxia, angiogenesis, and inflammation were significantly elevated in the vitreous of CRVO patients. Moreover, some markers known to indicate atherosclerosis may be related to a basic vascular disease underlying RVO. This would imply that local therapeutic targeting might not be sufficient for a long term therapy in a systemic disease but hypothetically reduce local changes as an initial therapeutic approach.

BACKGROUND

In monocytes of patients with autoimmune diabetes, we recently identified a gene expression fingerprint of two partly overlapping gene clusters, a PDE4B-associated cluster (consisting of 12 core proinflammatory cytokine/compound genes), a FABP5-associated cluster (three core genes), and a set of nine overlapping chemotaxis, adhesion, and cell assembly genes correlating to both PDE4B and FABP5.

OBJECTIVE

Our objective was to study whether a similar monocyte inflammatory fingerprint as found in autoimmune diabetes is present in autoimmune thyroid disease (AITD).

METHODS

Quantitative PCR was used for analysis of 28 genes in monocytes of 67 AITD patients and 70 healthy controls. The tested 28 genes were the 24 genes previously found abnormally expressed in monocytes of autoimmune diabetes patients plus four extra genes found in whole-genome analysis of monocytes of AITD patients reported here.

RESULTS

Monocytes of 24% of AITD and 50% of latent autoimmune diabetes of adults (LADA) patients shared an inflammatory fingerprint consisting of the set of 24 genes of the PDE4B, FABP5, and overlapping gene sets. This study in addition revealed that FCAR, the gene for the Fcalpha receptor I, and PPBP, the gene for CXCL7, were part of this proinflammatory monocyte fingerprint.

CONCLUSIONS

Our study provides an important tool to determine a shared, specific proinflammatory state of monocytes in AITD and LADA patients, enabling further research into the role of such proinflammatory cells in the failure to preserve tolerance in these conditions and of key fingerprint genes involved.

BACKGROUND

The angiogenic proteins angiopoietin (Ang)-1, Ang-2 and vascular endothelial growth factor (VEGF) are regulators of endothelial inflammation and integrity. Since platelets store large amounts of Ang-1 and VEGF, measurement of circulation levels of these proteins is sensitive to platelet number, in vivo platelet activation and inadvertent platelet activation during blood processing. We studied plasma Ang-1, Ang-2 and VEGF levels in malaria patients, taking the necessary precautions to avoid ex vivo platelet activation, and related plasma levels to platelet count and the soluble platelet activation markers P-selectin and CXCL7.

METHODS

Plasma levels of Ang-1, Ang-2, VEGF, P-selectin and CXCL7 were measured in CTAD plasma, minimizing ex vivo platelet activation, in 27 patients with febrile Plasmodium falciparum malaria at presentation and day 2 and 5 of treatment and in 25 healthy controls.

RESULTS

Levels of Ang-1, Ang-2 and VEGF were higher at day 0 in malaria patients compared to healthy controls. Ang-2 levels, which is a marker of endothelial activation, decreased after start of antimalarial treatment. In contrast, Ang-1 and VEGF plasma levels increased and this corresponded with the increase in platelet number. Soluble P-selectin and CXCL7 levels followed the same trend as Ang-1 and VEGF levels. Plasma levels of these four proteins correlated strongly in malaria patients, but only moderately in controls.

CONCLUSIONS

In contrast to previous studies, we found elevated plasma levels of Ang-1 and VEGF in patients with malaria resulting from in vivo platelet activation. Ang-1 release from platelets may be important to dampen the disturbing effects of Ang-2 on the endothelium. Evaluation of plasma levels of these angiogenic proteins requires close adherence to a stringent protocol to minimize ex vivo platelet activation.

BACKGROUND

Many studies have demonstrated that chemokines and their receptors play important roles in breast cancer. However, few of them focus on the concentration change of chemokines along breast cancer evolvement, especially for primary breast cancer.

OBJECTIVE

To investigate the effects of chemokines and their receptors on different stage of primary breast cancer, and to find correlationships between chemokines, between different clinico-pathological characters of patients or between chemokines and different clinico-pathological characters of patients.

METHODS

We evaluated and compared the concentration of 10 chemokines and receptors in serum of patients diagnosed as breast benign change, epithelial proliferation (present only or with atypia), in situ carcinoma and invasive carcinoma.

RESULTS

Our oneway ANOVA analysis results showed that in all cases from benign diseases to invasive carcinoma, the concentration of CXCL8, CXCR4 and CXCL12 was significantly different; in benign subgroups (benign change, benign change with proliferation, atypia), the concentration of CCL2 and CCR5 was significantly different; in invasive carcinoma cases, DARC concentration was significantly correlated with the relapse risk of patients.

CONCLUSIONS

The correlation analysis indicated the great crosstalk between chemokines and receptors in the course of primary breast cancer; Ki67 expression was associated with CXCL5 and CXCL7 concentration; tumor size was associated with CXCL8 concentration; and the correlation analysis between clinico-pathological characters of patients showed that pathological diagnosis was correlated with tumor size, relapse risk and Ki67 expression; nuclear grades was correlated with LN metastasis, ER status, PR status and the breast cancer genotype; LN metastasis was correlated with relapse risk. Our findings clearly indicated for the first time that the fluctuations of chemokines and receptors contributed to the evolving of primary breast cancer.

CXCL7, a chemokine highly expressed in platelets, orchestrates neutrophil recruitment during thrombosis and related pathophysiological processes by interacting with CXCR2 receptor and sulfated glycosaminoglycans (GAG). CXCL7 exists as monomers and dimers, and dimerization (~50 μM) and CXCR2 binding (~10 nM) constants indicate that CXCL7 is a potent agonist as a monomer. Currently, nothing is known regarding the structural basis by which receptor and GAG interactions mediate CXCL7 function. Using solution nuclear magnetic resonance (NMR) spectroscopy, we characterized the binding of CXCL7 monomer to the CXCR2 N-terminal domain (CXCR2Nd) that constitutes a critical docking site and to GAG heparin. We found that CXCR2Nd binds a hydrophobic groove and that ionic interactions also play a role in mediating binding. Heparin binds a set of contiguous basic residues indicating a prominent role for ionic interactions. Modeling studies reveal that the binding interface is dynamic and that GAG adopts different binding geometries. Most importantly, several residues involved in GAG binding are also involved in receptor interactions, suggesting that GAG-bound monomer cannot activate the receptor. Further, this is the first study that describes the structural basis of receptor and GAG interactions of a native monomer of the neutrophil-activating chemokine family.