Impact of diabetes on neutrophil-to-lymphocyte ratio and its relationship to coronary artery disease.

BACKGROUND: Coronary artery disease (CAD) is the leading cause of mortality among diabetic patients, and the neutrophil-to-lymphocyte ratio (NLR) has recently emerged from among inflammatory parameters as a potential indicator of vascular complications and poorer outcome in patients with diabetes. This study aimed to evaluate: 1) the impact of diabetes on NLR; and 2) the role of NLR on the extent of CAD among diabetic patients undergoing coronary angiography. METHODS: Consecutive patients undergoing coronary angiography were included. Diabetic status and main chemistry parameters were assessed at the time of admission. Significant CAD was defined as at least one vessel with stenosis>50%, while severe CAD was left main and/or three-vessel disease, as evaluated by quantitative coronary angiography (QCA). RESULTS: Diabetes was observed in 1377 of 3756 patients (36.7%); they were older, and displayed higher-risk cardiovascular profile and more complex CAD. Diabetic status was also associated with a significant increase in NLR (P=0.004). Among diabetics, higher NLR tertile values were related to ageing (P<0.001), dyslipidaemia (P<0.001), renal failure (P<0.001), body mass index (P<0.001), previous percutaneous coronary revascularization (P=0.004) and cerebrovascular events (P=0.003), acute presentation (P<0.001), treatment at admission with beta-blockers/statins/ASA (all P<0.001), diuretics (P=0.01) or clopidogrel (P=0.04), platelet count (P=0.03), white blood cell count, creatinine, glycaemia and C-reactive protein (P<0.001), and inversely related to haemoglobin, triglyceride levels (P<0.001) and smoking (P=0.03). NLR was associated with multivessel disease (P<0.001), degree of stenosis (P=0.01), type C lesions (P=0.02), coronary calcifications and intracoronary thrombus (P<0.001), but inversely with in-stent restenosis (P=0.003) and TIMI flow grade (P=0.02). Also, NLR was directly related to CAD prevalence (P<0.001; adjusted OR [95% CI]: 1.62 [1.27-2.07], P<0.001) and CAD severity (P<0.001; adjusted OR [95% CI]: 1.19 [1.00-1.43], P=0.05). CONCLUSION: NLR is increased among diabetic patients and, in such patients, is independently associated with the prevalence and severity of CAD. Further studies are now needed to confirm present results and to evaluate the underlying pathophysiological mechanisms behind our findings.

Human platelets express authentic CB1 and CB2 receptors.

In the last decade, the neurovascular effects exerted by endocannabinoids (eCBs) have attracted growing interest, because they hold the promise to open new avenues of therapeutic intervention against major causes of death in Western society. Several actions of eCBs are mediated by type-1 (CB1) or type-2 (CB2) cannabinoid receptors, yet there is no clear evidence of the presence of these proteins in platelets. To demonstrate that CB1 and CB2 are expressed in human platelets, we analyzed their protein level by Western blotting and ELISA, visualized their cellular localization by confocal microscopy, and ascertained their functionality by binding assays. We found that CB1, and to a lesser extent CB2, are expressed in highly purified human platelets. Both receptor subtypes were predominantly localized inside the cell, thus explaining why they might remain undetected in preparations of plasma membranes. The identification of authentic CB1 and CB2 in human platelets supports the potential exploitation of selective agonists or antagonists of these receptors as novel therapeutics to combat neurovascular disorders. It seems remarkable that some of these substances have been already used in humans to treat disease states.

The endocannabinoid 2-arachidonoylglycerol activates human platelets through non-CB1/CB2 receptors.

BACKGROUND: The endocannabinoid 2-arachidonoylglycerol (2-AG) is an endogenous lipid that acts through the activation of G-protein-coupled cannabinoid receptors and plays essential roles in many physiological contexts. In the cardiovascular system 2-AG is generated by both activated endothelial cells and platelets, and participates in the regulation of inflammation and thrombosis. Although human platelets actively metabolize endocannabinoids, 2-AG also binds to platelet surface and leads to cell activation. OBJECTIVE: To investigate the biological consequence of 2-AG interactions with human platelets and to clarify the role of cannabinoid receptors. METHODS: Gel-filtered platelets were stimulated with 2-AG in the presence or absence of various inhibitors. Platelet aggregation and secretion were measured in a lumiaggregometer. Calcium ion movements were measured in FURA-2 loaded platelets. Thromboxane A(2) (TxA(2)) generation was evaluated as Thromboxane B(2) accumulation with a commercial EIA assay. RESULTS: 2-AG induced platelet shape change, aggregation and secretion with a dose-dependent mechanism that required engagement of platelet TxA(2) receptors. 2-AG caused also cytosolic calcium increase; however, it was totally dependent on availability of TxA(2). Indeed 2-AG was able to induce a robust generation of TxA(2) through the cyclooxygenase pathway. Treatment of platelets with inhibitors of monoacylglycerol lipase and fatty acid amide hydrolase did not affect the activation induced by 2-AG. Moreover, neither CB(1) and CB(2) proteins nor CB(1)/CB(2) mRNAs were detected in platelets. CONCLUSIONS: 2-AG can be considered a new physiologic platelet agonist able to induce full platelet activation and aggregation with a non-CB(1)/CB(2) receptor-mediated mechanism.

Diacylglycerol kinase-alpha phosphorylation by Src on Y335 is required for activation, membrane recruitment and Hgf-induced cell motility.

Diacylglycerol (DAG) kinases (Dgk), which phosphorylate DAG to generate phosphatidic acid, act as either positive or negative key regulators of cell signaling. We previously showed that Src mediates growth factors-induced activation of Dgk-alpha, whose activity is required for cell motility, proliferation and angiogenesis. Here, we demonstrate that both hepatocytes growth factor (HGF) stimulation and v-Src transformation induce tyrosine phosphorylation of Dgk-alpha on Y335, through a mechanism requiring its proline-rich C-terminal sequence. Moreover, we show that both proline-rich sequence and phosphorylation of Y335 of Dgk-alpha mediate: (i) its enzymatic activation, (ii) its ability to interact respectively with SH3 and SH2 domains of Src, (iii) its recruitment to the membrane. In addition, we show that phosphorylation of Dgk-alpha on Y335 is required for HGF-induced motility, while its constitutive recruitment at the membrane by myristylation is sufficient to trigger spontaneous motility in absence of HGF. Providing the first evidence that tyrosine phosphorylation of Dgk-alpha is required for growth-factors-induced activation and membrane recruitment, these findings underscore its relevance as a rheostat, whose activation is a threshold to elicit growth factors-induced migratory signaling.

Activation of the small GTPase Rap2B in agonist-stimulated human platelets.

The activation of the small GTPase Rap2B in resting and agonist-stimulated human platelets was investigated. Both thrombin, that stimulates heterotrimeric G-protein-coupled receptors, and the GPVI ligand convulxin, that activates a tyrosine-kinase based signaling pathway, were able to induced the rapid and sustained binding of GTP to Rap2B. Similarly, a number of other agonists tested, previously known to activate the highly related protein Rap1B, were also able to stimulate Rap2B. In contrast, platelet antagonists that increase the intracellular concentration of cAMP did not signal to Rap2B. Thrombin- and convulxin-induced activation of Rap2B was not dependent on thromboxane A2, did not require the interaction of the protein with the cytoskeleton, and was not regulated by integrin alphaIIbbeta3-dependent outside-in signaling. When secreted ADP was neutralized, activation of Rap2B induced by thrombin, but not by convulxin, was significantly reduced. ADP itself was found to induce the rapid and sustained binding of GTP to Rap2B, and this effect was predominantly mediated by stimulation of the Gi-coupled P2Y12 receptor. Activation of Rap2B promoted by both thrombin and convulxin was regulated by intracellular Ca2+, while protein kinase C was found to be involved in convulxin- but not in thrombin-induced activation of Rap2B. Moreover, Rap2B activation induced by thrombin, but not by convulxin, was totally dependent on phosphatidylinositol 3-kinase activity. These results demonstrate that the small GTPase Rap2B is involved in platelet activation, and outline some important differences between the regulation of highly related GTPases Rap2B and Rap1B in human platelets.

Triggering receptor expressed on myeloid cells: role in the diagnosis of lung infections.

The triggering receptor expressed on myeloid cells (TREM)-1 is a recently described molecule, which plays an important role in myeloid cell-activated inflammatory responses. TREM-1 is expressed on blood neutrophils and monocytes, and also on alveolar macrophages, thus suggesting a potential role in lung inflammatory responses against infections. To investigate the differential expression of TREM-1 in lung infections, its levels were assessed in bronchoalveolar lavage specimens from patients with community-acquired pneumonia or tuberculosis. TREM-1 was also investigated in patients with interstitial lung diseases, as a model of noninfectious inflammatory disease of the lung. TREM-1 expression was significantly increased in lung neutrophils and in lung macrophages of patients with pneumonia (n=7; 387.9+/-61.4 and 660.5+/-18.3, respectively) compared with patients with pulmonary tuberculosis (n=7; 59.2+/-13.1 and 80.6+/-291.2) and patients with interstitial lung diseases (n=10; 91.8+/-23.3 and 123.9+/-22.8). In contrast, TREM-1 expression on peripheral blood neutrophils was no different among the three groups. In conclusion, these data suggest that triggering receptor expressed on myeloid cells-1 is selectively expressed in the lungs of patients with pneumonia caused by extracellular bacteria and not in patients with tuberculosis, providing a potential marker for differential diagnosis.

Unique chemotactic response profile and specific expression of chemokine receptors CCR4 and CCR8 by CD4(+)CD25(+) regulatory T cells.

Chemokines dictate regional trafficking of functionally distinct T cell subsets. In rodents and humans, a unique subset of CD4(+)CD25(+) cytotoxic T lymphocyte antigen (CTLA)-4(+) regulatory T cells (Treg) has been proposed to control peripheral tolerance. However, the molecular basis of immune suppression and the trafficking properties of Treg cells are still unknown. Here, we determined the chemotactic response profile and chemokine receptor expression of human blood-borne CD4(+)CD25(+) Treg cells. These Treg cells were found to vigorously respond to several inflammatory and lymphoid chemokines. Treg cells specifically express the chemokine receptors CCR4 and CCR8 and represent a major subset of circulating CD4(+) T cells responding to the chemokines macrophage-derived chemokine (MDC)/CCL22, thymus and activation-regulated chemokine (TARC)/CCL17, I-309/CCL1, and to the virokine vMIP-I (ligands of CCR4 and CCR8). Blood-borne CD4(+) T cells that migrate in response to CCL1 and CCL22 exhibit a reduced alloproliferative response, dependent on the increased frequency of Treg cells in the migrated population. Importantly, mature dendritic cells preferentially attract Treg cells among circulating CD4(+) T cells, by secretion of CCR4 ligands CCL17 and CCL22. Overall, these results suggest that CCR4 and/or CCR8 may guide Treg cells to sites of antigen presentation in secondary lymphoid tissues and inflamed areas to attenuate T cell activation.

The C-C chemokine receptors CCR4 and CCR8 identify airway T cells of allergen-challenged atopic asthmatics.

In vitro polarized human Th2 cells preferentially express the chemokine receptors CCR3, CCR4, and CCR8 and migrate to their ligands: eotaxin, monocyte-derived chemokine (MDC), thymus- and activation-regulated chemokine (TARC), and I-309. We have studied the expression of chemokines and chemokine receptors in the airway mucosa of atopic asthmatics. Immunofluorescent analysis of endobronchial biopsies from six asthmatics, taken 24 hours after allergen challenge, demonstrates that virtually all T cells express IL-4 and CCR4. CCR8 is coexpressed with CCR4 on 28% of the T cells, while CCR3 is expressed on eosinophils but not on T cells. Expression of the CCR4-specific ligands MDC and TARC is strongly upregulated on airway epithelial cells upon allergen challenge, suggesting an involvement of this receptor/ligand axis in the regulation of lymphocyte recruitment into the asthmatic bronchi. In contrast to asthma, T cells infiltrating the airways of patients with chronic obstructive pulmonary disease and pulmonary sarcoidosis produce IFN-gamma and express high levels of CXCR3, while lacking CCR4 and CCR8 expression. These data support the role of CCR4, of its ligands MDC and TARC, and of CCR8 in the pathogenesis of allergen-induced late asthmatic responses and suggest that these molecules could be considered as targets for therapeutic intervention.

Platelet activation by von Willebrand factor requires coordinated signaling through thromboxane A2 and Fc gamma IIA receptor.

Interaction of von Willebrand Factor with glycoprotein Ib-IX-V induces platelet activation through a still poorly defined mechanism. Previous studies have suggested a possible role for the low affinity receptor for immunoglobulin, Fc gamma RIIA, in GPIb-IX-V signaling. Here we show that binding of vWF to platelets induces the tyrosine phosphorylation of Fc gamma RIIA by a Src kinase. Treatment of platelets with the anti-Fc gamma RIIA monoclonal antibody IV.3 specifically inhibits vWF-induced but not thrombin-induced pleckstrin phosphorylation and serotonin secretion. Moreover, vWF fails to induce pleckstrin phosphorylation in mouse platelets, lacking Fc gamma RIIA, and serotonin secretion is impaired. Pleckstrin phosphorylation and serotonin secretion in human platelets stimulated with vWF are blocked by the cyclooxygenase inhibitor acetylsalicylic acid. However, release of arachidonic acid and synthesis of TxA(2) induced by vWF are not affected by the anti-Fc gamma RIIA monoclonal antibody IV.3. Similarly, vWF-induced tyrosine phosphorylation of Fc gamma RIIA, as well as of Syk and PLC gamma 2, occurs normally in aspirinized platelets. Inhibition of the tyrosine kinase Syk by piceatannol does not affect vWF-induced tyrosine phosphorylation of Fc gamma RIIA but prevents phosphorylation of PLC gamma 2. Pleckstrin phosphorylation and platelet secretion induced by vWF, but not by thrombin, are also inhibited by piceatannol. Pleckstrin phosphorylation is also sensitive to the phosphatidylinositol 3-kinase inhibitor wortmannin. These results indicate that PLC gamma 2 plays a central role in platelet activation by vWF and that the stimulation of this enzyme requires coordinated signals through endogenous TxA(2) and Fc gamma RIIA.

Fractalkine (CX3CL1) as an amplification circuit of polarized Th1 responses.

Fractalkine (FKN, CX3CL1) is a membrane-bound CX3C chemokine induced by primary proinflammatory signals in vascular endothelial cells (ECs). Here we examined the role of FKN in polarized Th1 or Th2 responses. Proinflammatory signals, including LPS, IL-1, TNF, and CD40 ligand, induced FKN, as did IFN-gamma, which had synergistic activity with TNF. IL-4 and IL-13 did not stimulate the expression of FKN and markedly reduced induction by TNF and IFN-gamma. TNF alone or combined with IFN-gamma also induced release of soluble FKN, which was inhibited by IL-4 and IL-13. In light of this differential regulation of FKN by the master cytokines that control polarized responses, we analyzed the interaction of FKN with natural killer (NK) cells and polarized T-cell populations. NK cells expressed high levels of the FKN receptor CX3CR1 and responded to FKN. CX3CR1 was preferentially expressed in Th1 compared with Th2 cells. Th1 but not Th2 cells responded to FKN. By immunohistochemistry, FKN was expressed on ECs in psoriasis, a Th1-dominated skin disorder, but not in Th2-driven atopic dermatitis. Similarly, ECs in Mycobacterium tuberculosis granulomatous lymphadenitis, but not those in reactive lymph node hyperplasia or in Castelman's disease, showed immunoreactive FKN. These results indicate that regulated expression of FKN in ECs participates in an amplification circuit of polarized type I responses.

Gene expression profiling in immune cells using microarray.

The recent development of DNA microarray, which offers the opportunity to study the expression of thousands of individual genes simultaneously in different biological systems, has provided new insights into the immune system. Examples discussed in this review include molecular descriptions of the differentiation program of T helper (Th) cells into Th1 and Th2 pathways and the genetic program underlying maturation of dendritic cells. It is anticipated that this new information can be used to understand gene function in both physiological and pathological conditions of the immune system.

Regulation of helper T cell differentiation and recruitment in airway inflammation.

CD4(+) helper T type 1 (Th1) and Th2 cells are critical mediators of inflammatory diseases. Although T cells represent only a fraction of the leukocytes that are found in the lung during inflammation, they play a critical role in coordinating the immune response to infectious agents and allergens. T cells have the ability to rapidly expand in response to specific stimuli and to differentiate into effector cells that, through the production of soluble factors such as cytokines and chemokines, communicate with other cells to initiate a cascade of inflammatory events. The objective of this review is to outline the cellular and molecular mechanisms involved in the generation and recruitment of Th1 and Th2 cells in the lung. Defining these mechanisms should lead to improved immunopharmacological strategies for prophylaxis and therapy.

Residual type 1 immunity in patients genetically deficient for interleukin 12 receptor beta1 (IL-12Rbeta1): evidence for an IL-12Rbeta1-independent pathway of IL-12 responsiveness in human T cells.

Genetic lack of interleukin 12 receptor beta1 (IL-12Rbeta1) surface expression predisposes to severe infections by poorly pathogenic mycobacteria or Salmonella and causes strongly decreased, but not completely abrogated, interferon (IFN)-gamma production. To study IL-12Rbeta1-independent residual IFN-gamma production, we have generated mycobacterium-specific T cell clones (TCCs) from IL-12Rbeta1-deficient individuals. All TCCs displayed a T helper type 1 phenotype and the majority responded to IL-12 by increased IFN-gamma production and proliferative responses upon activation. This response to IL-12 could be further augmented by exogenous IL-18. IL-12Rbeta2 was found to be normally expressed in the absence of IL-12Rbeta1, and could be upregulated by IFN-alpha. Expression of IL-12Rbeta2 alone, however, was insufficient to induce signal transducer and activator of transcription (Stat)4 activation in response to IL-12, whereas IFN-alpha/IFN-alphaR ligation resulted in Stat4 activation in both control and IL-12Rbeta1-deficient cells. IL-12 failed to upregulate cell surface expression of IL-18R, integrin alpha6, and IL-12Rbeta2 on IL-12Rbeta1-deficient cells, whereas this was normal on control cells. IL-12-induced IFN-gamma production in IL-12Rbeta1-deficient T cells could be inhibited by the p38 mitogen-activated protein kinase (MAP) kinase inhibitor SB203580 and the MAP kinase kinase (MEK) 1/2 inhibitor U0126, suggesting involvement of MAP kinases in this alternative, Stat4-independent, IL-12 signaling pathway.Collectively, these results indicate that IL-12 acts as a partial agonist in the absence of IL-12Rbeta1. Moreover, the results reveal the presence of a novel IL-12Rbeta1/Stat4-independent pathway of IL-12 responsiveness in activated human T cells involving MAP kinases. This pathway is likely to play a role in the residual type 1 immunity in IL-12Rbeta1 deficiency.

Selective inhibition of HIV replication in primary macrophages but not T lymphocytes by macrophage-derived chemokine.

Macrophage-derived chemokine (MDC) has been reported to inhibit different HIV-1 strains in activated peripheral blood mononuclear cells (T cell blasts), although other investigators have not confirmed these findings. Here we demonstrate that MDC inhibits the replication of CCR5-dependent (R5) HIV-1(BaL) in monocyte-derived macrophages (MDM), but not in T cell blasts, although with variable potency depending on donor variability. Analysis of HIV-1(BaL) proviral DNA synthesis in MDM indicated that the suppressive effect of MDC did not involve inhibition of early events such as entry or reverse transcription. Finally, an inverse correlation was observed between the levels of endogenous MDC secreted by uninfected MDM of different donors and the efficiency of different HIV strains, including two primary isolates with different coreceptor usage, to replicate in these cells. Thus, MDC represents an example of a chemokine inhibiting HIV replication in macrophages acting at one or more postentry levels in the virus life cycle.

Proliferative responses to selected peptides of IA-2 in identical twins discordant for Type 1 diabetes.

BACKGROUND: The aim of the study was to define T lymphocyte reactivity to selected peptides of an islet antigen IA-2, associated with Type 1 diabetes. METHODS: We used 10 peptides selected from the IA-2 molecule due to their predicted ability to bind to HLA-DRB1*0401, a Type 1 diabetes-associated allele. We tested 21 identical twin pairs discordant for the disease and 15 control subjects and then followed them prospectively; seven non-diabetic twins developed diabetes. RESULTS: Twins of identical pairs tended to respond to different peptides suggesting that the T cell response is, to a degree, shaped by non-genetically determined factors (p<0. 0001). However, there was no difference in the T cell responses between diabetic twins and either their non-diabetic identical twins or control subjects and the response was heterogenous. T cell responses did not differ in those seven non-diabetic twins who developed diabetes from those twins who did not. T cell responses to peptide 11 (amino acids 502-514) was immunodominant in diabetic twins as well as their non-diabetic twins and controls; responses were not correlated with HLA, IA-2 antibodies, age or duration of disease. CONCLUSION: We conclude that T cell responses to selected IA-2 peptides are not genetically determined, heterogeneous, not strictly HLA controlled and did not distinguish diabetic or prediabetic twins from non-diabetic twins or controls. The identification of an immunodominant T cell response to IA-2 peptide 502-514 raises the possibility that this, or similar, epitopes may be of therapeutic value in disease prevention.

Transcript imaging of the development of human T helper cells using oligonucleotide arrays.

Many pathological processes, including those causing allergies and autoimmune diseases, are associated with the presence of specialized subsets of T helper cells at the site of inflammation. Understanding the genetic program that controls the functional properties of T helper type 1 (Th1) versus T helper type 2 (Th2) cells may provide insight into the pathophysiology of inflammatory diseases. We compared the gene-expression profiles of human Th1 and Th2 cells using high-density oligonucleotide arrays with the capacity to display transcript levels of 6,000 human genes. Here we analyse the data sets derived from five independent experiments using statistical algorithms. This approach resulted in the identification of 215 differentially expressed genes, encoding proteins involved in transcriptional regulation, apoptosis, proteolysis, and cell adhesion and migration. A subset of these genes was further upregulated by exposure of differentiated Th1 cells to interleukin-12 (IL-12), as confirmed by kinetic PCR analysis, indicating that IL-12 modulates the effector functions of Th1 cells in the absence of antigenic stimulation. Functional assays and in vivo expression of selected genes have validated the biological relevance of our study. Our results provide new insight into the transcriptional program controlling the functional diversity of subsets of T helper cells.

Inhibition by IL-12 and IFN-alpha of I-309 and macrophage-derived chemokine production upon TCR triggering of human Th1 cells.

Th1 and Th2 cells, which produce distinct sets of cytokines, differentially express several chemokine receptors that may regulate their tissue-specific localization. Although the expression pattern and regulation of chemokines are likely to play a critical role in many immunopathological processes, they remain largely unknown. Here, we investigated the requirements for Th1 and Th2 cells to produce the Th2 cell-attracting chemokines thymus and activation-regulated chemokine (TARC), macrophage-derived chemokine (MDC) and I-309. TCR triggering of Th1 and Th2 cells leads to production of MDC and I-309 (CCR4 and CCR8 ligands, respectively), whereas TARC (CCR4 ligand) is selectively produced by Th2 cells. Secretion of these chemokines appears to be independent of endogenous production of IL-4 and IFN-gamma. IL-12 and IFN-alpha, cytokines that promote the differentiation of human Th1 cells, selectively inhibit secretion and mRNA expression of MDC and I-309 by Th1 cells. Suppression of I-309 secretion results in a decreased chemotactic effect on L1.2 cells transfected with human CCR8, indicating that IL-12 and IFN-alpha may inhibit the recruitment of CCR8-expressing cells such as Th2 cells. The inhibition of Th2 cell-attracting chemokines MDC and I-309 illustrates a novel mechanism by which IL-12 and IFN-alpha could promote and maintain an ongoing Th1 response.