Ca2+/calmodulin-dependent protein kinase II regulates cardiac Na+ channels

In heart failure (HF), Ca(2+)/calmodulin kinase II (CaMKII) expression is increased. Altered Na(+) channel gating is linked to and may promote ventricular tachyarrhythmias (VTs) in HF. Calmodulin regulates Na(+) channel gating, in part perhaps via CaMKII. We investigated effects of adenovirus-mediated (acute) and Tg (chronic) overexpression of cytosolic CaMKIIdelta(C) on Na(+) current (I(Na)) in rabbit and mouse ventricular myocytes, respectively (in whole-cell patch clamp). Both acute and chronic CaMKIIdelta(C) overexpression shifted voltage dependence of Na(+) channel availability by -6 mV (P < 0.05), and the shift was Ca(2+) dependent. CaMKII also enhanced intermediate inactivation and slowed recovery from inactivation (prevented by CaMKII inhibitors autocamtide 2-related inhibitory peptide [AIP] or KN93). CaMKIIdelta(C) markedly increased persistent (late) inward I(Na) and intracellular Na(+) concentration (as measured by the Na(+) indicator sodium-binding benzofuran isophthalate [SBFI]), which was prevented by CaMKII inhibition in the case of acute CaMKIIdelta(C) overexpression. CaMKII coimmunoprecipitates with and phosphorylates Na(+) channels. In vivo, transgenic CaMKIIdelta(C) overexpression prolonged QRS duration and repolarization (QT intervals), decreased effective refractory periods, and increased the propensity to develop VT. We conclude that CaMKII associates with and phosphorylates cardiac Na(+) channels. This alters I(Na) gating to reduce availability at high heart rate, while enhancing late I(Na) (which could prolong action potential duration). In mice, enhanced CaMKIIdelta(C) activity predisposed to VT. Thus, CaMKII-dependent regulation of Na(+) channel function may contribute to arrhythmogenesis in HF.     

Th1 and Th2 cytokine responses to academic stress.

Predominant Th2 profiles are associated with the worsening of asthma, and stress is speculated to induce a Th2 profile. The goals of this study were to examine the responses of the cytokines Th1 (IFN-gamma and IL-2) and Th2 (IL-4, IL-5, and IL-6) to a stressor and to look at the relationships between cytokine and psychological responses. Twenty-four students with and without a history of asthma completed questionnaires and gave blood samples during nonexam and exam periods. Cytokines were measured by ELISA from supernatants of stimulated mononuclear cells (MNC) and whole blood. During examinations, there were a significant decrease in IL-2 and a significant increase in IL-6 production (both cultures) and a significant decrease in IFN-gamma production (MNC cultures). Baseline IL-2 levels showed significant negative correlations with several stress and mood scores. Findings of this study indicate a down-regulation of Th1 and a selective up-regulation of Th2 cytokines during a stressful exposure.     

Reversal of chronic inflammatory pain by acute inhibition of Ca2+/calmodulin-dependent protein kinase II

Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) is a major protein kinase that is capable of regulating the activities of many ion channels and receptors. In the present study, the role of CaMKII in the complete Freund's adjuvant (CFA)-induced inflammatory pain was investigated. Intraplantarly injected CFA was found to induce spinal activity of CaMKII (phosphorylated CaMKII), which was blocked by KN93 [[2-[N-(2-hydroxyethyl)]-N-(4-methoxybenzenesulfonyl)]amino-N-(4-chlorocinnamyl)-N-methylbenzylamine)], a CaMKII inhibitor. Pretreatment with KN93 (i.t.) dose-dependently prevented the development of CFA-induced thermal hyperalgesia and mechanical allodynia. Acute treatment with KN93 (i.t.) also dose-dependently reversed CFA-induced thermal hyperalgesia and mechanical allodynia. The action of KN93 started in 30 min and lasted for at least 2 to 4 h. KN92 (45 nmol i.t.) [2-[N-(4-methoxybenzenesulfonyl)]amino-N-(4-chlorocinnamyl)-N-methylbenzylamine], an inactive analog of KN93, showed no effect on CFA-induced CaMKII activation, allodynia, or hyperalgesia. Furthermore, our previous studies identified trifluoperazine, a clinically used antipsychotic drug, to be a potent CaMKII inhibitor. Inhibition of CaMKII activity by trifluoperazine was confirmed in the study. In addition, trifluoperazine (i.p.) dose-dependently reversed CFA-induced mechanical allodynia and thermal hyperalgesia. The drug was also effectively when given orally. In conclusion, our findings support a critical role of CaMKII in inflammatory pain. Blocking CaMKII or CaMKII-mediated signaling may offer a novel therapeutic target for the treatment of chronic pain.     

Spinal CK2 regulates nociceptive signaling in models of inflammatory pain

Casein kinase 2 (CK2) is a widely expressed protein kinase. Over the last several years a long list of protein substrates has evolved, many of which have proven or hypothesized roles in nociceptive signal transmission. However, CK2 has not itself been demonstrated to participate in nociception prior to this time. We set out to test the hypothesis that spinal CK2 regulates nociception using several pain models. Our first studies focused on the ability of the selective CK2 inhibitors 4,5,6,7-tetrabromobenzotriazole (TBBT) and 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB) to reduce formalin-stimulated pain behaviors in mice. Both phases of the response to subcutaneous formalin were strongly inhibited by intrathecal administration of TBBT or DRB in dose-dependent fashion. Likewise, using the complete Freund's adjuvant (CFA) model of chronic inflammatory pain, TBBT was observed to strongly reduce mechanical allodynia. The inhibition of spinal CK2 with either inhibitor did not, however, alter withdrawal latencies in the hotplate thermal pain model while intrathecal morphine was very effective. Immunohistochemical studies demonstrated all three known CK2 subunits, alpha, alpha' and beta to be expressed in spinal cord tissue as did real-time PCR experiments. While mRNA levels for each of the subunits was transiently enhanced after formalin or CFA hindpaw injection, overall spinal cord protein levels were not elevated in a sustained fashion. Our results indicate that CK2 participates in inflammatory nociception both in the acute and chronic phases. Simple changes in the abundance of spinal CK2 subunits do not likely underlie these phenomena, however.     

TLR9 regulates Th1 responses and cooperates with TLR2 in mediating optimal resistance to Mycobacterium tuberculosis

To investigate the role of Toll-like receptor (TLR)9 in the immune response to mycobacteria as well as its cooperation with TLR2, a receptor known to be triggered by several major mycobacterial ligands, we analyzed the resistance of TLR9(-/-) as well as TLR2/9 double knockout mice to aerosol infection with Mycobacterium tuberculosis. Infected TLR9(-/-) but not TLR2(-/-) mice displayed defective mycobacteria-induced interleukin (IL)-12p40 and interferon (IFN)-gamma responses in vivo, but in common with TLR2(-/-) animals, the TLR9(-/-) mice exhibited only minor reductions in acute resistance to low dose pathogen challenge. When compared with either of the single TLR-deficient animals, TLR2/9(-/-) mice displayed markedly enhanced susceptibility to infection in association with combined defects in proinflammatory cytokine production in vitro, IFN-gamma recall responses ex vivo, and altered pulmonary pathology. Cooperation between TLR9 and TLR2 was also evident at the level of the in vitro response to live M. tuberculosis, where dendritic cells and macrophages from TLR2/9(-/-) mice exhibited a greater defect in IL-12 response than the equivalent cell populations from single TLR9-deficient animals. These findings reveal a previously unappreciated role for TLR9 in the host response to M. tuberculosis and illustrate TLR collaboration in host resistance to a major human pathogen.     

Th1/Th2 cells in inflammatory disease states: therapeutic implications

Inflammation is initiated as a protective response by the host, but can often result in systemic pathology. Among cells of the immune system, T lympho-cytes play a major role in the inflammatory response. T cell inflammation is characterised histologically by an infiltration of mononuclear cells. Key regulators of this response are a subset of T lymphocytes called T helper (Th) cells. These cells secrete soluble mediators called cytokines, which orchestrate the immune response. The appropriate regulation of Th cell immunity is critical in the control and prevention of diverse disease states. This review will focus on the role of Th cells in the inflammatory process involved in allergic disease, diabetes, infectious disease, rheumatoid arthritis, heart disease, multiple sclerosis and cancer. In the area of autoimmunity, in particular, a basic understanding of Th cells and cytokines has contributed to the development of clinically efficacious biological agents. This review also examines current and novel treatment strategies under investigation at present that regulate Th cell immunity, which may result in better treatments for immune-mediated diseases.     

Th1/Th2 cells in inflammatory disease states: therapeutic implications

Inflammation is initiated as a protective response by the host, but can often result in systemic pathology. Among cells of the immune system, T lymphocytes play a major role in the inflammatory response. T cell inflammation is characterised histologically by an infiltration of mononuclear cells. Key regulators of this response are a subset of T lymphocytes called T helper (Th) cells. These cells secrete soluble mediators called cytokines, which orchestrate the immune response. The appropriate regulation of Th cell immunity is critical in the control and prevention of diverse disease states. This review will focus on the role of Th cells in the inflammatory process involved in allergic disease, diabetes, infectious disease, rheumatoid arthritis, heart disease, multiple sclerosis and cancer. In the area of autoimmunity, in particular, a basic understanding of Th cells and cytokines has contributed to the development of clinically efficacious biological agents. This review also examines current and novel treatment strategies under investigation at present that regulate Th cell immunity, which may result in better treatments for immune-mediated diseases.     

Collaborative induction of inflammatory responses by dectin-1 and Toll-like receptor 2

Toll-like receptors (TLRs) mediate recognition of a wide range of microbial products including lipopolysaccharides, lipoproteins, flagellin, and bacterial DNA, and signaling through TLRs leads to the production of inflammatory mediators. In addition to TLRs, many other surface receptors have been proposed to participate in innate immunity and microbial recognition, and signaling through some of these receptors is likely to cooperate with TLR signaling in defining inflammatory responses. In this report we have examined how dectin-1, a lectin family receptor for beta-glucans, collaborates with TLRs in recognizing microbes. Dectin-1, which is expressed at low levels on macrophages and high levels on dendritic cells, contains an immunoreceptor tyrosine-based activation motif-like signaling motif that is tyrosine phosphorylated upon activation. The receptor is recruited to phagosomes containing zymosan particles but not to phagosomes containing immunoglobulin G-opsonized particles. Dectin-1 expression enhances TLR-mediated activation of nuclear factor kappa B by beta-glucan-containing particles, and in macrophages and dendritic cells dectin-1 and TLRs are synergistic in mediating production of cytokines such as interleukin 12 and tumor necrosis factor alpha. Additionally, dectin-1 triggers production of reactive oxygen species, an inflammatory response that is primed by TLR activation. The data demonstrate that collaborative recognition of distinct microbial components by different classes of innate immune receptors is crucial in orchestrating inflammatory responses.     

Th1/Th2 balance in systemic inflammatory response syndrome (SIRS)

The activation of a pro-inflammatory cascade after infection, major surgery, burn or trauma appears to be important in the development of subsequent immune dysfunction, susceptibility to sepsis and multiple organ failure. It is well known that T-cell plays a critical role in the systemic response to infection. Distinct patterns of cytokines are produced by two different types of T-helper cells (Th). Th1 lymphocytes produce IFN-gamma and IL-2, favoring cell mediated immunity; Th2 cells secrete IL-4, IL-5, IL-10, IL-13, favoring humoral immunity. Cytokines produced in systemic inflammatory response syndrome (SIRS) may effect Th subset predominance and subsequent immune responses. We measured Thl/Th2 balance in patients with severe sepsis, SIRS patients with non sepsis, and healthy subjects by flow cytometry. In patients with severe sepsis, Th2 antibody mediated (humoral) immune responses predominate. We believe that severe sepsis clearly induce polarization of T-helper lymphocyte activity with a clear shift in Th2 direction. This type of response may lead immunosuppression. Modulation of Th cell subset predominance may present a novel therapeutic option in the treatment of severe sepsis.     

Kinetic Th1/Th2 responses of transgenic mice with bacterial meningitis induced by Haemophilus influenzae

To investigate the kinetic Th1/Th2 immunopathogenic mechanisms of Haemophilus influenzae meningitis, we established a murine experimental model of meningitis and elucidated the Th1/Th2 immune responses in T1/T2 doubly transgenic mice based on a BALB/c background under the control of the IFN-gamma (interferon-gamma)/IL-4 (interleukin-4) promoters respectively. NTHi (non-typeable Haemophilus influenzae) meningitis was induced in these mice by inoculation with either a colonized (CNTHi) or invasive (INTHi) strain of NTHi. Mice inoculated with CNTHi displayed a less severe degree of disease in terms of clinical symptoms, mortality rate and brain histopathology. Conversely, INTHi-inoculated mice had more severe clinical symptoms. CNTHi-inoculated mice had a more significant Th1 response in terms of a higher percentage and longer maintenance of Th1 cells, and more production of IFN-gamma from strain-specific antigen-stimulated splenocytes than INTHi-inoculated mice. In contrast, INTHi-inoculated mice had a more significant Th2 response. This was due to a significant increase in IL-4-producing CD4(+) T-cells (Th2 cells) and more production of IL-4 from strain-specific antigen-stimulated splenocytes accompanied by a rapid decline of Th1 cells in INTHi-inoculated mice. In conclusion, the preferential Th1/Th2 trend in this murine model of NTHi meningitis is correlated with clinical severity as well as isolated characteristics of the pathogens themselves.     

Ets-1, a functional cofactor of T-bet, is essential for Th1 inflammatory responses

To mount an effective type 1 immune response, type 1 T helper (Th1) cells must produce inflammatory cytokines and simultaneously suppress the expression of antiinflammatory cytokines. How these two processes are coordinately regulated at the molecular level is still unclear. In this paper, we show that the proto-oncogene E26 transformation-specific-1 (Ets-1) is necessary for T-bet to promote interferon-gamma production and that Ets-1 is essential for mounting effective Th1 inflammatory responses in vivo. In addition, Ets-1-deficient Th1 cells also produce a very high level of interleukin 10. Thus, Ets-1 plays a crucial and unique role in the reciprocal regulation of inflammatory and antiinflammatory Th responses.     

高血压心肌梗死与Th1／Th2类细胞因子变化

目的探讨高血压心肌梗死患者Th1／Th2类细胞因子变化。方法临床及冠状动脉造影确诊的高血压心肌梗死患者45例为A（病例1）组,非高血压心肌梗死患者42例为B（病例2）组,冠状动脉造影无心肌梗死的高血压患者48例为C（病例3）组,常规体验健康者20例为D（对照）组。采用放射免疫法检测血管紧张素Ⅱ（Ang-Ⅱ）水平,ELISA法检测血清1干扰素（IFN-γ）和白细胞介素4（IL-4）水平。结果①病例1、2、3组Ang-Ⅱ[（94．3±35．4）、（74．4±31．2）ng／L和（63．4±28．9）ng／L]及IFN-γ[（24．3±5．4）、（19．4±3．9）ng／L和（12．1±3．8）]ng／L]较对照组Ang-Ⅱ[（43．4±11．2）ng／L]及IFN-γ[（4．1±1．4）ng／L]水平升高（P〈0．05,P〈0．01）;②病例1组IL-4水平[（14．4±10．7）ng／L与2组[（12．4±2．9）ng／L]、3组[（12．3±6．6）ng／L]及对照组[（11．4±3．9）ng／L]比较差异无统计学意义（P〉0．05）;③病例1、2和3组IFN-1与IL-4比值（0．77±0．33）、（0．62±0．24）和（0．58±0．26）较对照组（0．36±0．19）增大（P〈0．01,P〈0．05）;④Ang-Ⅱ水平与IFN-γ呈正相关（r=0．52,P〈0．01）,而与IL-4水平无相关性（r=0．19,P〉0．05）。结论高血压和非高血压心肌梗死均具有较高的Ang-Ⅱ、IFN—γ及IL-4水平,但高血压心肌梗死Ang-Ⅱ、IFN-γ及IL4水平更高,这是辅助性T细胞亚群Th1／Th2功能失衡的表现;高血压心肌梗死与Th1／Th2失衡,Th1细胞功能亢进密切相关,系统免疫状态和炎性内环境的改变可能对疾病的进展起重要作用。     

Na+/Ca2+ exchanger(NCX1) and salt-sensitive hypertension

Hypertension is the most common chronic disease, and is the leading risk factor for death caused by stroke, myocardial infarction, and end-stage renal failure. The critical importance of excess salt intake in the pathogenesis of hypertension is widely recognized. However, the molecular mechanisms underlying salt-sensitive hypertension remain obscure. Recent studies using selective inhibitors and genetically engineered mice provide compelling evidence that salt-sensitive hypertension is triggered by Ca2+ entry through Na+/Ca2+ exchanger type-1 (NCX1) in vascular smooth muscle. Intriguingly, endogenous Na+ pump inhibitors seem to be necessary for NCX1-mediated hypertension. These findings have enabled us to explain how high salt intake leads to hypertension, and further to describe the potential of vascular NCX1 as a new therapeutic or diagnostic target for salt-sensitive hypertension.     

Non-hematopoietic EPCR regulates the coagulation and inflammatory responses during endotoxemia

BACKGROUND: Activated protein C (APC) protects the host from severe sepsis. Endothelial protein C receptor (EPCR) is expressed on both hematopoietic leukocytes and non-hematopoietic endothelium, and plays a key role in protein C activation. OBJECTIVES: We explore the influence of EPCR deletion on the responses to lipopolysaccharide (LPS) and then determine whether the observed differences are due to loss of hematopoietic or non-hematopoietic EPCR. METHODS AND RESULTS: After LPS challenge, EPCR null (Procr(-/-)) mice exhibited more thrombin and cytokine generation, neutrophil sequestration in the lung and a higher mortality rate than Procr(+/-) mice. Procr(+/-) BM/Procr(-/-) (non-hematopoietic Procr(-/-)) and Procr(-/-) BM/Procr(+/-) (hematopoietic Procr(-/-)) chimeric mice were generated by bone marrow (BM) transplantation. Compared with control Procr(+/-) mice, non-hematopoietic Procr(-/-) mice exhibited reduced protein C activation by thrombin and exaggerated responses to LPS challenge, whereas Procr(+/-) mice and hematopoietic Procr(-/-) mice exhibited similar protein C activation by thrombin and similar responses to LPS challenge. CONCLUSIONS: EPCR deletion exaggerates the host responses to LPS primarily due to deficiency of EPCR on the non-hematopoietic cells.     

Downregulation of Th1 cytokine production accompanies induction of Th2 responses by a parasitic helminth, Schistosoma mansoni

In the mouse, infection with Schistosoma mansoni results in an egg-producing infection and associated disease, whereas vaccination with attenuated larval stages produces a substantial and specific immunity in the absence of egg-induced pathology. Preliminary data showing enhanced interleukin-5 (IL-5) production by T cells from infected mice and interferon gamma (IFN-gamma) synthesis by cells from vaccinated animals (7), suggested differential CD4+ subset stimulation by the different parasite stimuli. To confirm this hypothesis, lymphocytes from vaccinated or infected animals were compared for their ability to produce IFN-gamma and IL-2 (secreted by Th1 cells) as compared with IL-4 and IL-5 (characteristic Th2 cytokines). After stimulation with specific antigen or mitogen, T cells from vaccinated mice or prepatently infected animals responded primarily with Th1 lymphokines, whereas lymphocytes from patently infected mice instead produced Th2 cytokines. The Th2 response in infected animals was shown to be induced by schistosome eggs and directed largely against egg antigens, whereas the Th1 reactivity in vaccinated mice was triggered primarily by larval antigens. Interestingly, Th1 responses in mice carrying egg-producing infections were found to be profoundly downregulated. Moreover, the injection of eggs into vaccinated mice resulted in a reduction of antigen and mitogen-stimulated Th1 function accompanied by a coincident expression of Th2 responses. Together, the data suggest that coincident with the induction of Th2 responses, murine schistosome infection results in an inhibition of potentially protective Th1 function. This previously unrecognized downregulation of Th1 cytokine production may be an important immunological consequence of helminth infection related to host adaptation.     

Prostaglandin E2 regulates Th17 cell differentiation and function through cyclic AMP and EP2/EP4 receptor signaling

Prostaglandins, particularly prostaglandin E2 (PGE2), play an important role during inflammation. This is exemplified by the clinical use of cyclooxygenase 2 inhibitors, which interfere with PGE2 synthesis, as effective antiinflammatory drugs. Here, we show that PGE2 directly promotes differentiation and proinflammatory functions of human and murine IL-17–producing T helper (Th17) cells. In human purified naive T cells, PGE2 acts via prostaglandin receptor EP2- and EP4-mediated signaling and cyclic AMP pathways to up-regulate IL-23 and IL-1 receptor expression. Furthermore, PGE2 synergizes with IL-1β and IL-23 to drive retinoic acid receptor–related orphan receptor (ROR)-γt, IL-17, IL-17F, CCL20, and CCR6 expression, which is consistent with the reported Th17 phenotype. While enhancing Th17 cytokine expression mainly through EP2, PGE2 differentially regulates interferon (IFN)-γ production and inhibits production of the antiinflammatory cytokine IL-10 in Th17 cells predominantly through EP4. Furthermore, PGE2 is required for IL-17 production in the presence of antigen-presenting cells. Hence, the combination of inflammatory cytokines and noncytokine immunomodulators, such as PGE2, during differentiation and activation determines the ultimate phenotype of Th17 cells. These findings, together with the altered IL-12/IL-23 balance induced by PGE2 in dendritic cells, further highlight the crucial role of the inflammatory microenvironment in Th17 cell development and regulation.Prostaglandins, prostaglandin E2 (PGE2) in particular, play an important role in the regulation of inflammatory responses. PGE2 is a key mediator of pyrexia, hyperalgesia, and arterial dilation, which increases blood flow to inflamed tissues and, in combination with enhanced microvascular permeability, results in edema. The relevance of this pathway in promoting inflammation is supported by the clinical use of cyclooxygenase inhibitors, which interfere with prostaglandin synthesis and are used as effective antiinflammatory agents (1). However, PGE2 can also exert antiinflammatory properties and is a negative regulator of neutrophil, monocyte, and lymphocyte function, particularly of Th1 cells that produce IFN-γ (2). This apparent paradox has puzzled many investigators for decades. The interplay among PGE2, IL-23, and IL-1β biology may now provide an explanation of this paradox.Th17 cells have been recognized as a unique subset of effector T cells that are distinct from the Th1 and Th2 subsets (3–6), and they have been implicated as potent effectors of autoimmune disorders, such as multiple sclerosis, psoriasis, arthritis, and inflammatory bowel disease (IBD) (7–10). We and others have previously reported that IL-23 and IL-1β are crucial factors during development of human Th17 cells (9, 11, 12). In addition, IL-23 and the IL-23–dependent Th17 cell population play essential roles in chronic inflammation and autoimmunity (13).PGE2 has been shown to exacerbate inflammation and disease severity in murine models of IBD and collagen-induced arthritis through the IL-23–IL-17 pathway (14, 15). These effects have been attributed to actions of PGE2 on innate cells, as PGE2 enhances the production of IL-23 and IL-1β in macrophages and DCs, while down-regulating IL-12 production (16). A recent report has shown that PGE2, together with IL-23, favors the expansion of human Th17 cells from PBMCs, and that PGE2 enhances IL-17 production induced by IL-23 from memory CD4⁺ cells (17). However, the molecular mechanism of PGE2-mediated signaling during human Th17 cell development has not yet been examined.In this study, we show that PGE2 acts directly on both human and murine T cells to enhance Th17 development and effector cytokine production. In human T cells, PGE2 acts via the prostaglandin receptor EP2- and EP4-mediated signaling and cAMP pathways to up-regulate IL-23 and IL-1 receptor expression. Furthermore, PGE2 synergizes with IL-1β and IL-23 to drive retinoic acid receptor–related orphan receptor (ROR)-γt, IL-17, IL-17F, CCL20, and CCR6 expression, which is consistent with the previously reported Th17 phenotype (8, 18). While enhancing Th17 cytokine expression mainly through EP2, PGE2 differentially regulates IFN-γ production and inhibits production of the antiinflammatory cytokine IL-10 in both naive and memory Th17 cells predominantly through EP4. Hence, the combination of inflammatory cytokines and noncytokine immunomodulators, such as PGE2, during differentiation and activation determines the ultimate phenotype of Th17 cells. These findings, together with the altered IL-12/IL-23 balance induced by PGE2 in dendritic cells, further highlight the crucial role of the inflammatory microenvironment in Th17 cell development and regulation.