Dopamine‐induced inhibition of Na+‐K+‐ATPase activity requires integrity of actin cytoskeleton in opossum kidney cells

The present study evaluated the importance of the association between Na⁺‐K⁺‐ATPase and the actin cytoskeleton on dopamine‐induced inhibition of Na⁺‐K⁺‐ATPase activity. The approach used measures the transepithelial transport of Na⁺ in monolayers of opossum kidney (OK) cells, when the Na⁺ delivered to Na⁺‐K⁺‐ATPase was increased at the saturating level by amphotericin B. The maximal amphotericin B (1.0 μg mL^–1) induced increase in short‐circuit current (I_sc) was prevented by ouabain (100 μM ) or removal of apical Na⁺. Dopamine (1 μM ) applied from the apical side significantly decreased (29 ± 5% reduction) the amphotericin B‐induced increase in I_sc, this being prevented by the D₁‐like receptor antagonist SKF 83566 (1 μM ) and the protein kinase C (PKC) inhibitor chelerythrine (1 μM ). Exposure of OK cells to cytochalasin B (1 μM ) or cytochalasin D (1 μM ), inhibitors of actin polymerization, from both cell sides reduced by 31 ± 4% and 36 ± 3% the amphotericin B‐induced increase in I_sc and abolished the inhibitory effect of apical dopamine (1 μM ), but not that of the PKC activator phorbol‐12,13‐dibutyrate (PDBu; 100 nM ). Colchicine (1 μM ) failed to alter the inhibitory effects of dopamine. The relationship between Na⁺‐K⁺‐ATPase and the concentration of extracellular Na⁺ showed a Michaelis–Menten constant (K_m) of 44.1 ± 13.7 mM and a V_max of 49.6 ± 4.8 μA cm^–2 in control monolayers. In the presence of apical dopamine (1 μM ) or cytochalasin B (1 μM ) V_max values were significantly (P < 0.05) reduced without changes in K_m values. These results are the first, obtained in live cells, showing that the PKC‐dependent inhibition of Na⁺‐K⁺‐ATPase activity by dopamine requires the integrity of the association between actin cytoskeleton and Na⁺‐K⁺‐ATPase.     

Effects of different types of K+ channel modulators on the spontaneous myogenic contraction of guinea‐pig urinary bladder smooth muscle

In the present study, effects of different types of K⁺ channel modulators on the spontaneous rhythmic contractile activity were examined in guinea‐pig urinary bladder smooth muscle (UBSM). Guinea‐pig UBSM exhibited myogenic rhythmic contraction in the presence of atropine (1 μM ), phentolamine (1 μM ), propranolol (1 μM ), suramin (10 μM ) and tetrodotoxin (1 μM ). Nisoldipine (100 nM ) or diltiazem (10 μM ) substantially diminished UBSM contractile activity. Nisoldipine‐resistant component of UBSM rhythmic contraction was further inhibited by gadolinium (200 μM ). Iberiotoxin (50 nM ), a selective blocker of large‐conductance, voltage‐gated Ca²⁺‐activated K⁺ (K_Ca) (BK) channel, dramatically increased both contraction amplitude and frequency whereas NS‐1619 (30 μM ), which increases BK channel activity, decreased them. Apamin (100 nM ), a selective blocker of small‐conductance, K_Ca (SK) channel, increased contraction amplitude but decreased frequency. A blocker of voltage‐gated K⁺ (K_v) channel, 4‐aminopyridine (100 μM ), significantly increased contraction frequency. E‐4031, a blocker of a novel inwardly rectifying K⁺ channel, i.e. the human ether‐a‐go‐go‐related gene (HERG) K⁺ channel, significantly increased contraction amplitude. Glibenclamide (1–10 μM ) (K_ATP channel blocker) and Ba²⁺ (10 μM ) (conventional K_ir channel blocker) did not exhibit conspicuous effects on spontaneous contractile activity of UBSM. These findings imply that two types of K_Ca (BK and SK) channels have prominent roles as negative feedback elements to limit extracellular Ca²⁺ influx‐mediated guinea‐pig UBSM contraction by regulating both amplitude and frequency. It was also suggested that both non‐K_Ca type of K⁺ (K_v and HERG‐like K⁺) channels may contribute to the regulation of UBSM myogenic rhythmic contraction.     

Antigen‐specific over‐expression of human cartilage glycoprotein 39 on CD4+CD25+ forkhead box protein 3+ regulatory T cells in the generation of glucose‐6‐phosphate isomerase‐induced arthritis

Human cartilage gp‐39 (HC gp‐39) is a well‐known autoantigen in rheumatoid arthritis (RA). However, the exact localization, fluctuation and function of HC gp‐39 in RA are unknown. Therefore, using a glucose‐6‐phosphate isomerase (GPI)‐induced model of arthritis, we investigated these aspects of HC gp‐39 in arthritis. The rise in serum HC gp‐39 levels was detected on the early phase of GPI‐induced arthritis (day 7) and the HC gp‐39 mRNA was increased significantly on splenic CD4⁺T cells on day7, but not on CD11b⁺cells. Moreover, to identify the characterization of HC gp‐39⁺CD4⁺T cells, we assessed the analysis of T helper (Th) subsets. As a result, HC gp‐39 was expressed dominantly in CD4⁺CD25⁺ forkhead box protein 3 (FoxP3)⁺ refulatory T cells (T_reg), but not in Th1, Th2 or Th17 cells. Furthermore, to investigate the effect of HC gp‐39 to CD4⁺T cells, T cell proliferation assay and cytokine production from CD4⁺T cells using recombinant HC gp‐39 was assessed. We found that GPI‐specific T cell proliferation and interferon (IFN)‐γ or interleukin (IL)‐17 production were clearly suppressed by addition of recombinant HC gp‐39. Antigen‐specific over‐expression of HC gp‐39 in splenic CD4⁺CD25⁺ FoxP3⁺ T_reg cells occurs in the induction phase of GPI‐induced arthritis, and addition of recombinant HC gp‐39 suppresses antigen‐specific T‐cell proliferation and cytokine production, suggesting that HC gp‐39 in CD4⁺ T cells might play a regulatory role in arthritis.     

CD4+ CD25+ Treg regulate the contribution of CD8+ T-cell subsets in repopulation of the lymphopenic environment

Peripheral T‐cell expansion is of major relevance for immune function after lymphopenia. In order to promote regeneration, the process should result in a peripheral T‐cell pool with a similar subpopulation structure as before lymphopenia. We investigated the repopulation of the CD8⁺ central‐memory T cells (T_CM) and effector‐memory T cells (T_EM) pools after adoptive transfer of sorted CD8⁺ T cells from naïve, T_CM and T_EM subsets into T‐cell‐deficient hosts. We show that the initial kinetics of expansion are distinct for each subset and that the contribution to the repopulation of the CD8⁺ T‐cell pool by the progeny of each subset is not a mere function of its initial expansion. We demonstrate that CD4⁺CD25⁺ Treg play a major role in the repopulation of the CD8⁺ T‐cell pool and that CD8⁺ T‐cell subsets impact on each other. In the absence of CD4⁺CD25⁺ Treg, a small fraction of naïve CD8⁺ T cells strongly proliferates, correlating with further expansion and differentiation of co‐expanding CD8⁺ T cells. CD4⁺CD25⁺ Treg suppress these responses and lead to controlled repopulation, contributing decisively to the maintenance of recovered T_CM and T_EM fractions, and leading to repopulation of each pool with progeny of its own kind.     

Circulating CD14+CD16+ monocyte levels predict tissue invasive character of cholangiocarcinoma

Chronic inflammation as a risk factor for cancer development is driven in part by monocyte/macrophages, which in many cancers exhibit pro‐tumorigenic activity. In this study we identified elevation in CD14⁺CD16⁺, a minor blood monocyte subpopulation in cholangiocarcinoma (CCA) patients, compared to normal and biliary disease patient specimens. Tumour association was suggested by the observation that this elevated level decreased to normal after tumour resection. Moreover, the elevated level of CD14⁺CD16⁺ monocytes in CCA patient blood correlated with degree of MAC387‐positive (recent blood‐derived macrophage migrant‐specific marker) tumour‐associated macrophage infiltration as determined by immunohistochemistry. These CD14⁺CD16⁺ monocytes were suggested to enhance tumour progression as this subpopulation possesses (i) high expression of adhesion molecules (CD11c, CD49d, and CD54) and scavenger receptor (CD163), which enable them to adhere strongly to endothelial cells, and (ii) that peripheral blood monocytes from CCA patients express high levels of growth and angiogenic factor‐related genes (epiregulin, VEGF‐A and CXCL3). Elevation of peripheral CD14⁺CD16⁺ monocyte levels was associated with features associated with poor prognosis CCA parameters (non‐papillary type and high number of tissue macrophages). These data indicate that the CD14⁺CD16⁺ monocytes from CCA patients with pro‐tumorigenic characteristics may associate with rapid tumour progression and poor patient outcome. If confirmed in subsequent studies, the level of CD14⁺CD16⁺ monocytes may serve as a marker for disease activity in CCA patients and serve as a target for pathogenic macrophage specific drug development.     

Short‐term anti‐CD25 monoclonal antibody administration down‐regulated CD25 expression without eliminating the neogenetic functional regulatory T cells in kidney transplantation

CD4⁺CD25⁺ forkhead box P3 (FoxP3)⁺regulatory T (T_reg) cells are generated and play a key role in the induction and maintenance of transplant tolerance in organ recipients. It has been proposed that interleukin (IL)-2/IL-2 receptor (IL-2R) signalling was essential for the development and proliferation of antigen-activated T cells that included both effector T cells and T_reg cells. Basiliximab (Simulect™), a chimeric monoclonal antibody directed against the α-chain of the IL-2R (CD25), can be expected to not only affect alloreactive effector T cells, but also reduce the number and function of T_reg cells. We therefore examined the effect of basiliximab induction therapy on the number and function of the T_reg cells in renal recipients. Basiliximab decreased the percentage of CD4⁺CD25⁺T cells, but failed to influence the percentage of CD4⁺FoxP3⁺ T_reg cells. The cellular CD25 expression was decreased significantly by basiliximab injection, but CD4⁺CD25⁺ T cells was not depleted from the circulating pool through monoclonal antibody activation-associated apoptosis. Functional analysis revealed that inhibitory function of T_reg cells from recipients with basiliximab injection was not significantly different from recipients without injection. These data indicate that the functional T_reg population may not be influenced by short-term basiliximab treatment.     

The regulation of regulation: interleukin‐10 increases CD4+ CD25+ regulatory T cells but impairs their immunosuppressive activity in murine models with schistosomiasis japonica or asthma

CD4⁺ CD25⁺ Foxp3⁺ regulatory T (Treg) cells play an important role in maintaining immune homeostasis. Interleukin‐10 (IL‐10), a cytokine with anti‐inflammatory capacities, also has a critical role in controlling immune responses. In addition, it is well known that production of IL‐10 is one of the suppression mechanisms of Treg cells. However, the action of IL‐10 on Treg cells themselves remains insufficiently understood. In this study, by using a Schistosoma japonicum‐infected murine model, we show that the elevated IL‐10 contributed to Treg cell induction but impaired their immunosuppressive function. Our investigations further suggest that this may relate to the up‐regulation of serum transforming growth factor (TGF‐β) level but the decrease in membrane‐bound TGF‐β of Treg cells by IL‐10 during S. japonicum infection. In addition, similar IL‐10‐mediated regulation on Treg cells was also confirmed in the murine model of asthma. In general, our findings identify a previously unrecognized opposing regulation of IL‐10 on Treg cells and provide a deep insight into the precise regulation in immune responses.     

Expression and function of Na+/Ca2+ exchangers 1 and 3 in human macrophages and monocytes

The Na⁺/Ca²⁺ exchanger (NCX) is a membrane transporter that can switch Na⁺ and Ca²⁺ in either direction to maintain the homeostasis of intracellular Ca²⁺. Three isoforms (NCX1, NCX2, and NCX3) have been characterized in excitable cells, e.g. neurons and muscle cells. We examined the expression of these NCX isoforms in primary human lung macrophages (HLM) and blood monocytes. NCX1 and NCX3, but not NCX2, are expressed in HLM and monocytes at both mRNA and protein levels. Na⁺‐free medium induced a significant increase in intracellular calcium concentration ([Ca²⁺]_i) in both cell types. This response was completely abolished by the NCX inhibitor 5‐(N‐4‐chlorobenzyl)‐20,40‐dimethylbenzamil (CB‐DMB). Moreover, inhibition of NCX activity during Ca²⁺‐signaling induced by histamine caused a delay in restoring baseline [Ca²⁺]_i. Na⁺‐free medium induced TNF‐α expression and release in HLM comparable to that caused by LPS. TNF‐α release induced by Na⁺‐free medium was blocked by CB‐DMB and greatly reduced by RNAi‐mediated knockdown of NCX1. These results indicate that human macrophages and monocytes express NCX1 and NCX3 that operate in a bidirectional manner to restore [Ca²⁺]_i, to generate Ca²⁺‐signals, and to induce TNF‐α production. Therefore, NCX may contribute to regulate Ca²⁺ homeostasis and proinflammatory functions in human macrophages and monocytes.     

T‐cell help dependence of memory CD8+ T‐cell expansion upon vaccinia virus challenge relies on CD40 signaling

Due to their capacity to differentiate into long‐lived memory cells, CD8⁺ T cells are able to resolve subsequent infections faster than during the primary response. Among other factors, CD4⁺ T cells play a crucial role during primary and secondary CD8⁺ T‐cell responses. However, the timing and mechanisms by which they influence CD8⁺ T cells may differ in primary and secondary responses. Here, we demonstrate that during both primary and secondary vaccinia virus infection, CD4⁺ T cells are necessary to promote CD8⁺ T‐cell responses. While CD4⁺ T cells contributed to memory CD8⁺ T‐cell development, they were even more important during memory recall responses during challenge, as absence of CD4⁺ T cells during challenge resulted in markedly decreased proliferation and increased apoptosis. T‐cell help during primary and secondary responses was mediated via CD40 signaling, with DCs being an integral part of that pathway. As opposed to primary CD8⁺ T‐cell responses where only a combination of agonistic CD40 signaling and provision of IL‐2 could substitute for T‐cell help, agonistic CD40 triggering alone was sufficient to rescue memory CD8⁺ T‐cell responses in absence of T‐cell help in the context of vaccinia virus infection.     

IL‐15‐dependent CD8+CD122+ T cells ameliorate experimental autoimmune encephalomyelitis by modulating IL‐17 production by CD4+ T cells

Interleukin‐15 (IL‐15) is an inflammatory cytokine whose role in autoimmune diseases has not been fully elucidated. Th17 cells have been shown to play critical roles in experimental autoimmune encephalomyelitis (EAE) models. In this study, we demonstrate that blockade of IL‐15 signaling by TMβ‐1 mAb treatment aggravated EAE severity. The key mechanism was not NK‐cell depletion but depletion of CD8⁺CD122⁺ T cells. Adoptive transfer of exogenous CD8⁺CD122⁺ T cells to TMβ‐1‐treated mice rescued animals from severe disease. Moreover, transfer of preactivated CD8⁺CD122⁺ T cells prevented EAE development and significantly reduced IL‐17 secretion. Naïve effector CD4⁺CD25^? T cells cultured with either CD8⁺CD122⁺ T cells from wild‐type mice or IL‐15 transgenic mice displayed lower frequencies of IL‐17A production with lower amounts of IL‐17 in the supernatants when compared with production by effector CD4⁺CD25^? T cells cultured alone. Addition of a neutralizing antibody to IL‐10 led to recovery of IL‐17A production in Th17 cultures. Furthermore, coculture of CD8⁺CD122⁺ T cells with effector CD4⁺ T cells inhibited their proliferation significantly, suggesting a regulatory function for IL‐15 dependent CD8⁺CD122⁺ T cells. Taken together, these observations suggest that IL‐15, acting through CD8⁺CD122⁺ T cells, has a negative regulatory role in reducing IL‐17 production and Th17‐mediated EAE inflammation.     

CD4+ CD30+ T cells perpetuate IL-5 production in Dermatophagoides pteronyssinus allergic patients

BACKGROUND: Airway allergic diseases are regulated by interleukin (IL)-5, which causes infiltration of eosinophils into the bronchial epithelium, and by IL-4 which increases serum immunoglobulin E (IgE) production and promotes CD30 expression on Th cells. CD30 generates a costimulatory signal involved in apoptosis or cell proliferation, depending on the microenvironment. Our aims were: (i) to analyze if CD4+ CD30+ T cells from allergic patients proliferate in response to Dermatophagoides pteronyssinus, and (ii) if upon stimulation this cell population produces IL-4 and IL-5. METHODS: Peripheral blood mononuclear cell (PBMC) from 17 allergic rhinitis and mild allergic asthma patients and 12 healthy nonallergic individuals were stimulated with allergen in the presence or absence of anti-IL-4, anti-IL-5 or anti-IL-4Ralpha monoclonal antibodies (mAbs). TdT-mediated dUTP nick end-labeling (TUNEL) assay, 7-aminoactinomycin-D (7-AAD) intercalation, and flow cytometry were used to determine the CD4+ CD30+ blasts percentage, cell proliferation, apoptosis, and intracellular cytokines after 7 culture days. RESULTS: Cell proliferation induced with allergen showed that 90% of the allergen-stimulated blasts were CD4+, 50% of which were CD30+. Allergen-stimulated PBMC showed a progressive increase (mean: from 7% to 23%) of CD4+ CD30+IFN-gamma+ and CD4+ CD30+IL-4+ blasts which diminished (mean: 6%) after 5 culture days. In contrast, CD4+ CD30+IL-5+ blasts showed a continuous progression (from 12% to 24%) that maintained after 7 culture days. The vast majority of CD4+ CD30+ blasts were negative to 7-AAD or TUNEL. Additionally, a significant decrease (34%) was observed in the number of CD4+ CD30+ blasts when IL-4 was neutralized. CONCLUSIONS: These data suggest that specific allergen stimulation of PBMC isolated from allergic patients generates a nonapoptotic CD4+ CD30+ blast subset that produces IL-5.     

Interrupting CD28 costimulation before antigen rechallenge affects CD8+ T‐cell expansion and effector functions during secondary response in mice

The role of CD28‐mediated costimulation in secondary CD8⁺ T‐cell responses remains controversial. Here, we have used two tools — blocking mouse anti‐mouse CD28‐specific antibodies and inducible CD28‐deleting mice — to obtain definitive answers in mice infected with ovalbumin‐secreting Listeria monocytogenes. We report that both blockade and global deletion of CD28 reveal its requirement for full clonal expansion and effector functions such as degranulation and IFN‐γ production during the secondary immune response. In contrast, cell‐intrinsic deletion of CD28 in transferred TCR‐transgenic CD8⁺ T cells before primary infection leads to impaired clonal expansion but an increase in cells able to express effector functions in both primary and secondary responses. We suggest that the proliferation‐impaired CD8⁺ T cells respond to CD28‐dependent help from their environment by enhanced functional differentiation. Finally, we report that cell‐intrinsic deletion of CD28 after the peak of the primary response does not affect the establishment, maintenance, or recall of long‐term memory. Thus, if given sufficient time, the progeny of primed CD8⁺ T cells adapt to the absence of this costimulator.     

Memory lapses in graft-versus-host disease

“Faster, better, more” is the conventional benchmark used to define responses of memory T cells when compared with their naïve counterparts. In this issue of the European Journal of Immunology, Mark and Warren Shlomchik and colleagues [Eur. J. Immunol. 2011. 41 : 2782–2792] make the intriguing observation that murine memory CD4⁺ T‐cell populations enriched for alloreactive precursors are fully capable of rejecting allogeneic skin grafts but yet are incapable of inducing significant graft‐versus‐host disease. These observations add to the emerging concept that memory CD4⁺ T‐cell development is more nuanced and complex than predicted by conventional models. In particular, the data suggest that it may be just as important to consider what naïve or effector cells have “lost” in their transition to memory.     

Preserved immune system in long-term asymptomatic vertically HIV-1 infected children

The objective of this study was to study immune system status in long‐term asymptomatic (LTA) HIV‐1‐infected children. A cross‐sectional study was used, involving HIV‐1‐infected children over 7 years of age who were rated into two groups according to their clinical and immunological classification: (a) LTA: 7 asymptomatic HIV‐1‐infected children in A1; (b) Rapid progressor (RP): 14 age‐matched C3 HIV‐1‐infected children. The control group consisted of 17 age‐matched uninfected children. The characterization of CD4+ T‐cell subsets was determined by three‐colour flow cytometry. The proliferative response and cytokine production by activated peripheral blood T‐cells were also measured. IL‐7 levels were measured in serum. Thymic production of T‐cells was quantified by TCR rearrangement excision circles (TRECs). The LTA children showed similar proliferative responses to PHA, PWM and anti‐CD3+ anti‐CD28, but lower responses to tetanus toxoid and streptokinase, in comparison with the controls but always higher responses in comparison with the RP group. The production of TNF‐α and IFN‐γ was similar in the LTA and control groups, and both were higher than the levels in the RP group. The LTA group showed a lower percentage of memory CD4+ T‐cells (CD4+ CD45RO+, CD4+ CD45RA‐CD62L+) than the control and RP groups. The LTA group also showed lower percentages of CD4+ CD7‐ cells than the controls. As for naïve CD4+ T‐cells (CD4+ CD45RA+ CD62L+), CD4+ CD45RA+ and CD4+ CD62L+ cells, the LTA group showed higher values than the control and RP groups. The LTA group showed higher percentages of CD4+ HLA‐DR+ CD38+ than the controls, but lower values than the RP group. In contrast, the LTA group had percentages of CD4+ HLA‐DR‐CD38+ T‐cells higher than both the control and RP groups, whereas CD4+ CD38+ levels were only higher in the LTA group in comparison with the controls. CD4+ HLA‐DR+ CD38‐ and CD4+ HLA‐DR+ cell numbers were lower in the LTA group in comparison with the RP group. We found almost normal values of TRECs and IL‐7 in the LTA group, but lower values in the RP group. Moreover, we found an inverse relation between TREC levels and IL‐7 in plasma from HIV‐infected children. Asymptomatic HIV‐1 infected children have a well preserved immune system similar to that of control uninfected children in spite of HIV‐infection for more than 7 years. Moreover, our results identified new markers of HIV disease, such as TRECs and IL‐7, that could be used to monitor disease.