Relation of exocytotic release of γ-aminobutyric acid to Ca2+ entry through Ca2+ channels or by reversal of the Na+/Ca2+ exchanger in synaptosomes

The specific inhibitor of the -aminobutyric acid (GABA) carrier, NNC-711, {1-[(2-diphenylmethylene) amino]oxyethyl}-1,2,5,6-tetrahydro-3-pyridinecarboxylic acid hydrochloride, blocks the Ca²⁺-independent release of [³H]GABA from rat brain synaptosomes induced by 50 mM K⁺ depolarization. Thus, in the presence of this inhibitor, it was possible to study the Ca²⁺-dependent release of [³H]GABA in the total absence of carrier-mediated release. Reversal of the Na⁺/Ca²⁺ exchanger was used to increase the intracellular free Ca²⁺ concentration ([Ca²⁺]_i) to test whether an increase in [Ca²⁺]_i alone is sufficient to induce exocytosis in the absence of depolarization. We found that the [Ca²⁺]_i may rise to values above 400 nM, as a result of Na⁺/Ca²⁺ exchange, without inducing release of [³H]GABA, but subsequent K⁺ depolarization immediately induced [³H]GABA release. Thus, a rise of only a few nanomolar Ca²⁺ in the cytoplasm induced by 50 mM K⁺ depolarization, after loading the synaptosomes with Ca²⁺ by Na⁺/Ca²⁺ exchange, induced exocytotic [³H]GABA release, whereas the rise in cytoplasmic [Ca²⁺] caused by reversal of the Na⁺/Ca²⁺ exchanger was insufficient to induce exocytosis, although the value for [Ca²⁺]_i attained was higher than that required for exocytosis induced by K⁺ depolarization. The voltage-dependent Ca²⁺ entry due to K⁺ depolarization, after maximal Ca²⁺ loading of the synaptosomes by Na⁺/Ca²⁺ exchange, and the consequent [³H]GABA release could be blocked by 50 M verapamil. Although preloading the synaptosomes with Ca²⁺ by Na⁺/Ca²⁺ exchange did not cause [³H]GABA release under any conditions studied, the rise in cytoplasmic [Ca²⁺] due to Na⁺/Ca²⁺ exchange increased the sensitivity to external Ca²⁺ of the exocytotic release of [³H]GABA induced by subsequent K⁺ depolarization. Thus, our results show that the vesicular release of [³H]GABA is rather insensitive to bulk cytoplasmic [Ca²⁺] and are compatible with the view that GABA exocytosis is triggered very effectively by Ca²⁺ entry through Ca²⁺ channels near the active zones.     

Immune tolerance induced by intravenous transfer of immature dendritic cells via up-regulating numbers of suppressive IL-10+ IFN-γ+-producing CD4+ T cells

Dendritic cells (DCs) regulate immunity and immune tolerance in vivo. However, the mechanisms of DC-mediated tolerance have not been fully elucidated. Here, we demonstrate that intravenous (i.v.) transfer of bone marrow-derived DCs pulsed with myelin oligodendrocyte glycoprotein (MOG) peptide blocks the development of experimental autoimmune encephalomyelitis in C57BL/6J mice. i.v. transfer of MOG-pulsed DCs leads to the down-regulation of the production of IL-17A and IFN-γ and up-regulation of IL-10 secretion. The development of regulatory T cells (Tregs) is facilitated via up-regulation of FoxP3 expression and production of IL-10. The number of suppressive CD4⁺IL-10⁺IFN-γ⁺ T cells is also improved. The expression of OX40, CD154, and CD28 is down-regulated, but the expression of CD152, CD80, PD-1, ICOS, and BTLA is up-regulated on CD4⁺ T cells after i.v. transfer of immature DCs. The expression of CCR4, CCR5, and CCR7 on CD4⁺ T cells is also improved. Our results suggest that immature DCs may induce tolerance via facilitating the development of CD4⁺FoxP3⁺ Tregs and suppressive CD4⁺IL-10⁺IFN-γ⁺ T cells in vivo.     

Regulation of Con A – dependent cytokine production from CD4+ and CD8+ T lymphocytes by autosecretion of histamine

Objectives: Previously we have shown that both CD4+ T cells and CD8+ T cells produce histamine when activated with Con A. The aim of this study was to examine whether cytokine production by these cells is regulated by autosecretion of histamine.Materials: CD4+ and CD8+ T cells were separated from spleen cells of C57BL/6 mice and mice lacking the H1 receptor (H1R) or H2R, using anti – CD4+ – and anti – CD8+ – coupled magnetic beads, respectively.Results: Depletion of the H1R resulted in decreases in the release of IL-2 and IL-10 from both CD4+ and CD8+ cells and increases in the release of IL-4 from CD4+ T cells and IFN- from CD8+ cells. Mice lacking the H2R showed up – regulation of IFN- secretion from CD8+ cells and of IL-4 from CD4+ and CD8+ T cells. Release of IL-2 and IL-10 from CD4+ as well as CD8+ cells was down – regulated in these mice. Both CD4+ and CD8+ T cell fractions synthesized histamine, which was enhanced in the H1R - deficient CD8+ T cells. Treatment of the cells with -fluoromethyl-histidine, a specific inhibitor of HDC, or histaminase increased IFN- from CD8+ cells, whereas it had no appreciable effect on IL-4 secretion from CD4+ cells.Conclusions: These results suggest that cytokine production by CD4+ and CD8+ T lymphocytes is regulated by autosecretion of histamine.Received 4 July 2003; returned for revision 23 September 2003; returned for final revision 13 October 2003, accepted by M. Parnham 17 October 2003     

Preferential Activation of CD4+Vβ5.2/5.3+Intestinal Intraepithelial Lymphocytes in the Inflamed Lesions of Crohn's Disease

To analyze the nature of intestinal intraepithelial lymphocytes (iIELs) in inflammatory intestinal disease, we established T cell lines of iIELs isolated from endoscopic biopsied ileal and colonic mucosa of Crohn's disease patients. Seven T cell lines from the inflamed terminal ileum of 13 patients, but none of 16 T cell lines from normal terminal ileum, have shown a deviation of T cell receptor variable region gene usage and were enriched in the proportion of CD4⁺Vβ5.2/5.3⁺cells. CD4⁺Vβ5.2/5.3⁺cells in the T cell lines were not increased after stimulation with purified protein derivatives or 65-kDa heat-shock protein but significantly increased after stimulation with staphylococcal enterotoxins C1 and D. Those cells showed increased cytolytic activity against target cells cross-linked by anti-Vβ5.2/5.3 and produced a large amount of interferon-γ. These results indicated that CD4⁺Vβ5.2/5.3⁺iIELs were preferentially activated in the inflamed lesions of Crohn's and may play a possible role in the triggering and progression of human inflammatory intestinal disease.     

Higher Percentage of CD3+CD154+ T Lymphocytes Predicts Efficacy of TNF-α Inhibitors in Active Axial SpA Patients

The objective of this study was to evaluate which subtypes of T lymphocytes (CD3⁺CD28⁺ and CD3⁺CD154⁺) could predict clinical efficacy after TNF-α inhibitor treatment in active axial SpA patients. Patients who fulfilled Assessment of SpondyloArthritis international Society (ASAS) criteria for axial SpA had a BASDAI of ≥40 mm. All patients received TNF-α inhibitor treatment for 12 weeks. ASAS20 was used to evaluate the effect of the treatment at week 12. We detected the percentage of CD3⁺CD28⁺ and CD3⁺CD154⁺ T lymphocytes on lymphocyte cells in the peripheral blood in patients and healthy controls. We evaluated whether the percentage of the above subtypes of T lymphocytes could predict clinical efficacy by ROC curve analysis. Fifty-eight healthy controls and 74 active axial SpA patients were included. Mean age was 26.28?±?9.08 and 26.95?±?8.13 years for healthy controls and patients, respectively (p?=?0.767). The percentage of CD3⁺CD154⁺ T lymphocytes was significantly higher in axial SpA patients than in healthy controls (1.62?±?1.89 % vs 0.79?±?0.52 %, p?+CD154⁺ T lymphocytes was significantly higher in HLA-B27(⁺) patients than HLA-B27(^?) ones (HLA-B27⁺ vs HLA-B27^?:1.77?±?1.95 % vs 0.41?±?0.27 %, p?=?0.005). Compared with baseline, the percentage of CD3⁺CD154⁺ T lymphocytes significantly decreased to 0.87?±?0.49 % at week 12 (p?+CD154⁺ T lymphocytes could predict clinical efficacy of SpA patients with TNF-α inhibitor treatment (AUC?=?0.733, p?=?0.014). High percentage of CD3⁺CD154⁺ is over-expressed on lymphocytes in peripheral blood of active SpA patients and can be down-regulated by TNF-α inhibitor therapy. High-percentage of CD3⁺CD154⁺ T lymphocytes may predict clinical efficacy of TNF-α inhibitor treatment in active axial SpA patients.     

Improved Mycobacterium tuberculosis clearance after the restoration of IFN-γ+TNF-α+CD4+T cells: Impact of PD-1 inhibition in active tuberculosis patients

Prolonged therapy, drug toxicity, noncompliance, immune suppression, and alarming emergence of drug resistance necessitate the search for therapeutic vaccine strategies for tuberculosis (TB). Such strategies ought to elicit not only IFN-γ, but polyfunctional response including TNF-α, which is essential for protective granuloma formation. Here, we investigated the impact of PD-1 inhibition in facilitating protective polyfunctional T cells (PFTs), bacillary clearance, and disease resolution. We have observed PD-1 inhibition preferentially rescued the suppressed PFTs in active tuberculosis patients. In addition, polyfunctional cytokine milieu favored apoptosis of infected MDMs over necrosis with markedly reduced bacillary growth (≪CFU) in our in vitro monocyte-derived macrophages (MDMs) infection model. Furthermore, the animal study revealed a significant decline in the bacterial burden in the lungs and spleen of infected mice after in vivo administration of α-PD-1 along with antitubercular treatment. Our findings suggest that rescuing polyfunctional immune response by PD-1 inhibition works synergistically with antituberculosis chemotherapy to confer improved control over bacillary growth and dissemination. In summary, our data strongly indicate the therapeutic potential of α-PD-1 as adjunct immunotherapy that can rejuvenate suppressed host immunity and enhance the efficacy of candidate therapeutic vaccine(s).     

Regulation of the Na+2Cl–K+ cotransporter in isolated rat colon crypts

The Na(+2)Cl(-)K+ cotransporter accepts NH4+ at its K+-binding site. Therefore, the rate of cytosolic acidification after NH4+ addition to the bath (20 mmol/l) measured by BCECF fluorescence can be used to quantify the rate of this cotransporter. In isolated colon crypts of rat distal colon (RCC) addition of NH4+ led to an initial alkalinization, corresponding to NH3 uptake. This was followed by an acidification, corresponding to NH4+ uptake. The rate of this uptake was quantified by exponential curve fitting and is given in arbitrary units (delta fluorescence ratio units/1000 s). In pilot experiments it was shown that the pH signal caused by the Na(+)2Cl(-)K+ co-transporter could be amplified if the experiments were carried out in the presence of bath Ba2+ to inhibit NH4+ uptake via K+ channels. Therefore all subsequent experiments were performed in the presence of 1 mmol/l Ba2+. In the absence of any secretagogue, preincubation of RCC in a low-Cl- solution (4 mmol/l) for 10 min enhanced the uptake rate significantly from 1.70+/-0.11 to 2.54+/-0.27 U/1000 s (n=20). The addition of 100 mmol/l mannitol (hypertonic solution) enhanced the rate significantly from 1.93+/-0.17 to 2.84+/-0.43 U/1000 s (n=5). Stimulation of NaCl secretion by a solution containing 100 micromol/l carbachol (CCH) led to a small but significant increase in NH4+ uptake rate from 2.06+/-0.34 to 2.40+/-0.30 U/1000 s (n= 11). The increase in uptake rate observed with stimulation of the cAMP pathway by isobutylmethylxanthine (IBMX) and forskolin (100 micromol/l and 5 micromol/l, respectively) was from 2.39+/-0.24 to 3.06+/-0.36 U/1000 s (n=24). Whatever the mechanism used to increase the NH4+ uptake rate, azosemide (500 micromol/l) always reduced this rate to control values. Hence three manoeuvres enhanced loop-diuretic-inhibitable uptake rates of the Na(+)2Cl(-)K+ cotransporter: (1) lowering of cytosolic Cl- concentration; (2) cell shrinkage; (3) activation of NaCl secretion by carbachol and (4) activation of NaCl secretion by cAMP. The common denominator of all four activation pathways may be a transient fall in cell volume.     

Evidence for the involvement of K+ channels and K+-Cl– cotransport in the regulatory volume decrease of newborn rat cardiomyocytes

In order to delineate ion transport mechanisms involved in volume homeostasis of freshly isolated newborn rat ventricular myocytes, we investigated the effects of ion substitutions and pharmacological maneuvers upon (1) isotonic volume, (2) hypotonically induced initial swelling, and (3) the subsequent regulatory volume decrease (RVD), as determined by electronic cell sizing. Cardiomyocytes exposed to hypotonic medium (176 mosmol/l) swelled by 51+/-1% of isotonic volume, and they underwent a partial regulatory volume decrease (RVD), reaching a maximum regulation after 30 min (51+/-1% of initial swelling), with a half-time (t1/2) of 6+/-1 min (n=60). RVD was associated with significant cardiomyocyte K+ loss (12+/-4% at 5 min and 15+/-2% of isotonic control after 30 min: n=6, P<0.001), 71% of which was Cl- dependent (P<0.05). Within the 30-min experimental time frame, ouabain, a Na+/K+ pump inhibitor, had no significant effect on RVD (despite an inhibitory trend), cell swelling or on isotonic volume (n=6). Bumetanide (50 microM), a Na+-K+-Cl- co-transport blocker, induced a significant reduction of isotonic cell volume (3+/-2%, n=6. P<0.05), potentiated initial swelling by 16+/-1% (n=8, P<0.02), and it partially inhibited RVD (24+/-11% at 30 min, n=6), whereas Na+ omission had no significant effect on isotonic cell volume, cell swelling or RVD. The effects of bumetanide on initial swelling and RVD were prevented by gadolinium ion (10 microM), a stretch-activated cation channel blocker (n=5). Quinidine (500 microM), a non-selective Ca(2+)-activated potassium channel blocker with no side-effects on K(+)-Cl(-) cotransport, did not modify initial cell swelling, but inhibited RVD (50+/-3% at 5 min, n=9, P<0.01; 22+/-3% at 30 min), an effect which was cancelled by external Ca2+ chelation with EGTA (n=5), and reproduced by tetraethylammonium (TEA, 20 mM), another K+ channel blocker. 4,4'-Diisothiocyanatostilbene 2,2'-disulfonic acid (DIDS, 100 microM), a non-selective swelling-activated Cl- channel blocker with marginal side-effects on K(+)-Cl(-)cotransport, did not modify initial swelling, but inhibited RVD to the same extent as quinidine (42+/-3% at 5 min, and 23+/-3% at 30 min, n=15, P<0.05), whereas hypotonic Cl(-)-free solution had no effect on isotonic volume, but potentiated initial swelling by 16+/-2% (P<0.05) and fully inhibited RVD (n=5, P<0.001). R(+)-[(2-n-Butyl-6,7-dichloro-2-cyclopentyl-2,3-dihydro-1-oxo-1H-inde n-5yl)-oxy] acetic acid) (DIOA, 80 microM), a K(+)-Cl- cotransport blocker (with inhibitory potency toward Ca(2+)-activated K+ channels), inhibited 87+/-5% of the RVD process at 5 min (P<0.001) and 56+/-16% at 30 min (P<0.001), whereas it had a small effect on isotonic volume (+4%, P<0.01) and initial cell swelling (+2%, N.S.; n=9). In contrast to quinidine, DIOA was able to inhibit Ca(2+)-omission-resistant RVD (full inhibition at 5 min, and 56+/-9% at 30 min; P<0.01, n=5). In conclusion, our results suggest that at least three distinct ion transport mechanisms are involved in the RVD in newborn rat cardiomyocytes: (1) K+ and Cl-channels, (2) K(+)-Cl- cotransport, and (3) Na(+)-K(+)-Cl- co-transport.     

Identification of the association of CD28+CD244+ Tc17/IFN-γ cells with chronic hepatitis C virus infection

CD8⁺ T cells play multiple and complex immunological roles including antiviral, regulatory, and exhaustive effects in hepatitis C virus (HCV) infected patients. Some CD8⁺ T-cell subsets were confirmed to be closely related to HCV infection such as T_CM, T_EM, T_EMRA, Tc17, and CD8⁺ Treg. Herein, we report a new subset of interleukin (IL)-17/interferon (IFN)-γ producing CD8⁺ T (Tc17/IFN-γ) cells that markedly correlate with CD28⁺CD244⁺ cells, IL-17 levels, and HCV RNA in HCV patients. During early treatment with peg-IFN-a2a plus ribavirin, the imbalance of these Tc17/IFN-γ cells could be partially restored, together with normalized serum alanine aminotransferase but not aspartate transaminase. Also, we analyzed the dynamic change of the percentage of this T cells subset in patients with different outcome after 4-week course of treatment with peg-IFN-a2a plus ribavirin and found that the percentage of CD8⁺CD28⁺CD244⁺ T cells significantly decreased in recovered patients but not in nonrecovered patients. In vitro, CD28⁺CD244⁺ T cells were the only CD8⁺ T-cell group that secreted both IL-17 and IFN-γ in this axis and blockade with anti-CD244 antibodies significantly reduced cytokine production. Taken together, this study demonstrates that the frequency and regulatory functions of CD28⁺CD244⁺ Tc17/IFN-γ cells may play an important role in persistent HCV infection.     

Differential sensitivity of naïve and subsets of memory CD4+ and CD8+ T cells to hydrogen peroxide-induced apoptosis

CD4+ and CD8+ memory T cells are identified into central and effector memory subsets, which are characterized by distinct homing patterns and functions. In this investigation, we show that na?ve and central memory CD4+ and CD8+ T cells are sensitive to hydrogen peroxide (H2O2)-induced apoptosis, whereas effector memory CD4+ and CD8+ T cells are relatively resistant to H2O2-induced apoptosis. Apoptosis in na?ve and central memory CD4+ and CD8+ is associated with the release of cytochrome c and activation of caspase-9 and caspase-3, upregulation of Bax and voltage-dependent anion channel (VDAC) expression, and decreased intracellular glutathione (GSH). In vitro GSH and a superoxide dismutase mimetic Mn(III) tetrakis (1-methyl-4-pyridyl) porphyrin inhibited H2O2-induced apoptosis in both na?ve and central memory CD4+ and CD8+ T cells. Furthermore, VDAC inhibitor 4,4'-diisothiocynostilbene-2,2'-disulfonic acid blocked H2O2-induced apoptosis. These data demonstrate that H2O2 induces apoptosis preferentially in human na?ve and central memory CD4+ and CD8+ T cells via the mitochondrial pathway by regulating intracellular GSH and the expression of Bax and VDAC.     

Functional up-regulation of basolateral Na+-dependent HCO3 − transporter NBCn1 in medullary thick ascending limb of K+-depleted rats

The electroneutral, Na⁺-coupled HCO₃^– co-transporter (NBCn1) is expressed in the basolateral membrane of rat medullary thick ascending limb (mTAL) segments and is up-regulated strongly during metabolic acidosis. We have suggested previously that NBCn1-mediated HCO₃^– uptake across the basolateral cell membrane is an important buffering mechanism during the transcellular transport of NH₄⁺ in the mTAL. To investigate this further we treated rats with a low-K⁺ diet for 4 days, a manoeuvre known to increase proximal tubular ammoniagenesis and delivery of luminal NH₄⁺ to the mTAL. Hypokalaemia strongly increased immunolabelling of basolateral NBCn1 in the mTAL of the inner stripe of the outer medulla. Immunoblotting revealed an eightfold up-regulation of NBCn1 protein in K⁺-depleted rats. Subsequently we used in vitro perfusion of isolated mTAL and BCECF imaging to investigate Na⁺-coupled HCO₃^– influx in normal and K⁺-depleted rats. Hypokalaemia induced a threefold up-regulation of Na⁺-coupled HCO₃^– influx (1.64±0.28 vs. 0.47±0.05 pH units/minute, n=8). The Na⁺-dependent alkalinization was also significantly larger in K⁺-depleted rats (0.38±0.04 vs. 0.23±0.03 pH units). These data indicate that K⁺-depleted rats respond with a strong up-regulation of NBCn1 protein and function, probably to cope with the higher tubular load of NH₄⁺, and strengthen our previous suggestion that NBCn1 is an important player in the excretion of NH₄⁺. They also indicate that it is the delivery of luminal NH₄⁺ rather than systemic changes of pH that determine the expression of NBCn1.     

30+ years of P300 brain–computer interfaces

Brain–computer interfaces (BCIs) directly measure brain activity with no physical movement and translate the neural signals into messages. BCIs that employ the P300 event-related brain potential often have used the visual modality. The end user is presented with flashing stimuli that indicate selections for communication, control, or both. Counting each flash that corresponds to a specific target selection while ignoring other flashes will elicit P300s to only the target selection. P300 BCIs also have been implemented using auditory or tactile stimuli. P300 BCIs have been used with a variety of applications for severely disabled end users in their homes without frequent expert support. P300 BCI research and development has made substantial progress, but challenges remain before these tools can become practical devices for impaired patients and perhaps healthy people.     

Control of the development of CD8αα+ intestinal intraepithelial lymphocytes by TGF-β

The molecular mechanisms that direct the development of TCRαβ+CD8αα+ intestinal intraepithelial lymphocytes (IELs) are not thoroughly understood. Here we show that transforming growth factor-β (TGF-β) controls the development of TCRαβ+CD8αα+ IELs. Mice with either a null mutation in the gene encoding TGF-β1 or T cell-specific deletion of TGF-β receptor I lacked TCRαβ+CD8αα+ IELs, whereas mice with transgenic overexpression of TGF-β1 had a larger population of TCRαβ+CD8αα+ IELs. We observed defective development of the TCRαβ+CD8αα+ IEL thymic precursors (CD4?CD8?TCRαβ+CD5+) in the absence of TGF-β. In addition, we found that TGF-β signaling induced CD8α expression in TCRαβ+CD8αα+ IEL thymic precursors and induced and maintained CD8α expression in peripheral populations of T cells. Our data demonstrate a previously unrecognized role for TGF-β in the development of TCRαβ+CD8αα+ IELs and the expression of CD8α in T cells.     

Cytoprotective effects of high dose of α-galactosylceramide against activation-induced CD4+ T and CD8+ T cell death as an adjuvant

Objective: To investigate the cytoprotective effects of high dose of α-galactosylceramide (α-GC) on the activation-induced CD4+ T and CD8+ T cell death. Methods: Experimental autoimmune encephalomyelitis (EAE) was induced using adoptive transfer of MOGCD4+ cells treated using α-GC into recipient C57BL/6 mice while the MOGCD4+ cells treated using 0.5% polysorbate were set as vehicle group, based on which to investigate the effects of α-GC on activation induced CD4+ T cell death. Additionally, an EG7 tumor-bearing mice model is established using adoptive transfer of CD8+ T cells, based on which to investigate the effect of α-GC on the apoptosis of CD8+ T cells. Results: A higher induction rate was noticed after adoptive transfer of MOGCD4+ cells treated using α-GC together with the severity of EAE compared with the conventional methods. Longer survival duration was noted in the green fluorescent protein (GFP) labeled MOGT in the α-GC group compared with the vehicle group (P < 0.05). Severe inflammatory cell infiltration and myelinoclasis was noted in the white matter of nervous system in the α-GC group. In the EG7 tumor model, more adoptive CD8+ T cells were survived in α-GC group compared with that of vehicle group. The growth of tumor mass was significantly inhibited in α-GC group. Conclusions: high dose of α-GC could be used as an adjuvant for inhibiting activation-induced CD4+ T and CD8+ T cell death. Our study could provide helpful information for the development of adoptive cell therapy with reduced programmed cell death.     

Localization of Na+–K+-ATPase α/β, Na+–K+–2Cl-cotransporter 1 and aquaporin-5 in human eccrine sweat glands

In order to evaluate the function of the repaired or regenerated eccrine sweat glands, we must first localize the proteins involved in sweat secretion and absorption in normal human eccrine sweat glands. In our studies, the cellular localization of Na⁺–K⁺-ATPase α/β, Na⁺–K⁺–2Cl-cotransporter 1 (NKCC1) and aquaporin-5 (AQP5) in eccrine sweat glands were detected by immunoperoxidase labeling. The results showed that Na⁺–K⁺-ATPase α was immunolocalized in the cell membrane of the basal layer and suprabasal layer cells of the epidermis, the basolateral membrane of the secretory coils, and the cell membrane of the outer cells and the basolateral membrane of the luminal cells of the ducts. The localization of Na⁺–K⁺-ATPase β in the secretory coils was the same as Na⁺–K⁺-ATPase α, but Na⁺–K⁺-ATPase β labeling was absent in the straight ducts and epidermis. NKCC1 labeling was seen only in the basolateral membrane of the secretory coils. AQP5 was strongly localized in the apical membrane and weakly localized in the cytoplasm of secretory epithelial cells. The different distribution of these proteins in eccrine sweat glands was related to their functions in sweat secretion and absorption.     

Accumulation of CCR5+ T cells around RANTES+ granulomas in Crohn's disease: a pivotal site of Th1-shifted immune response?

Immunological abnormalities are implicated in the pathogenesis of inflammatory bowel disease (IBD), that is, Crohn's disease and ulcerative colitis. In particular, Crohn's disease is considered to be a T helper type 1 (Th1)-shifted disease. Chemokines and their receptors are involved in various immune responses including Th1- and Th2 responses. In this study, we analyzed chemokines and their receptors by immunohistochemistry, using frozen sections derived from 33 patients with Crohn's disease and 24 with ulcerative colitis. In inflamed mucosa, small mononuclear cells predominantly expressed CCR5 and CXCR3, the receptors selectively expressed on Th1 cells, without significant differences between Crohn's disease and ulcerative colitis. We then focused on the noncaseating granulomas that are characteristic of Crohn's disease. Granuloma cells, observed in all the layers of intestinal tissues, were positive for RANTES/CCL5 protein along their cell membranes. Lymphocytes surrounding granulomas were mostly CCR5+ and CXCR3+ T cells with CD4+ and CD8+ cells at similar frequencies. Granuloma cells were positive for RANTES mRNA by in situ hybridization. By contrast, lymphoid aggregates in Crohn's disease and lymphoid follicles in the normal intestinal mucosa were characterized by abundant B cells, a predominance of CD4+ T cells over CD8+ T cells, and low frequencies of cells expressing CCR5 or CXCR3. Together with the notion that granuloma cells are possible antigen-presenting cells, our results suggest that the noncaseating granulomas could be one of the crucial sites of Th1-shifted immune responses in Crohn's disease.