Analysis of regulatory T cell associated forkhead box P3 expression in the lungs of patients with sarcoidosis.

In pulmonary sarcoidosis, the typical T helper 1-mediated immune response in the lungs has been proposed to be co-ordinated by regulatory T cells; however, their exact role needs to be clarified. We used real-time polymerase chain reaction to study genes involved in regulatory T cell functions in CD4+ T cells isolated from bronchoalveolar lavage fluid (BALF) of patients (n = 24) and healthy subjects (n = 7). The genes included the transcription factor forkhead box P3 (FoxP3), interleukin (IL)-10, transforming growth factor-beta1 and chemokine receptor 2 (CCR2). The same genes were also studied in isolated BALF CD4+ T cell receptor AV2S3+ and AV2S3(-) T cells of patients with lung-restricted AV2S3 T cell expansions (n = 12). Intracellular staining of the FoxP3 protein was performed additionally in 14 patients and nine healthy subjects. mRNA expression of FoxP3, CCR2 and IL-10 was decreased significantly in BALF CD4+ T cells of patients. Flow cytometric analysis of CD4+ T cells also demonstrated a decreased frequency of FoxP3+ cells in the BALF and blood of sarcoidosis patients as well as a reduced intensity (mean fluorescence intensity) of FoxP3 expression in BALF FoxP3+ cells of patients. BALF CD4+AV2S3+ T cells expressed significantly lower levels of FoxP3 and CCR2 mRNA versus BALF CD4+AV2S3- T cells. The main conclusion of our study is that there is a reduced expression of regulatory T cell associated genes in BALF CD4+ T cells in sarcoidosis. In addition, our data suggest an effector function of AV2S3+ lung-accumulated T cells in sarcoidosis.

Changes in immune regulation in response to examination stress in atopic and healthy individuals.

BACKGROUND: Stress can aggravate the allergic inflammation, but determinants of disturbed immune regulation are largely unknown. OBJECTIVE: To determine systemic immunological, local inflammatory and functional airway responses to stress in healthy and atopic individuals. METHODS: Forty-one undergraduate students, 22 with allergy of whom 16 had asthma, and 19 healthy controls, were studied in a low-stress period and in association with a large exam. Subjects completed questionnaires on stress and health behaviours, underwent lung function tests, bronchial methacholine challenge, measurements of exhaled nitric oxide and urine cortisol. Blood cells were phenotyped, and cytokines from mononuclear blood cells were analysed. RESULTS: Perceived stress and anxiety increased in both groups during the exam period while cortisol increased only in the atopy group. Cytokine production decreased broadly in response to stress in both groups, which was paralleled by an increase in the proportion of regulatory T cells (CD4(+)CD45RO(+)CD25(bright)). Interestingly, atopic individuals, but not controls, reacted with a decreased T-helper type 1/T-helper type 2 (Th1/Th2) ratio and a decrease in natural killer (NK) cell numbers in response to stress. In control subjects only, exhaled nitric oxide decreased and forced expiratory volume in one second increased during stress. CONCLUSION: Atopic and non-atopic subjects shared some immune changes in response to stress, such as a dramatic decline in cytokines and an increase in the number of regulatory T cells in peripheral blood. However, other stress-induced immune changes were unique to atopic individuals, such as a skewed Th1/Th2 ratio and reduced NK cell numbers, indicating that some pathogenic mechanisms in atopics may be more strongly affected by stress than others.

Organic dust-induced interleukin-12 production activates T- and natural killer cells.

Exposure in swine confinement buildings causes intense airway inflammation and lymphocyte activation, as assessed by bronchoalveolar lavage. To further clarify the T-cell activation, the present in vitro study focused on intracellular cytokine production following exposure to organic dust from swine houses. Whole blood from healthy donors was incubated with swine dust, phytohaemagglutinin (positive control) or Roswell Park Memorial Institute 1640 medium (negative control), and the production of intracellular interferon (IFN)-gamma, tumour necrosis factor (TNF)-alpha, interleukin (IL)4 and IL-12 was analysed by flow cytometry. Cells were double stained for specific cell surface markers on T-cells (CD3+, CD4+, CD8+), natural killer (NK) cells (CD56+ CD16+) and monocytes (defined as CD14+ cells). Following 1 h of incubation of whole blood with swine dust, CD14+ cells produced high levels of TNF-alpha and IL-12, whereas CD3+, CD4+ and CD8+ T-cells and CD56+ CD16+ NK cells required a longer incubation time (22 h) to produce IFN-gamma and TNF-alpha. When antibodies that block the IL-12 receptor were added to whole blood incubated with swine dust, NK cell production of IFN-gamma was attenuated and CD69 expression on CD3+ cells decreased. In conclusion, this study indicates that swine dust can, at least in part, stimulate phagocytic cells to activate natural killer cells and T-lymphocytes through the production of interleukin-12.

Lymphocyte activation after exposure to swine dust: a role of humoral mediators and phagocytic cells.

Exposure to swine dust causes intense airway inflammation with multifold increase in inflammatory cells and secretion of pro-inflammatory cytokines. This in vitro study focuses on the swine-dust activation of lymphocytes in whole blood, in phagocyte-depleted whole blood and in peripheral blood mononuclear cells (PBMC), in order to investigate whether phagocytic cells and/or soluble mediators are involved in the activation of T-cells following exposure to organic dust from a swine confinement house. T-cell activation was analysed by flow cytometry with double staining for CD3 and the activation marker CD69. Swine dust (50 microg) incubated (24 h) with heparinized whole blood was shown to activate 27.6% of the T-cells, while swine dust incubated with whole blood depleted from phagocytic cells or PBMC only activated 4.5%, and 4.8% of the T-cells, respectively. Plasma separated from whole blood preincubated with swine dust for 24 h stimulated as much as 32.4% of PBMC T-cells and contained high levels of interleukin (IL)-12 (14 pg x mL) and interferon (IFN)-gamma (2284 pg x mL(-1)), while plasma from PBMC incubated with swine dust contained low levels of IL-12 (2 pg x mL(-1)) and IFN-gamma (196 pg x mL(-1)). This study demonstrates that activation of T-cells by organic dust from a swine confinement building seems to require phagocytic cells, most likely acting through the release of soluble mediators. Also, conditioned plasma from swine-dust exposed whole blood, which was capable of activating T-cells, contained high concentrations of interleukin-12 and interferon-y.

T-cell activation by organic dust in vitro.

Inhalation of swine dust causes intense airway inflammation with a multifold increase of inflammatory cells and lymphocyte activation as assessed by bronchoalveolar lavage. To further investigate the mechanism for lymphocyte activation the present in vitro study focuses on the lymphocyte response to swine dust in whole blood. Various concentrations of phytohaemagglutinin (PHA) (final concentrations: 3.16, 10.0, 3.16 and 100 microg ml(-1)) and swine dust (final concentrations: 10.0, 31.6, 100 and 316 microg ml(-1)) were added to heparinized whole blood from healthy donors. The blood samples were incubated in duplicate, using the homologous unstimulated blood as control, for 4, 24, 48 and 72 h in a water bath at 37 degrees C. The cells were stained with fluorochrome conjugated monoclonal antibodies. For analysis of T-cell activation CD3 was double-stained together with the activation markers CD69, CD25 and HLADR. Cell count percentages were analysed by flow cytometry. Soluble IL-2sRalpha in plasma was analysed using commercial sandwich ELISA technique. At baseline CD69, CD25 and HLA-DR were expressed in < 1%, approx 5% and < 1% of the T-cells respectively. We found a dose response relationship between swine dust exposure and the expression of all three T-cell activation markers which appeared at different time-points. Maximal expression of CD69 (8%, P<0.05) and CD25 (15%, P<0.001) was found after 24h of activation. HLA-DR was significantly expressed after 48h (8%) and maximally expressed after 72 h of activation (13%, P<0.05). The soluble IL-2sRalpha in plasma was maximally expressed after 24-48 h (1200 pg ml(1) and 1500 pg ml(-1), respectively. In conclusion, T-cells were activated by swine dust in vitro. Thus, our previous findings of T-cell activation following swine dust exposure, in vivo may be an effect of the dust either directly on T-cells or on other cells which in turn contribute to the T-cell activation.

Inhalation of cold air increases the number of inflammatory cells in the lungs in healthy subjects.

Prolonged exposure to cold air may induce a chronic asthma-like condition in healthy subjects as has been demonstrated in cross-country skiers. In the present controlled study, our aim was to elucidate further the link between cold air exposure and airway inflammation by assessing the cellular influx and mediator levels within the airways following acute exposure to cold air. Bronchoalveolar (BAL) and nasal lavages were performed after exposure to cold air (-23 degrees C) and normal indoor air (+22 degrees C) during a light, intermittent work for 2 h in a cross-over design in eight healthy, nonsmoking, subjects. Analyses of inflammatory cell number, cell activation markers, pro-inflammatory cytokines, albumin and interleukin (IL)-8 in lavage fluids were performed. The number of granulocytes and of alveolar macrophages in BAL fluid was significantly higher after cold air exposure (p<0.05). No increase in BAL fluid lymphocytes and no signs of lymphocyte activation in BAL fluid were found. The concentration of IL-8 was unchanged. There were no signs of granulocyte activation (myeloperoxidase, eosinphilic cationic protein) in BAL fluid. Cold air did not influence the number of inflammatory cells or the concentration of albumin and IL-8 in nasal lavage fluid. In conclusion, exposure to cold air induces an increased number of granulocytes and macrophages in the lower airways in healthy subjects without influencing other inflammatory indices such as cellular activation, plasma leakage and pro-inflammatory cytokines. These findings support the hypothesis that cold air could be of pathogenetic importance in the asthma-like condition previously found in cross-country skiers.

Budesonide but not terbutaline decreases phagocytosis in alveolar macrophages.

Alveolar macrophages are the most common cells in bronchoalveolar lavage fluid. The macrophages participate in the inflammatory response and defence of the airways by secretion of mediators and by phagocytizing foreign particles such as bacteria and viruses. beta-Agonists and glucocorticosteroids are the most frequently used drugs in asthma. Alveolar macrophages have beta 2-adrenoceptors on their surface but the functional role of these receptors is unknown. Glucocorticosteroids interact with mediator release from macrophages. However, nothing is known about the effects of those drugs on the phagocytic capacity of alveolar macrophages. Therefore, the present study has investigated phagocytosis of alveolar macrophages from nine healthy volunteers after incubation with a beta 2-agonist, terbutaline (10(-8), 10(-6) and 10(-4) M) and a glucocorticosteroid, budesonide (10(-9), 10(-7) and 10(-5) M). Alveolar macrophages were incubated with FITC-labelled Escherichia coli, and the drugs and phagocytosis was assessed by flow cytometry. Phagocytosis was measured as the proportion of phagocytizing cells and mean fluorescence intensity (MFI). MFI was highly correlated with phagocytized E. coli per cell assessed by fluorescence microscopy (r = 0.996). The proportion of phagocytizing macrophages (control) was [median (25th-75th) percentiles] 46% (40-63) and 29% (18-60), and MFI were 174 (154-205) and 122 (90-271) in the terbutaline and budesonide experiments, respectively. Terbutaline did not affect the phagocytosis significantly, while budesonide decreased the phagocytic capacity (percent phagocytizing cells and MFI) in a dose-dependent manner (P < 0.01). At the highest budesonide concentration (10(-5) M), phagocytosis was approximately half of the control situation. In conclusion, this in vitro study indicate that a glucocorticosteroid decreases phagocytosis in alveolar macrophages in a concentration that may be relevant in the airway lining fluid. Further investigations regarding the effect on other micro-organisms and in vivo effects are necessary to further elucidate these findings.

Increased number of activated lymphocytes in human lung following swine dust inhalation.

Inhalation of swine dust causes intense alveolar inflammation, with recruitment of inflammatory cells, predominantly neutrophils, but also alveolar macrophages and lymphocytes. The present study focuses on the lymphocyte response to inhaled swine dust. Twenty four healthy, nonsmoking, nonallergic subjects were exposed to swine dust for 3 h in a swine confinement building. Bronchoalveolar lavage (BAL) was performed before and 24 h after the start of exposure, and blood samples were drawn before, and at 7 and 24 h after exposure. Total and differential cell counts were carried out. Monoclonal antibodies recognizing T-cells, T-cell subsets, T-cell activation markers, and B-cells were analysed by flow cytometry. The number of granulocytes increased more than 50 times and alveolar macrophages and lymphocytes increased two- to three-fold in BAL fluid. The exposure did not alter the proportion of T-cells but increased the number of activated T-cells in BAL fluid. The interleukin-2 (IL-2) receptor (CD25), human leucocyte antigen-DR (HLA-DR) major histocompatibility complex (MHC) class II and the early activation marker CD69 were expressed by 8.4% (25-75th percentiles 6.4-9.6%), 9.9% (8.2-21.6%) and 22.0% (18.1-24.3%) of the lymphocytes prior to exposure, and 11.6% (9.0-16.4%) (p < 0.01), 18.8% (12.9-30.4%) (p < 0.01) and 42.1% (38.4-47.3%) (p < 0.05), respectively, after the exposure. In peripheral blood, the concentration of T-cells decreased after exposure and B-cells increased slightly but significantly. The ratio naive/memory T-cells (CD45RA/RO) did not change in blood. In conclusion, 3 h of swine dust inhalation led to an influx of lymphocytes into the lower airways and increased expression of lymphocyte activation markers on the cell surface in previously unexposed subjects. The finding suggests a role for T-cells, in conjunction with other cells, in the inflammatory response to inhaled swine dust.

Renal interstitial pressure and tubuloglomerular feedback control in rats during infusion of atrial natriuretic peptide (ANP).

Atrial natriuretic peptide (ANP), injected at physiological concentrations, is known to induce both natriuresis and diuresis. It has been suggested by some investigators that these changes result from an increasing glomerular filtration rate (GFR), but others have been unable to demonstrate an increased GFR. The tubuloglomerular feedback (TGF) mechanism is an important regulator of GFR, and the sensitivity of TGF is decreased during ANP administration. Furthermore, resetting of TGF is, in most instances, related to changes in renal interstitial hydrostatic and oncotic pressures. It is also known that ANP may increase capillary permeability which may change renal interstitial pressure. The present study was performed to examine renal interstitial pressures and the TGF mechanism during ANP infusion. In accordance with previous studies, TGF sensitivity was found to be decreased. The tubular flow rate which elicited half the maximal drop in stop-flow pressure (Psf) was increased from 18.5 to 25.7 nl min-1. In contrast, ANP infusion resulted in a decreased interstitial hydrostatic pressure and an increased interstitial oncotic pressure. From previous experiments, such changes in interstitial pressures would be expected to increase TGF sensitivity. The changes in interstitial pressure cannot, therefore, directly explain the resetting of the feedback mechanism. In conclusion, the present paper shows a decreased renal net interstitial pressure after intravenous administration of ANP.

Glomerular filtration and tubular secretion of MAG-3 in the rat kidney.

Technetium-99m mercaptoacetyltriglycine (MAG-3) has recently been introduced as a new radiopharmaceutical for dynamic renal scintigraphy. To elucidate the mechanism of renal excretion, micropuncture experiments were performed in rat kidneys for direct measurements of glomerular filtration and tubular secretory capacity. Fluid of Bowman space was collected from superficial glomeruli and analyzed for its contents of [99mTc]MAG-3, [125I]hippurate and [3H]inulin during constant infusion of these compounds. The ratio of activity of ultrafiltrate to that of arterial plasma was 0.23 for MAG-3, 0.68 for hippurate and 1.04 for inulin which demonstrates that the filtrated amount of MAG-3 is only 23% of that of inulin, presumably because of higher plasma protein binding which was also measured in vitro and found to be 80 +/- 1.5% for MAG-3 and 32 +/- 2% for [125I]hippurate. Proximal and distal tubules were also micropunctured and their tubular fluid as well as the final urine analyzed for the activity of hippurate and MAG-3. The tubular fluid to plasma ratio values along the nephron and in the final urine were all lower for MAG-3 than for hippurate, indicating a lower secretory capacity. From measurements of whole renal clearance, GFR and plasma protein binding the filtered amount of MAG-3 was 0.26 and of hippurate 0.87 ml/min.g kidney weight (p less than 0.001) and the secreted amount 2.01 and 2.38 ml/min.g kidney weight (p less than 0.05), respectively. We conclude that MAG-3 is predominantly excreted by tubular secretion and that the lower renal clearance of MAG-3 as compared with that of hippurate is a result both of a substantially decreased glomerular filtration and of a lower tubular secretion.

Back-leak of pelvic urine to the bloodstream.

The aim of the present investigation was to measure the back-leak of pelvic urine to the blood circulation. In normopenic hydronephrotic, dehydrated hydronephrotic and dehydrated control kidneys the back-leak was estimated from a servocontrolled machine which regulated infused saline to keep a present pelvic pressure constant. The disappearance of fluid from the renal pelvis could be measured at different pressure levels, and a pressure-dependent outflow of fluid was found. From these measurements a back-leak conductance could be calculated which proved to be independent of pressure. In the lower pressure range (15-20 mmHg) there was a significantly lower conductance in the dehydrated controls compared with the dyhydrated hydronephrotic kidneys, while in the higher pressure range (25-30 mmHg) no difference was found. From electron microscopical studies the pyelorenal back-leak of fluid in both hydronephrotic and control animals seemed to be most pronounced in the fornix region, as documented by a heavy presence of horseradish peroxidase in the intracellular spaces there. Other experiments with radioactively labelled inulin, which was injected into the pelvic cavity, indicated that most of the back-leak occurred via the renal blood vessels and not through the lymphatic system. The importance of this back-leak was evident from the measurements of the total kidney glomerular filtration rate (GFR) at a slightly increased pelvic pressure, where some of the urine with radioactive tracer flows back to circulation. The back-leak of pelvic urine could also affect the concentration mechanism by removing diluted urine which had flowed over the renal papilla, and through water and urea diffusion increased papillary interstitial osmolarity.

Measurements of macula densa cell volume changes in isolated and perfused rabbit cortical thick ascending limb. I. Isosmotic and anisosmotic cell volume changes.

A method to measure time-dependent volume changes in macula densa (MD) cells is described. Cell volume is calculated from cell height measurements for which an image-splitting eyepiece is used. This paper presents the results of experiments designed to investigate the behaviour of the macula densa cells in anisosmotic media, to evaluate the cell volume response to sudden decreases in luminal or peritubular osmolarity and to examine the effect of different luminal NaCl concentrations on the steady-state isosmotic cell volume and on the regulatory volume response to anisosmotic media. The result showed that induced alteration in macula densa cell volume did not change macula densa surface area, but only cell height. The mean control cell height was 13.3 microns +/- 0.4. When MD cells were exposed to a luminal osmolarity of 180 mosM (control 300 mosM) they swelled only to 1.19 +/- 0.02 of the control value and with furosemide present to 1.13 +/- 0.02 or with low NaCl to 1.13 +/- 0.01. While after 5 min of exposure values were 1.15 +/- 0.03, 0.99 +/- 0.02 and 1.02 +/- 0.02, respectively. Addition of furosemide (10(-4) M) to the luminal perfusate (300 mosM) resulted in a rapid decrease in cell height to 0.8 +/- 0.02 in relation to control. When furosemide was removed cell volume was restituted (0.98 +/- 0.03). When luminal perfusate was replaced by mannitol and (12 mM Na+, 7 mM Cl-) cell volume decreased to 0.83 +/- 0.02 of the control value.(ABSTRACT TRUNCATED AT 250 WORDS)

Measurements of macula densa cell volume changes in isolated and perfused rabbit cortical thick ascending limb. II. Apical and basolateral cell osmotic water permeabilities.

Cortical thick ascending limbs containing macula densa plaques were dissected and perfused in vitro. Macula densa cell osmotic water permeability of the apical and basolateral membranes were measured by setting up osmotic steps across them in less than 0.1 s and following the ensuing time-dependent cell volume changes. The results of this study are in accordance with the view that the macula densa cells have a relatively low permeability to water. Apical and basolateral osmotic water permeabilities are 2.4 and 30.4 x 10(-4) cm3 s-1 osMolar-1 cm-2 basement membrane area, respectively. No infoldings were taken into consideration. These water permeabilities were not affected by maximal and supramaximal doses of vasopressin. This paper provides new insight into the physiological behaviour of this small, and almost inaccessible, sensing epithelial disc of cells which improves the understanding of its participation in the juxtaglomerular feedback response.

Tubuloglomerular feedback in animals with unilateral, partial ureteral occlusion.

Previous experiments have shown that the sensitivity of the tubuloglomerular feedback system (TGF) is reduced by volume expansion in normal rats. This reduction in sensitivity is probably mediated by changes in the renal interstitial pressure. The present study was designed to investigate the TGF control system during volume expansion in rats with chronic, partial ureteral occlusion--hydronephrosis. Hydronephrosis was induced on the left or right side according to the method described by Ulm and Miller, in weanling Sprague-Dawley rats three weeks old. Three to six weeks later the rats were prepared for whole kidney and micropuncture experiments. Sham-operated animals were used as controls. Net interstitial pressure (that is, interstitial hydrostatic pressure minus interstitial oncotic pressure) was higher in the hydronephrotic, volume expanded animals than in the volume expanded controls. From findings in earlier investigations this increase in interstitial pressure would have been expected to reduce TGF sensitivity but this sensitivity was increased in the hydronephrotic kidneys, as indicated by a reduction in the turning point, the tubular flow rate at which 50% of the maximal stop-flow pressure response was observed (14.4 nl/min, sham-operated control 33.4 nl/min). Strong activity of the TGF mechanism was also indicated by a large proximal-distal difference in the single-nephron glomerular filtration rate (11.9 nl/min versus 3.3 nl/min in sham-operated controls) in the hydronephrotic kidney during volume expansion. Thus, in hydronephrotic kidneys in the latter condition the TGF mechanism was highly sensitive and activated to reduce the glomerular filtration rate. This mechanism may protect the diseased kidney from high intrapelvic pressures which otherwise could damage the kidney during saline volume expansion.

Renal and extrarenal handling of a new imaging compound (99m-Tc-MAG-3) in the rat.

We investigated the renal clearance, the extrarenal clearance, the biodistribution, the plasma extraction ratio and the imaging quality on a scintillation camera of a new substance: 99mTc-mercaptoacetyltriglycine (MAG-3) in rats. Simultaneously 125I-hippurate (OIH) and 51Cr-EDTA were used as reference substances. In scintillation camera studies, 123I-hippurate served as reference. MAG-3 was prepared by kit labelling without further purification, as it would be used in clinical studies. However in three separate rats, HPLC purified MAG-3 was used. High renal clearance and extraction ratio was found but these were less than that for OIH (1.95 ml/min per 100 g BW vs 2.76 ml/min per 100 g BW and 64% vs 85% respectively). The extrarenal clearance was higher for MAG-3 than for OIH (0.20 ml/min per 100 g BW vs 0.04 ml/min per 100 g BW), presumably because of bile excretion. HPLC purification increased the renal excretion of MAG-3: the clearance was 2.53 ml/min per 100 g BW, the extraction fraction 75%, but the extrarenal clearance was unchanged. The imaging quality was comparable to that of 123I-hippurate in early pictures, but extrarenal activity, presumably representing bile, was observed in late pictures. We conclude that MAG-3 has some potential for the replacement of OIH but clinical studies are necessary to test whether the same conditions and limitations exist in humans.

Renal interstitial pressure measured in the subcapsular space using an in vivo oncometer.

Continuous measurements of renal interstital pressure are of importance for several reasons. The present paper describes an in vivo oncometer developed for this purpose. A piece of dialysis tubing was filled with a 0.15 M NaCl solution containing 5 g albumin 100 ml-1. For detecting the hydrostatic pressure inside the tubing, a thin catheter and a silver wire were inserted into it and both ends of the tubing were sealed with glue. The catheter and the silver wire were connected to a servo-nulling pressure device. The oncometer was then placed under the renal capsule. The pressure inside the dialysis tubing was pi onc+Psc-pi sc, and since pi onc was known, the net interstitial pressure (Pnet, i.e. Psc-pi sc) could be measured continuously in the subcapsular space. Measurements were made during (I) intravenous bolus injection of 2 ml of 0.9% NaCl, (2) saline expansion of 5% of body weight, and (3) elevation of renal venous pressure to 20 mmHg by clamping the renal vein. In the control situation, Pnet averaged-3.3 mmHg, a value in good accordance with findings in earlier studies in which the hydrostatic and oncotic pressure components have been measured separately. Following bolus injection of fluid, Pnet increased transiently by 2.6 mmHg, whereas volume expansion produced a permanent increase in Pnet of almost the same magnitude. During elevated renal venous pressure Pnet was unaffected, except for a minor increase on clamping and a minor decrease on release of the clamp. The results show that reproducible and accurate measurements of Pnet in the renal subcapsular space can be made with an in vivo oncometer.