Jan Wahlström

PDE4D plays a critical role in the control of airway smooth muscle contraction

The airways of mice deficient in the cAMP phosphodiesterase PDE4D gene are refractory to muscarinic cholinergic stimulation. This study was undertaken to determine whether altered smooth muscle contractility causes the PDE4D−/− phenotype. A major disruption in contractility was observed in isolated PDE4D−/− tracheas, with a 60% reduction in maximal tension and a fivefold decrease in sensitivity to muscarinic cholinergic agonists. Conversely, responses to KCl or arginine vasopressin were unaffected. PDE4D is the predominant PDE4 form in tracheal extracts and PDE4D mRNA is expressed in smooth muscle where muscarinic binding sites are most abundant. Cyclic AMP accumulation in response to acute Gsα‐coupled receptor stimulation was increased up to fourfold in the airway of PDE4D−/− mice when compared with wild‐type. This increase in cAMP was associated with an increased sensitivity to PGE2‐induced relaxation of the PDE4D−/− tracheas. Furthermore, a blockade of prostanoid accumulation in PDE4D−/− tracheas restored the response to muscarinic cholinergic stimulation in vitro and in vivo. These results demonstrate that PDE4D plays a key role in balancing relaxant and contracting cues in airway smooth muscle, suggesting that natural mutations in the PDE4D gene have profound effects on airway tone.—Me´hats, C., Jin, S.‐L. C., Wahlstrom, J., Law, E., Umetsu, D., Conti, M. PDE4D plays a critical role in the control of airway smooth muscle contraction. FASEB J. 17, 1831–1841 (2003)

T Cell Responses to Mycobacterial Catalase-Peroxidase Profile a Pathogenic Antigen in Systemic Sarcoidosis

Sarcoidosis is a systemic granulomatous disease associated with local epithelioid granulomas, CD4+ T cells, and Th1 cytokines. The tissue Ags that drive this granulomatous inflammation are uncertain. In this study, we used IFN-γ-ELISPOT assays and flow cytometry to assess lung and blood T cell responses to the candidate pathogenic Ag, Mycobacterium tuberculosis catalase-peroxidase (mKatG) in patients with sarcoidosis from two centers. Despite differences in patient phenotypic, genetic, and prognostic characteristics, we report that T cell responses to mKatG were remarkably similar in these cohorts, with higher frequencies of mKatG-reactive, IFN-γ-expressing T cells in the blood of sarcoidosis patients compared with nontuberculosis sensitized healthy controls, and (in a subset) in greater numbers than T cells reactive to purified protein derivative. In sarcoidosis, mKatG-reactive CD4+ Th1 cells preferentially accumulated in the lung, indicating a compartmentalized response. Patients with or without Löfgren syndrome had similar frequencies of mKatG specific IFN-γ-expressing blood T cells. Circulating mKatG-reactive T cells were found in chronic active sarcoidosis but not in patients with inactive disease. Together, these results demonstrate that T cell responses to mKatG in sarcoidosis fit a profile expected for a pathogenic Ag, supporting an immunotherapeutic approach to this disease.

Phenotypic analysis of lymphocytes and monocytes/macrophages in peripheral blood and bronchoalveolar lavage fluid from patients with pulmonary sarcoidosis

BACKGROUND The granulomatous inflammation in sarcoidosis is driven by the interplay between T cells and macrophages. To gain a better understanding of this process the expression by these cells of cell surface activation markers, co-stimulatory molecules, and adhesion molecules was analysed. METHODS CD4+ and CD8+ T lymphocytes from peripheral blood (PBL) or bronchoalveolar lavage (BAL) fluid, as well as paired peripheral blood monocytes and alveolar macrophages from 27 patients with sarcoidosis were analysed by flow cytometry. RESULTS CD26, CD54, CD69, CD95, and gp240 were all overexpressed in T cells from BAL fluid compared with those from PBL in both the CD4+ and CD8+ subsets, while CD57 was overexpressed only in BAL CD4+ cells. In contrast, CD28 tended to be underexpressed in the BAL T cells. Monocyte/macrophage markers included CD11a, CD11b, CD11c, CD14, CD16, CD54, CD71, CD80 and CD86 and HLA class II. CD11a expression in alveolar macrophages (and peripheral blood monocytes) was increased in patients with active disease and correlated positively with the percentage of BAL lymphocytes. Expression of CD80 in macrophages correlated with the BAL CD4/CD8 ratio. CONCLUSIONS Our data indicate substantial activation of both CD4+ and CD8+ lung T cells in sarcoidosis. There were also increased numbers of BAL lymphocytes whose phenotypic characteristics have earlier been associated with clonally expanded, replicatively senescent cells of the Th1 type.

Identification of HLA-DR–bound peptides presented by human bronchoalveolar lavage cells in sarcoidosis

Sarcoidosis is an inflammatory disease of unknown etiology, most commonly affecting the lungs. Activated CD4+ T cells accumulate in the lungs of individuals with sarcoidosis and are considered to be of central importance for inflammation. We have previously shown that Scandinavian sarcoidosis patients expressing the HLA-DR allele DRB1*0301 are characterized by large accumulations in the lungs of CD4+ T cells expressing the TCR AV2S3 gene segment. This association afforded us a unique opportunity to identify a sarcoidosis-specific antigen recognized by AV2S3+ T cells. To identify candidates for the postulated sarcoidosis-specific antigen, lung cells from 16 HLA-DRB1*0301pos patients were obtained by bronchoalveolar lavage. HLA-DR molecules were affinity purified and bound peptides acid eluted. Subsequently, peptides were separated by reversed-phase HPLC and analyzed by liquid chromatography-mass spectrometry. We identified 78 amino acid sequences from self proteins presented in the lungs of sarcoidosis patients, some of which were well-known autoantigens such as vimentin and ATP synthase. For the first time, to our knowledge, we have identified HLA-bound peptides presented in vivo during an inflammatory condition. This approach can be extended to characterize HLA-bound peptides in various autoimmune settings.

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‐β1 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.

Reduced Th1 response in the lungs of HLA-DRB1*0301 patients with pulmonary sarcoidosis

To investigate why human leukocyte-associated antigen-DRB1*0301 (HLA-DRB1*0301) positive Scandinavian patients have a better prognosis than HLA-DRB1*0301 negative patients, the present authors examined patterns of cytokine expression in bronchoalveolar lavage (BAL) cells and BAL fluid (BALF) from patients with pulmonary sarcoidosis and controls. Using real-time PCR, the mRNA expression of selected cytokines in BAL cells from newly diagnosed, untreated nonsmoking patients (n = 25) and controls (n = 11) was quantified. Cytokine protein levels in BALF from patients (n = 34) and controls (n = 11) were assessed using cytometric bead array. The patients were evaluated and stratified into two subgroups: HLA-DRB1*0301 positive (all with an acute onset) and HLA-DRB1*0301 negative (all with an insidious onset). When comparing patients and controls, BAL cells of the patients expressed significantly higher levels of interferon (IFN)-γ and interleukin (IL)-10 mRNA. There were significantly decreased IFN-γ and tumour necrosis factor (TNF)-α mRNA levels, and a tendency toward higher levels of transforming growth factor-β1 mRNA in HLA-DRB1*0301 positive compared with HLA-DRB1*0301 negative patients. Protein levels of IL-1β, IL-2, IL-6, IL-12p70 and TNF-α in BALF were significantly higher in patients. HLA-DRB1*0301 positive patients exhibited tendencies to lower levels of most cytokines in BALF. In conclusion, the present data show a reduced expression of T-helper cell type-1 cytokines in human leukocyte-associated antigen-DRB1*0301 positive patients, which may relate to their good prognosis.

Lung restricted T cell receptor AV2S3+ CD4+ T cell expansions in sarcoidosis patients with a shared HLA-DRbeta chain conformation.

Background: Sarcoidosis is a systemic disease of unknown aetiology frequently affecting the lungs. CD4+ T cells, in particular, accumulate in the lungs, implicating them in the pathogenesis of the disease. Methods: T cell receptor (TCR) variable (V) gene expression on bronchoalveolar lavage (BAL) fluid T cells and the HLA DR alleles of 121 Scandinavian patients with sarcoidosis was determined. Results: As expected from our previous results, almost every DRB1*0301 (i.e. DR17) positive patient (67/69) had significantly increased numbers of AV2S3+ CD4+ T cells in the BAL fluid but normal levels in peripheral blood (that is, lung restricted expansions) compared with only six of 52 DRB1*0301 negative patients. Detailed genotypic HLA analysis showed that these six DRB1*0301 negative patients with lung restricted AV2S3+ T cell expansions had another HLA allele in common—the HLA-DRB3*0101 allele (also called DR52a)—which was not found in any other DRB1*0301 negative patient. A new group of sarcoidosis patients was therefore identified, characterised by a strict correlation between a distinct HLA allele and lung accumulated T cells expressing a particular TCR V segment. Furthermore, the HLA-DRB1*0301 and HLA-DRB3*0101 encoded molecules showed similarities, with identical amino acid sequences in regions important for antigen binding which may enable them to bind and present the same or similar antigenic peptides. Conclusions: HLA-DRB3*0101 as well as DRB1*0301 positive sarcoidosis patients may have the capacity to present specific sarcoidosis associated antigens in such a way that AV2S3+ CD4+ T cells are stimulated preferentially, generating lung restricted AV2S3+ T cell expansions.

Allergen-Specific MHC Class II Tetramer+ Cells Are Detectable in Allergic, but Not in Nonallergic, Individuals

Allergen-specific cells are present in very low frequency in peripheral blood of humans, and differ in function in allergic and nonallergic individuals. We report in this study that soluble class II MHC tetramers can be used to directly identify and study such allergen epitope-specific CD4+ T cells in humans. We identified the major antigenic epitope of rye grass allergen Lol p 1 in HLA-DRB1*0401 individuals using HLA-DR*0401 transgenic mice and peripheral blood cells from HLA-DR*0401 individuals. Using DRB1*0401 tetramers loaded with this major epitope of Lol p 1, we detected allergen-specific CD4+ T cells in the peripheral blood of DRB1*0401 rye grass allergic individuals after ex vivo expansion with allergen. These tetramer-positive cells produced IL-4, but little IFN-γ. In contrast, we were unable to detect rye grass tetramer-positive cells in cultures from HLA-DR*0401 nonallergic individuals, even after expansion with IL-2. Thus, our results suggest that rye grass allergen-specific T cells in DR*0401 nonallergic subjects are present at very low levels (e.g., because of deletion or suppression), differ in a fundamental way in their requirement for ex vivo expansion (e.g., they may be anergic), or use TCRs distinct from those of allergic individuals. Thus, analysis using DRB1*0401 tetramers loaded with a major epitope of Lol p 1 indicates that allergen-specific CD4+ T cells in nonallergic individuals are distinct from those in allergic subjects.

Different inflammatory cell pattern and macrophage phenotype in chronic obstructive pulmonary disease patients, smokers and non-smokers

Smokers exhibit airway inflammation and increased number of alveolar macrophages (AM), but not all develop chronic obstructive pulmonary disease (COPD). We hypothesized that AMs in COPD patients have an altered functional capacity mirrored in a different phenotype. Sixteen steroid‐naive COPD patients [forced expiratory volume in 1 s (FEV1) < 70% of predicted] underwent bronchoalveolar lavage (BAL). Age‐ and smoking‐matched non‐obstructive smokers (n = 10) and healthy non‐smokers (n = 9) served as controls. Nine COPD patients had a BAL cell yield sufficient for flow cytometry analysis, where expression of AM cell surface markers reflecting various functions was determined. AMs from COPD patients showed decreased expression of CD86 (co‐stimulation) and CD11a (adhesion) compared to smokers’ AMs (P < 0·05). Furthermore, smokers’ AMs showed lower (P < 0·05) expression of CD11a compared to non‐smokers. AM expression of CD11c was higher in the COPD and smokers groups compared to non‐smokers (P < 0·05). The expression of CD54 (adhesion) was lower in smokers’ AMs compared to non‐smokers (P < 0·05), whereas CD16 was lower (P < 0·05) in COPD patients compared to non‐smokers. The AM expression of CD11b, CD14, CD58, CD71, CD80 and human leucocyte antigen (HLA) Class II did not differ between the three groups. The AM phenotype is altered in COPD and further research may develop disease markers. The lower AM expression of CD86 and CD11a in COPD implies a reduced antigen‐presenting function. Some alterations were found in smokers compared to non‐smokers, thus indicating that changes in AM phenotype may be associated with smoking per se. The functional relevance of our findings remains to be elucidated.

Coeliac disease and risk of tuberculosis: a population based cohort study

Background: Coeliac disease (CD) is an autoimmune disease often characterised by malnutrition and linked to a number of complications such as an increased risk of lymphoma, adverse pregnancy outcome, and other autoimmune diseases. Tuberculosis (TB) affects a large proportion of the world population and is more common in individuals with malnutrition. We investigated the risk of TB in 14 335 individuals with CD and 69 888 matched reference individuals in a general population based cohort study. Methods: Cox proportional hazards method was used to calculate the risk of subsequent TB in individuals with CD. In a separate analysis, the risk of CD in individuals with prior TB was calculated using conditional logistic regression. Results: CD was associated with an increased risk of subsequent TB (hazard ratio (HR) 3.74, 95% CI 2.14 to 6.53; p<0.001). Similar risk estimates were seen when the population was stratified for sex and age at CD diagnosis. Individuals with CD were also at increased risk of TB diagnosed in departments of pulmonary medicine, infectious diseases, paediatrics, or thoracic medicine (HR 4.76, 95% CI 2.23 to 10.16; p<0.001). The odds ratio for CD in individuals with prior TB was 2.50 (95% CI 1.75 to 3.55; p<0.001). Conclusions: CD is associated with TB. This may be due to malabsorption and lack of vitamin D in persons with CD. Individuals with TB and gastrointestinal symptoms should be investigated for CD.