Multiparameter phenotyping of T-cell subsets in distinct subgroups of patients with pulmonary sarcoidosis.

OBJECTIVES: Sarcoidosis is an inflammatory disorder in which elevated numbers of activated T cells are found in the lung. HLA-DRB1*0301(pos) (DR3(pos) ) patients are characterized by good prognosis and an accumulation of lung CD4(pos) T cells expressing the T-cell receptor (TCR) gene segment AV2S3. Our aim was to phenotype lung and blood T-cell subsets in distinct patient groups to better understand the function of these subsets. DESIGN: Bronchoalveolar lavage (BAL) fluid and whole blood were obtained from a total of 22 patients with sarcoidosis, of whom 11 were DR3(pos) . Using eight-colour flow cytometry, phenotyping of T cells was performed with regard to CD3, CD4, CD8, CD25, CD27, CD45RO, CD57, CD69, CD103, FOXP3 and TCR AV2S3. RESULTS: DR3(pos) patients had fewer FOXP3(pos) (regulatory) CD45RO(pos) (memory) BAL T cells than DR3(neg) patients. Fewer AV2S3(pos) T cells were FOXP3(pos) , compared with AV2S3(neg) cells, thus indicating an effector function and not a regulatory role for this subset. Fewer lung and blood AV2S3(pos) T cells were CD25(pos) CD27(pos) , and more were CD25(neg) CD27(neg) and CD69(pos) , compared with AV2S3(neg) T cells, indicating a higher degree of differentiation and activation in both compartments. CONCLUSION: Our main findings were a lower proportion of regulatory T cells in DR3(pos) patients, together with the accumulation of AV2S3(pos) T cells with a highly activated effector phenotype in the lungs of these patients. This may provide for efficient elimination of a harmful antigen in DR3(pos) patients and could thus help to explain the spontaneous recovery typically seen in these patients.

Increased levels of nerve growth factor in the airways of patients with sarcoidosis.

OBJECTIVES: Nerve growth factor (NGF) is a potent neuronal growth factor with inflammatory properties that recently has been proposed to be of importance in airway pathology. A role for NGF in the inflammatory granulomatous lung disease sarcoidosis is not well elucidated. The aims of this study were to investigate the secreted levels of NGF in bronchoalveolar lavage fluid (BALF) from sarcoidosis patients compared with patients with resolved disease, patients with another granulomatous disease--chronic beryllium disease (CBD)--and healthy subjects and also to investigate the relationship between NGF levels and markers of inflammation. METHODS AND RESULTS: NGF levels in BALF from 56 patients with active sarcoidosis (22 with Löfgren's syndrome), nine subjects with resolved sarcoidosis, six patients with CBD, and 31 healthy subjects were compared. A 10-fold elevation of NGF levels was found in patients with active sarcoidosis compared with subjects with clinically resolved sarcoidosis, patients with CBD and healthy subjects. In sarcoidosis patients, positive correlations between concentrations of NGF and lymphocytes, eosinophils and interferon-gamma, interleukin (IL)-4, IL-10, IL-12 were found. CONCLUSIONS: We demonstrate that secreted levels of NGF are markedly enhanced in the airways in active pulmonary sarcoidosis. Furthermore, a relationship between NGF and pulmonary inflammation in sarcoidosis is supported.

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)-gamma and interleukin (IL)-10 mRNA. There were significantly decreased IFN-gamma and tumour necrosis factor (TNF)-alpha mRNA levels, and a tendency toward higher levels of transforming growth factor-beta1 mRNA in HLA-DRB1*0301 positive compared with HLA-DRB1*0301 negative patients. Protein levels of IL-1beta, IL-2, IL-6, IL-12p70 and TNF-alpha 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.

Ageing before mating and quinacrine ameliorate the expression of abnormal oocyte (abo) in homozygous Drosophila melanogaster females.

Several studies have shown that the characteristically skewed sex ratio among the progeny of abo homozygous females, derived from heterozygous stocks and mated to attached XY males, can be modified during homozygous stock-keeping. This amelioration seems to have a complex mechanistic background, and both loss of the blood transposon from chromosome 2, where abo is located, and amplification of a specific heterochromatic element (ABO) have been suggested to work in this direction. There is also an increased frequency of non-disjunction associated with abo, beside the poor recovery of X0 males. Experiments were performed to see if there was a coordinated loss of both phenotypic expressions during homozygous stock-keeping and if non-disjunction was amenable to modification by quinacrine. We found an unexpected spontaneous amelioration of the phenotypic expressions of abo despite heterozygous stock-keeping. The spontaneous amelioration of non-disjunction and male lethality under heterozygous condition was coordinated while the process initiated by homozygosity slowly decreased non-disjunction and rapidly increased male recovery over generations, which may point to a mechanistic difference between the amelioration processes. Quinacrine was found to ameliorate the skewed sex ratio but did not affect non-disjunction. In these experiments larval and adult treatment, respectively, were employed and the respective controls revealed that also ageing before mating significantly increased male recovery and reduced non-disjunction. Ageing before mating and quinacrine seemed to act additively on male recovery, suggesting independent action, while interaction could be suspected between quinacrine and some ameliorating factor associated with brood. Some of the results also suggest that quinacrine acts indirectly and does not substitute for the abo gene product. Due to the action of quinacrine in other biological systems it is speculated that the compound compensates for a biochemical aberration in abo/abo females or their progeny, showing some relation to phospholipase activity and/or actin polymerisation state.

Studies on the role of CD43 in human B-cell activation and differentiation.

The monoclonal antibody (MoAb) B1B6 to human leucocyte sialoglycoprotein, CD43, induces aggregation of T cells and delivers progression signals early during activation of both T and B cells in the presence of primary activators of protein kinase C. In this report we further studied the role of CD43 in human B-cell activation and differentiation. About 5-10% of resting tonsillar B cells are CD43+. In the presence of TPA or antibodies to CDw40, the proportions of CD43+ cells drastically increased. The expression was optimal on day 3 of culture, when up to 80% and 50%, respectively, were CD43+. Whereas MoAb B1B6 together with TPA induced a three- to fivefold higher proliferative response as compared to TPA alone, antibodies to CDw40 did not synergize with MoAb B1B6 in B-cell proliferation. Tonsillar populations depleted of CD43+ B cells responded with lower proliferation to TPA alone or to TPA and B1B6 or anti-CDw40 antibodies. MoAb B1B6 did not affect the production of IgM or IgG as induced by pokeweed mitogen in the presence of autologous T cells, from either peripheral blood or tonsillar B cells. Neither did it affect the IgG production from the CD43+ BSF-2 sensitive Epstein-Barr virus-transformed lymphoblastoid cell line CESS. The results show that CD43 is upregulated on B cells during activation. Furthermore, CD43+ B cells are included in the population which responds to signals delivered by TPA, anti-CD43 or anti-CDw40 antibodies, and the proliferation of this population is not merely due to an expansion of the small population of CD43+ cells present among these cells. Moreover, the epitopes recognized by MoAb B1B6 are not involved in the differentiation of and ultimate Ig-secretion from activated B cells.

Enhancement of human B-cell proliferation by a monoclonal antibody to CD43.

Monoclonal antibodies (MoAb) to human leucocyte sialoglycoprotein, CD43, have been shown to deliver mitogenic signals to human T cells or to enhance T-cell proliferation induced by concanavalin A, anti-CD3 antibodies or phorbol ester. In this paper, we studied the effects of anti-CD43 MoAb B1B6 on the activation of human B cells. Anti-CD43 MoAb B1B6 was not mitogenic by itself for human B cells. However, when added together with TPA, both resting and in vivo activated tonsillar B cells, containing 5-10% and about 35% CD43+ respectively, responded with three- to fivefold higher proliferation compared to that obtained with TPA alone. A peak in the proliferative response was reached on day 3. Optimal proliferation was obtained when the antibody was present from the start of culturing. Addition of MoAb B1B6 together with a calcium ionophore, ionomycin, did not induce B-cell proliferation. Neither did mAb B1B6 sustain the growth of B cells that were already in the cell cycle, i.e. precultured with phorbol ester (PDB) and ionomycin for 3 days. The results are similar to those obtained with antibodies to CD22 and CD23 and show that early progression signals are delivered to resting B cells through CD43 in the presence of primary activators of protein kinase C.

Induction of CD43 expression during activation and terminal differentiation of human B cells.

Only a small population (25-30%) of human peripheral blood B lymphocytes expresses large sialoglycoprotein (LSGP) (CD43). However, in the presence of autologous T cells and pokeweed mitogen (PWM) a majority (50-90%) of the immunoglobulin-producing cells (cIg+ cells) that develop from these B cells express CD43 is detected with anti-CD43 monoclonal antibodies (MoAb) B1B6, and the proportion of CD43+cIg+ cells increases with time of culture. Furthermore, a relatively larger proportion (60-80%) of the IgG-producing cIg+ cells are CD43+ compared with IgM-containing cIg+ cells (30-50%). In human tonsils, significantly more CD43+ cells (35%) are found in the in vivo-activated fraction of B cells than in the fraction of resting B cells (5%). A majority of the cIg+ cells that develop from the resting or the in vivo-activated tonsillar B cells in a PWM-induced B-cell differentiation system are CD43+ (80-100%). Furthermore, tonsillar B cells depleted of CD43+ cells give rise to cIg+ cells, of which the majority are CD43+, and the proportion of such cells increases with time of culture (60-90%). Taken together, these results indicate that LSGP belongs to a group of B-cell membrane molecules that are induced and upregulated upon activation and differentiation.

Functional characterization of human B cells carrying the lymphocyte large sialoglycoprotein gp150.

Human B cell-enriched populations were prepared from buffy coats of healthy donors. By means of affinity chromatography, the B cells were separated into two fractions, one enriched in and the other depleted of cells expressing gp150, the large sialoglycoprotein of lymphocytes. In the presence of autologous T cells, monocytes and pokeweed mitogen B cell populations enriched for gp150+ cells gave rise to significantly more plasma cells (cIg+ cells) and secreted significantly more IgG than gp150-depleted populations. In contrast, more or an equal amount of IgM was secreted in cultures containing gp150-depleted cells. The differences between the fractions could not be ascribed to uneven distribution of T3+ cells, OKM1+ cells or B1+ (CD20) cells. However, the gp150-enriched population contained significantly more B2+ (CD21) cells than the gp150-depleted population. These results suggest that the gp150+ B cells differ from gp150- B cells, not only in their responsiveness to T cell differentiation signals but also in their commitment to Ig heavy chain isotype secretion.

Helper function of human T cells with different affinity for Helix pomatia A hemagglutinin in a tetanus toxoid-induced B cell differentiation system.

T cells from human peripheral blood were enriched in T4+ cells by lysis of T8+ cells with the monoclonal antibody OKT8 plus complement. The T4+ subset was separated into 4 fractions differing in avidity for the lectin Helix pomatia A hemagglutinin (HP). The fractions were studied for their capacity to help autologous B cells to differentiate and mature into immunoglobulin (Ig) synthesis and secretion after activation with tetanus toxoid (TT) in vitro. To ensure the antigen specificity of induction, very low doses of TT (1-100 ng/ml) were used for activation of the lymphocytes, all obtained from previously sensitized donors. The T4+ cells with low avidity for HP (fractions HP-I and HP-II) exerted little help for B cell differentiation. Removal of these cells enhanced the helper function of the remaining T4+ cells, indicating that fractions HP-I and HP-II contained suppressor cells. In contrast, efficient B cell help was provided by T4+ cells with high avidity for HP (fractions HP-III and HP-IV). However, these fractions differed in the quality of help provided. Thus, while HP-III cells induced IgG secretion, HP-IV cells mainly induced IgM secretion. Moreover, while the Ig secreted after help from HP-III cells was TT-specific antibody, the Ig secreted by B cells in the presence of autologous HP-IV cells was polyclonal, probably reflecting induction of B cells differing in their responsiveness to signals provided by different types of T cells. The results indicate that T4+ cells vary in their stage of differentiation as seen by differences in expression of the HP marker and that differences in HP-marker expression appear to be associated with differences in cellular functions.

The use of supernatants from neuraminidase and galactose oxidase (NAGO)-treated lymphocytes as a source of T cell growth factor.

Human peripheral blood or tonsil lymphocytes produce T cell growth factor (TCGF), when activated with neuraminidase (NA) and galactose oxidase (GO). Partial purification of NAGO-TCGF on Sepharose G-100 columns gave a TCGF-active fraction within the same molecular weight range as the conventional lectin-induced TCGF (approximately 15,000 Da). Human T cells, activated in mixed lymphocyte culture (MLC) with irradiated allogeneic EB-virus transformed B-cells (LCL) could be maintained in continuous culture for several months with retained functional activities. The cells showed similar growth patterns when cultured in the presence of either NAGO-TCGF or PHA-TCGF. The growing cells were characterized by means of monoclonal antibodies. After 4 weeks of culture 98% of these were OKT3+ and 87% were also OKT8+. The cytolytic activities of the cultures were tested in cell-mediated lympholysis (CML) against allogeneic LCL as target cells, in natural cytotoxicity (NK) against K562 cells and in antibody dependent cytotoxicity (ADCC) against bovine erythrocytes. Cultures displaying one or several of these functions were obtained. The results indicate, that TCGF obtained from supernatants of NAGO-activated lymphocytes is as potent as the T cell growth promoting factor obtained by lectin stimulation. One major advantage of using NAGO-generated TCGF is that contamination with lectin is avoided.