Development of thymic and splenic dendritic cell populations from different hemopoietic precursors.

The antigen-presenting dendritic cells (DCs) found in mouse lymphoid tissues are heterogeneous. Several types of DCs have been identified on the basis of the expression of different surface molecules, including CD4, CD8alpha, and DEC-205. Previous studies by the authors showed that the mouse intrathymic lymphoid-restricted precursors (lin(-)c-kit(+)Thy-1(low)CD4(low)) can produce DCs in the thymus and spleen upon intravenous transfer, suggesting a lymphoid origin of these DCs. In the current study, the potential for DC production by the newly identified bone marrow (BM) common lymphoid precursors (CLPs), common myeloid precursors (CMPs), and committed granulocyte and macrophage precursors was examined. It was found that both the lymphoid and the myeloid precursors had the potential to produce DCs. All the different DC populations identified in mouse thymus and spleen could be produced by all these precursor populations. However, CLPs produced predominantly the CD4(-)CD8alpha(+) DCs, whereas CMPs produced similar numbers of CD4(-)CD8alpha(+) and CD4(+)CD8alpha(-) DCs, although at different peak times. On a per cell basis, the CLPs were more potent than the CMPs at DC production, but this may have been compensated for by an excess of CMPs over CLPs in BM. Overall, this study shows that the expression of CD8alpha does not delineate the hemopoietic precursor origin of DCs, and the nature of the early precursors may bias but does not dictate the phenotype of the DC product.

Molecular cloning of a C-type lectin superfamily protein differentially expressed by CD8alpha(-) splenic dendritic cells.

Dendritic cells (DC) are potent antigen presenting cells that activate naive T cells. It is becoming increasingly clear that DC are not a homogeneous cell population, but comprise different subpopulations that differ in ontogeny and function. To further the molecular characterisation of DC, we screened for genes that were differentially expressed amongst DC subsets and could therefore give insight into their varying biological functions. Using Representational Difference Analysis (RDA) we identified a gene (CIRE) that is expressed at higher levels in the myeloid-related CD8alpha(-) DC than in the lymphoid-related CD8alpha(+) DC. CIRE is a 238 amino acid type II membrane protein, of approximately 33 kDa in size, whose extracellular region contains a C-type lectin domain. Northern blot analysis revealed that CIRE is almost exclusively expressed in DC and was not detected in organs such as heart, brain, kidney, liver, and thymus. T cells failed to express message for CIRE, whilst B cells expressed very low levels. These data here further substantiated by Northern blot analysis of 18 cell lines of various origins (myeloid, macrophage, B and T cell) where only one cell line, which was of myeloid origin and could give rise to DC, expressed mRNA for CIRE. Semi-quantitative RT-PCR suggested that CIRE is down-regulated upon activation. CIRE shares 57% identity with human DC-SIGN, a molecule that has been shown to be the ligand of ICAM-3 and that is also a receptor that binds HIV and facilitates trans-infection of T cells.

c-Rel regulates interleukin 12 p70 expression in CD8(+) dendritic cells by specifically inducing p35 gene transcription.

Interleukin 12 (IL-12) is a 70-kD proinflammatory cytokine produced by antigen presenting cells that is essential for the induction of T helper type 1 development. It comprises 35-kD (p35) and 40-kD (p40) polypeptides encoded by separate genes that are induced by a range of stimuli that include lipopolysaccharide (LPS), DNA, and CD40 ligand. To date, the regulation of IL-12 expression at the transcriptional level has mainly been examined in macrophages and restricted almost exclusively to the p40 gene. Here we show that in CD8(+) dendritic cells, major producers of IL-12 p70, the Rel/nuclear factor (NF)-kappaB signaling pathway is necessary for the induction of IL-12 in response to microbial stimuli. In contrast to macrophages which require c-Rel for p40 transcription, in CD8(+) dendritic cells, the induced expression of p35 rather than p40 by inactivated Staphylococcus aureus, DNA, or LPS is c-Rel dependent and regulated directly by c-Rel complexes binding to the p35 promoter. This data establishes the IL-12 p35 gene as a new target of c-Rel and shows that the regulation of IL-12 p70 expression at the transcriptional level by Rel/NF-kappaB is controlled through both the p35 and p40 genes in a cell type-specific fashion.

Molecular cloning of F4/80-like-receptor, a seven-span membrane protein expressed differentially by dendritic cell and monocyte-macrophage subpopulations.

A novel dendritic cell (DC) surface molecule termed F4/80-like-receptor (FIRE) has been selected based on its differential expression between DC subsets. The gene encoding FIRE has been cloned and sequenced, and mAbs specific for FIRE have been produced. FIRE is a seven-transmembrane-spanning molecule with two epidermal growth factor-like domains in the extracellular region. It is a novel member of the epidermal growth factor/transmembrane-7 protein subfamily and shows similarity to the macrophage marker F4/80. FIRE is expressed by CD8- DC, but not by CD8+ DC, and it is down-regulated on DC activation. It is expressed by blood monocytes and by some tissue macrophages, but not by most macrophage cell lines or by lymphoid cells. FIRE is a useful marker of myeloid cells with a DC developmental potential.

Paradoxical effects of IL-12 in leishmaniasis in the presence and absence of vaccinating antigen.

Protective immunity against Leishmania major requires parasite-specific CD4+T helper cells, the development of which is promoted by interleukin 12 (IL-12). In this study we investigated the use of IL-12 DNA to enhance the protective immunity induced by prophylactic vaccination with the L. major Parasite Surface Antigen 2 (PSA-2) DNA. A plasmid was constructed in which the two murine IL-12 subunits p35 and p40 were secreted as a biologically active single chain cytokine. The immunomodulatory effects of this IL-12 DNA were examined by codelivery with PSA-2 DNA in susceptible BALB/c and resistant C3H/He mice and subsequent infection with L. major promastigotes. Surprisingly, administration of IL-12 DNA alone had a protective effect, while coadministration of IL-12 with PSA-2 DNA abrogated protection. This effect of IL-12 DNA was dose dependent and affected by the timing of administration in relation to PSA-2 DNA. The effect of IL-12 on protection was associated with a reduced number of INF-gamma-producing T cells early in infection. A further understanding of this paradoxical effect of IL-12 and possibly other cytokines on protective immunity may be important for their use as adjuvants for Leishmania DNA vaccines.

Differential production of IL-12, IFN-alpha, and IFN-gamma by mouse dendritic cell subsets.

Dendritic cells (DC) not only stimulate T cells effectively but are also producers of cytokines that have important immune regulatory functions. In this study we have extended information on the functional differences between DC subpopulations to include differences in the production of the major immune-directing cytokines IL-12, IFN-alpha, and IFN-gamma. Splenic CD4(-)8(+) DC were identified as the major IL-12 producers in response to microbiological or T cell stimuli when compared with splenic CD4(-)8(-) or CD4(+)8(-) DC; however, all three subsets of DC showed similar IL-12 regulation and responded with increased IL-12 p70 production if IL-4 was present during stimulation. High level CD8 expression also correlated with extent of IL-12 production for DC isolated from thymus and lymph nodes. By using gene knockout mice we ruled out any role for CD8alpha itself, or of priming by T cells, on the superior IL-12-producing capacity of the CD8(+) DC. Additionally, CD8(+) DC were identified as the major producers of IFN-alpha compared with the two CD8(-) DC subsets, a finding that suggests similarity to the human plasmacytoid DC lineage. In contrast, the CD4(-)8(-) DC produced much more IFN-gamma than the CD4(-)8(+) or the CD4(+)8(-) DC under all conditions tested.

Development of dendritic cells in culture from human and murine thymic precursor cells.

The earliest T-precursor population in the adult murine thymus can give rise to dendritic cells (DC) in culture if stimulated with a cocktail of cytokines that includes interleukin (IL)-3, but not with cytokine mixes based on granulocyte-macrophage colony stimulating factor (GM-CSF), normally used to generate myeloid-derived DC. This and other evidence led to the proposal that two different lineages of DC exist, one lymphoid-related and the other myeloid-related. To determine whether this selective response to cytokines was restricted to murine DC, early human thymic T-precursors were isolated and their capacity to generate DC in response to various cytokines directly compared to their murine counterparts. In contrast to cultures of murine thymic precursors, CD34+CD1a- lineage marker negative (Lin-) precursor cells from the human thymus proliferated and generated DC with both the IL-3-containing cytokine mix lacking GM-CSF and with GM-CSF based cytokine mixes. These CD34+CD1a-Lin- human precursor cells also gave rise to NK cells under appropriate culture conditions, but produced no granulocyte, monocyte, eosinophil, megakaryocyte or erythroid cells in standard soft-agar colony-forming cell assays. Thus, although apparently lymphoid-restricted, the human thymic DC precursors responded to the myeloid factor GM-CSF as well as to the cytokines selective for murine lymphoid-related DC.

Human thymus contains 2 distinct dendritic cell populations.

In this study, 2 distinct populations of mature dendritic cells (DCs) were identified in the human thymus. The major population is CD11b-, CD11c+, and CD45RO(low) and does not express myeloid-related markers. It displays all the characteristics of mature DCs with a typical dendritic morphology, high surface levels of HLA-DR, CD40, CD83, and CD86, and expression of DC-lysosome-associated membrane glycoprotein messenger RNA (mRNA). In addition, CD11b- thymic DCs do not express macrophage inflammatory protein-1alpha (MIP-1alpha) mRNA, but express thymus-expressed chemokine (TECK) mRNA and are able to secrete bioactive interleukin 12 (IL-12) upon stimulation. In contrast, the minor and variable thymic DC population is CD11b+, CD11c(high), and CD45RO(high) and comprises CD83+CD14- mature and CD83- CD14+ immature DCs. It expresses macrophage-colony stimulating factor receptor, MIP-1alpha mRNA and high amounts of decysin mRNA after CD40 activation, but does not express TECK and is a weak bioactive IL-12 producer. Also identified were the IL-3Ralpha(high) plasmacytoid cells, which are present in the thymic cortex and medulla. Upon culture with IL-3, granulocyte/macrophage-colony stimulating factor, and CD40 ligand, the plasmacytoid cells can adopt a phenotype resembling that of freshly isolated CD11b- thymic DCs. However, these plasmacytoid-derived DCs fail to secrete bioactive IL-12; therefore, conclusions cannot be made about a direct relation between thymic plasmacytoid cells and CD11b- DCs. Whereas CD11b+ thymic DCs appear to be related to tonsillar germinal-center DCs, the major CD11b- IL-12-secreting human thymus DC population has similarities to mouse CD11b- CD8+ DCs.

Interleukin (IL)-4 is a major regulatory cytokine governing bioactive IL-12 production by mouse and human dendritic cells.

Interleukin (IL)-12 may be secreted as a bioactive T helper type 1 (Th1) cell-inducing heterodimer, as a monomer, or as an antagonistic homodimer. We analyzed the IL-12 produced by mouse splenic dendritic cells (DCs), human thymic DCs, and cultured human monocyte-derived DCs. IL-12 production required both a microbial or T cell-derived stimulus and an appropriate cytokine milieu. The different IL-12 forms were differentially regulated by the cytokines present rather than the stimulus used. IL-4 alone or together with granulocyte/macrophage colony-stimulating factor or interferon gamma effectively enhanced the production of the bioactive heterodimer and selectively reduced the antagonistic homodimer of IL-12. Therefore, IL-4, the major Th2-driving cytokine, provides a negative feedback causing DCs to produce the major Th1-inducing cytokine, bioactive IL-12.

Regulation of T cell cytokine production by dendritic cells.

Previous work has established that the dendritic cells (DC) of mouse spleen regulate the IL-2 production, and hence the extent of proliferation, of the CD8 T cells they activate. It is now reported here that interaction of primary CD8 T cells with splenic CD8alpha- DC induced much higher production of IL-3, IFN-gamma and granulocyte-macrophage colony-stimulating factor (GM-CSF), as well as IL-2, than did interaction with CD8alpha+ splenic DC. Furthermore, the CD8alpha- DC also induced higher levels of IL-2, IL-3 and IL-10 production in primary CD4 T cells, compared with that induced by CD8alpha+ DC. These quantitative differences did not involve qualitative shifts in the type of cytokine produced. Interleukin-4 production remained low in all the primary T cell cultures and restimulation experiments in secondary cultures did not reveal any bias in the cytokine production profile. When exogenous IL-2 was added to the primary cultures to ensure equal proliferation in response to CD8alpha- or CD8alpha+ DC, the higher level of production of IL-3, IFN-gamma and GM-CSF induced by CD8alpha- DC was maintained. Thus, this general control of T cell cytokine production by splenic DC involves factors additional to those that govern activation of T cells into cell cycle.

CD4 and CD8 expression by dendritic cell subtypes in mouse thymus and spleen.

The dendritic cells (DC) of mouse spleen and thymus were examined for expression of CD4 and CD8. Provided care was taken to avoid selective extraction or selective depletion of DC subpopulations, three main types of DC were detected in mouse spleen: a major new population of CD4+8- DEC-205low CD11bhigh DC, together with the previously described CD4-8- DEC-205low CD11bhigh DC and CD4-8alphaalpha+ DEC-205high CD11blow DC. The CD4 on the surface of the CD4+ splenic DC subpopulation was produced by the DC themselves, and CD4 RNA transcripts were present. Likewise, the CD8alpha on the surface of the splenic CD8+ DC was shown to be a product of the DC themselves, in agreement with earlier evidence. All three spleen DC types would be considered as mature, based on expression of CD80, CD86, and CD40 as well as on T cell stimulating function. Mouse thymuses appeared to contain two DC types; both were DEC-205highCD11blow, but they differed in the level of CD8alphaalpha expression. However, as well as this authenticated marker expression, immunofluorescent staining was also found to reflect a series of artifacts, due to the autofluorescence of contaminating cells and due to pickup of CD4 and CD8alphabeta. By constructing mice chimeric for the hemopoietic lineages using mixtures of wild-type bone marrow with CD4null or CD8alphanull bone marrow, a marked pickup by thymic DC of Ags derived from thymocytes was demonstrated.

[Stress reduction through listening to music: effects on stress hormones, hemodynamics and mental state in patients with arterial hypertension and in healthy persons]. / Stressreduktion durch Musikhören. Einfluss auf Stresshormone, Hämodynamik und psychisches Befinden bei Patienten mit arterieller Hypertonie und bei Gesunden.

Stress hormones, tissue-plasminogen activator (t-PA) antigen, left-ventricular diastolic function and mood immediately before and after listening to three different kinds of music (a waltz by J. Strauss, a piece of modern classic by H. W. Henze, and meditative music by R. Shankar) were measured in 20 healthy persons (10 women, 10 men; mean age 25 [20-33] years) and 20 hypertensives (8 women, 12 men; mean age 57.5 [25-72] years). To recognise haemodynamic effects, mitral flow by Doppler ultrasound was used as a measure of left-ventricular diastolic function. Atrial filling pressure (AFF) was calculated from the flow integral (VTI) of the early E and the late A waves. The Zerssen scale was used to estimate the immediate mood of the subjects. In hypertensives the levels of cortisol (74 vs 78 ng/ml; P < 0.05) and t-PA antigen (4.3 vs 4.5 ng/ml; P < 0.05) were lower after than before the Strauss waltz. The muscle by Henze lowered the concentrations of cortisol (70 vs 84 ng/ml; P < 0.05), noradrenaline (203 vs 224 ng/l; P < 0.05) and t-PA antigen (4.1 vs 4.6 ng/ml; P < 0.05). After listening to the piece by Shankar the concentrations of cortisol (71 vs 78 ng/ml; P < 0.05), adrenaline 14.5 vs 24.5 ng/ml; P < 0.05) and t-PA antigen (4.2 vs 4.3 ng/ml; P < 0.05) were lower. In healthy subjects AFF (29 vs 26%; P < 0.05) rose after the Strauss music, VTI-E fell (69 vs 73 mm; P < 0.05, while natriuretic peptide rose (63 vs 60 pg/ml; P < 0.05.(ABSTRACT TRUNCATED AT 250 WORDS)

Immediate physiological responses of healthy volunteers to different types of music: cardiovascular, hormonal and mental changes.

A group of 20 healthy volunteers [10 women, 10 men; median age 25 (20-33) years] were examined by means of pulsed wave Doppler echocardiography, blood sample analysis and psychological testing before and after listening to three different examples of music: a waltz by J. Strauss, a modern classic by H. W. Henze, and meditative music by R. Shankar. To assess small haemodynamic changes, mitral flow, which reflects left ventricular diastolic behaviour, was measured by Doppler ultrasound. Heart rate, arterial blood pressure and plasma concentrations of adrenocorticotropic hormone, cortisol, prolactin, adrenaline, noradrenaline, atrial natriuretic peptide (ANP) and tissue plasminogen activator (t-PA) were determined simultaneously. Transmitral flow profile is characterized by early E-wave and late atrial induced A-wave. Velocity-time integrals were measured and the atrial filling fraction was calculated. The mental state was measured by using a psychological score (Zerssen) with low values (minimum 0) for enthusiastic and high values (maximum 56) for depressive patterns. Music by J. Strauss resulted in an increase of atrial filling fraction (AFF; 29% vs 26%; P < 0.05) and ANP (63 pg.ml-1 vs 60 pg.ml-1; P < 0.05). The mental state was improved (Zerssen: 6.5 vs 11 points; P < 0.05). After the music of H. W. Henze prolactin values were lowered (7.7 ng.ml-1 vs 9.1 ng.ml-1; P < 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

[Hemodynamic effect of molsidomine in coronary patients with heart failure with clinically manifest nitrate tolerance]. / Hämodynamische Wirkung von Molsidomin bei herzinsuffizienten Koronarkranken mit klinisch manifester Nitrattoleranz.

Objective of the present study was to investigate the hemodynamic response to molsidomine in nitrate tolerance state. In 13 out of 16 patients (5 women, 11 men, 62 [53/71] years [median, 25%/75%-percentiles]) with chronic heart failure (NYHA stage II-III; median angiographic ejection fraction (EF) 55%) and coronary artery disease (stenosis of at least 75%) the development of tolerance under the continuous infusion of high doses of nitroglycerin (10 mg/h) was observed. Tolerance was defined as a benefit loss of at least 50% of the initial nitroglycerin effect with respect to the pulmonary capillary wedge pressure. Compared to the state of tolerance to nitroglycerin the infusion of 10 mg molsidomine over 15 minutes resulted in significant changes of the median values (25%/75%-percentiles) of mean right atrial pressure from 16 (12/21) to 9 (5/12) mmHg (p < 0.01), mean pulmonary artery pressure from 37 (30/40) to 24 (20/30) mmHg (p < 0.001), mean pulmonary capillary wedge pressure from 22 (18/25) to 15 (10/22) mmHg (p < 0.01) and cardiac output from 4.1 (3.5/4.7) to 5.2 (4.2/5.6) l/min (p < 0.01). This action of molsidomine corresponded to a complete overcoming (> 100%) of the benefit loss observed during the development of nitrate tolerance with respect to all above-mentioned hemodynamic parameters. Under parallel maintainance of nitroglycerin infusion (10 mg/h) these hemodynamic effects of molsidomine, i.e. at least 90% of the peak effect, lasted for 147 (130/182) minutes (median, 25%/75%-percentiles). Baseline values, i.e. a loss of at least 75% of the molsidomine effect, were only reached after 363 (319/412) minutes (median, 25%/75%-percentiles).(ABSTRACT TRUNCATED AT 250 WORDS)

Left ventricular filling behaviour in ultra endurance and amateur athletes: a stress Doppler-echo study.

To evaluate the impact of different training levels on left ventricular (LV) filling dynamics, Doppler mitral flow was derived in 25 amateur endurance-trained athletes (amateurs) aged 31 +/- 9 years, with a personal marathon record > 200 min, and in 26 ultra endurance athletes (top athletes) aged 32 +/- 8 years, with a personal marathon record < 170 min, during bicycle exercise in supine position. In particular atrial filling fraction as the relative share of atrial contribution to LV filling was measured. During exercise (150 watt) atrial filling fraction increased significantly more in amateurs from 25% to 34% compared to top athletes from 25% to 29% (p < 0.001). Two min post exercise atrial filling fraction already reached baseline values in top athletes (25%), while it remained significantly elevated in amateurs compared to baseline values (29%, p < 0.001). Only ten min post exercise atrial filling fraction showed baseline values in amateurs (26%). Rate pressure product was not significantly different at all levels of exercise. Thus, while atrial filling fraction rose in both study groups during exercise, it returned earlier to baseline values in top athletes than in amateurs. This indicates a better cardiac adaptation to physical stress and a better diastolic performance during exercise in endurance athletes with a higher training level.

[Effects of endurance training on left ventricular filling dynamics: a study using stress Doppler echocardiography]. / Auswirkungen des Ausdauertrainings auf die linksventrikuläre Füllungsdynamik: Eine Untersuchung mittels Stress-Dopplerechokardiographie.

To evaluate the impact of endurance training on left-ventricular (LV) filling dynamics Doppler mitral flow was derived in 23 amateur endurance-trained athletes (AT) aged 31 (24-39) years with a personal marathon record greater than or equal to 200 min, and in 20 ultra-endurance athletes (UEA) aged 38 (28-42) years with a personal marathon record less than 200 min during bicycle exercise in supine position. Twenty-two untrained healthy volunteers (UT) aged 27 (24-30) years served as control. In particular, atrial filling fraction (AFF) as the relative share of atrial contribution to LV filling was measured. At rest AFF was significantly higher in UT (29%) as compared to AT (25%) and UEA (25%). During exercise (150 watt) atrial fraction increased significantly more in UT (37%) as compared to AT (34%) and UEA (29%) (p less than 0.01). At this point of measurement UEA had significantly lower values for AFF than AT (p less than 0.001). Two min post exercise atrial filling fraction already reached baseline values in UEA (24%) and AT (26%), while it remained significantly elevated in UT as compared to baseline values (38%, p less than 0.001). Ten min post exercise atrial filling fraction showed still elevated values in UT (32%), but decreased under baseline values in UEA (23%). No differences in heart rate between the two athlete groups at all times of measurement were observed. Thus, while atrial filling fraction rose in all study groups during exercise, it returned earlier to baseline values in athletes than in untrained subjects. This indicates a better cardiac adaptation to physical stress and a better diastolic performance during exercise in endurance-trained athletes, being even more pronounced in ultra-endurance athletes.