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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online January 2, 2003 as doi:10.1096/fj.02-0755fje. |
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* Laboratory of Immunology, Institute of Hematologic Research, National Academy of Medicine, and
# Laboratory of Immunogenetics, Department of Microbiology, Buenos Aires University School of Medicine, Buenos Aires, Argentina
2Correspondence: IIHEMA, Academia Nacional de Medicina, Pacheco de Melo 3081, 1425 Buenos Aires, Argentina. E-mail: geffnerj{at}fibertel.com.ar
SPECIFIC AIMS
The renin-angiotensin system (RAS) has different actions in biological processes ranging from development and reproduction to cardiovascular and renal functions. These actions are mainly mediated by the octapeptide hormone angiotensin II (AII), which interacts with two major receptor subtypes, AT1 and AT2. In this work, we analyzed the effect of the angiotensinergic system on the differentiation and function of both human and mouse dendritic cells by studying the effect induced by AT1 and AT2 receptor antagonists.
PRINCIPAL FINDINGS
1. Human monocytes produce AII and express AT1 and AT2 receptors
In a first set of experiments, we analyzed by flow cytometry the expression of AT1 and AT2 receptors for AII in freshly isolated monocytes, using specific rabbit polyclonal IgG antibodies. We found that monocytes express both receptors, expression of AT1 being higher than AT2 receptors: mean fluorescence intensity (MFI): 11 ± 5, 18 ± 5, and 44 ± 8, isotype control, AT2, and AT1, respectively (mean±SE, n=6, P<0.05, AT2 or AT1 vs. isotype). We also found that monocytes produce AII, concentrations in cell supernatants (2.5x105 cells/mL) collected after 48 h of culture of 376 ± 112 pM (n=7).
2. Blockade of AT1 or AT2 receptors during differentiation of human DC induces opposite phenotypic changes
Having shown that monocytes produce AII and express AT1 and AT2 receptors, we next tested the ability of AT1 and AT2 receptor antagonists to modulate the differentiation of DC. Monocytes were cultured with IL-4 plus GM-CSF (10 ng/mL) for 7 days, in the absence (controls) or presence of 25 µg/mL of candesartan, losartan, or irbesartan, three AT1 receptor antagonists, or PD123319, an AT2 receptor antagonist. As expected, control DC (C-DC) appear as nonadherent clumps or isolated floating cells (not shown) and express high levels of CD1a (MFI=706±165 and 9±5, CD1a and isotype control, respectively, n=10), indicating phenotypic differentiation of monocytes into DC. In contrast to C-DC, cells differentiated in the presence of candesartan (Ca-DC), losartan (Lo-DC), or irbesartan (Ir-DC) result in the generation of a large proportion of plastic adherent monocyte/macrophage-like cells. In fact, almost 50% of these cells (47±8, 53±8, and 42±7%, for Ca-DC, Lo-DC, and Ir-DC, respectively, n=6–7) were firmly attached to the plate surface. These cells express very low levels of CD1a (80–90% lower than C-DC) and produce O2- in response to phorbol myristic acid (PMA, 10 nM) at levels 8- to 10 fold higher than C-DC. Together, these data suggest that AT1 receptor antagonists hamper development of DC, favoring the differentiation of monocytes into a macrophage-like profile. Despite this profile, however, these cells were largely low/negative for CD14 expression. By contrast, cells differentiated in the presence of PD123319 (PD-DC) appear as nonadherent clumps or isolated floating cells, as C-DC, and express high levels of CD1a (57% higher than C-DC). Under all conditions tested, cell survival at day 7 was 
90%.
As described above, the autocrine production of AII by monocytes yielded concentrations of AII in the culture medium lower than 1 nM. We then asked whether monocyte exposure to higher concentrations of AII would modify the phenotypic profile of DC. In the presence of exogenous AII (10 nM), DC (AII-DC) develop as nonadherent cells and express high levels of CD1a (30% higher than C-DC).
Expression of CD40 and HLA-DR, but not CD86, was lower (P<0.05) in Ca-DC than C-DC, by contrast, all three markers were found to be higher (P<0.05) in PD-DC and AII-DC than C-DC.
3. Blockade of AT1 and AT2 receptors during differentiation of human DC induces opposite effects on their ability to endocyte HRP and stimulate allogeneic T cell proliferation
Differentiation of human monocytes into immature DC leads to a marked increase in cellular endocytic capacity. Experiments were performed to analyze the endocytic activity of DC differentiated in the presence of either AII receptor antagonists or exogenous AII. We used horseradish peroxidase (HRP), which is internalized via a mannose receptor-mediated mechanism and usually used to quantitate antigen uptake in bulk populations. Figure 1
A shows that Ca-DC and Lo-DC displayed low levels of endocytosis, whereas PD-DC as well as AII-DC expressed the highest endocytic capacity. The ability of DC to stimulate allogeneic T cell proliferation was then evaluated. Figure 1B
shows that Ca-DC and Lo-DC mediated lower MLR responses than C-DC, whereas PD-DC and AII-DC mediated higher responses than controls.
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4. AT1 and AT2 receptor antagonists also mediate opposite effects on the differentiation of mouse DC from bone marrow cultures
We next examined the effect of AT1 and AT2 receptor antagonists on the differentiation of mouse DC (mDC) from bone marrow cells cultured with GM-CSF (200 U/mL) for 9 days. Mouse DC differentiated in the presence of losartan (25 µg/mL, Lo-mDC) expressed levels of CD11c, CD40, and Ia lower (P<0.05) than control mDC (C-mDC). By contrast, cells differentiated in the presence of PD 123319 (25 µg/mL, PD-mDC) or exogenous AII (10 nM, AII-mDC) expressed these three markers at higher levels (P<0.05) than control cells. Endocytosis of HRP was found to be inhibited in Lo-mDC (% inhibition compared with controls=49±6, n=7, P<0.05) whereas it was improved in PD-mDC and AII-mDC (% enhancement compared with controls=118±9 and 133±8, respectively, n=7, P<0.05). We also examined the ability of DC obtained from BALB/c mice to stimulate allogeneic T cell responses (BALB/c X C57BL, ratio 1:10). As expected, the highest MLR responses were observed for AII-mDC and PD-mDC (% enhancement compared with controls=31±7, and 39±9, respectively, n=7, P<0.05). However, contrasting with the results observed in human DC, Lo-mDC and C-mDC mediated similar responses.
5. Blockade of AT1 and AT2 receptors during differentiation of mouse DC induced opposite effects on their ability to induce specific antibody responses when transferred to nonimmunized recipients
To evaluate the ability of DC to induce in vivo antibody responses, DC (C-mDC, Lo-mDC, PD-mDC, and AII-mDC) were pulsed with ovalbumin (OA) and injected intravenously (i.v.) into naive syngeneic mice. The animals were bled 14 days later and specific antibodies were measured. Figure 2
shows that Lo-mDC were poor stimulators of the antibody response, whereas PD-mDC and AII-mDC induced higher responses than C-mDC.
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CONCLUSIONS
Our results suggest the existence of an autocrine loop through which AII supports the normal differentiation of DC from human monocytes by interacting with the predominant class of AII receptor expressed in these cells, the AT1 receptor. Blockade of AT1 receptors not only hampers DC differentiation induced by GM-CSF plus IL-4, but also switches the differentiation of monocytes into macrophage-like cells (see Fig. 3
). As described for most of the actions exerted by AII on cardiovascular hemodynamics and cell growth, our results support the notion that activation of AT2 receptor by AII during the differentiation of DC antagonizes the effects induced through AT1 receptors. A similar mechanism appears to be operative in the differentiation of mouse DC. In summary, our results undercover a mechanism through which AII may affect the development of adaptive immunity by influencing the differentiation of myeloid DC. Further studies are required to define the relevance of our observations in vivo as well as the mechanisms through which the angiotensinergic system influences the development of DC.
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FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0755fje; to cite this article, use FASEB J. (January 2, 2003) 10.1096/fj.02-0755fje 
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