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(Journal of Leukocyte Biology. 2002;72:93-100.)
© 2002 by Society for Leukocyte Biology

Decrease in cAMP levels modulates adhesion to fibronectin and immunostimulatory ability of human dendritic cells

Dalia Burzyn*, Carolina C. Jancic*, Sandra Zittermann*, María I. Keller Sarmiento{dagger}, Leonardo Fainboim*, Ruth E. Rosenstein{dagger} and H. Eduardo Chuluyan*

* Laboratorio de Inmunogenética, Hospital de Clínicas "José de San Martín", and
{dagger} Departamento de Bioquímica Humana, Facultad de Medicina, Universidad de Buenos Aires, Argentina

Correspondence: Dr. H. Eduardo Chuluyan, Laboratorio de Inmunogenética, Piso 3, Sala 4, Hospital de Clínicas "José de San Martín", Facultad de Medicina, Universidad de Buenos Aires, Avenida Córdoba 2351 (C.P. 1120), Buenos Aires, Argentina. E-mail: chulu{at}interar.com.ar


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The aim of the present study was to analyze the early events elicited by tumor necrosis factor {alpha} (TNF-{alpha}) on monocyte-derived dendritic cells (moDC) adhesion to fibronectin (FN) and the involvement of cAMP in the signal transduction mechanism. The intracellular concentration of cAMP and moDC adhesion to FN decreased after TNF-{alpha} treatment. An inverted dose-dependency for TNF-{alpha} effect was observed for adhesion and cAMP levels. The presence of a phosphodiesterase (PDE) inhibitor (IBMX) and cAMP analogs (8Br-cAMP, Db-cAMP) reversed the observed TNF-{alpha} effects. The role of cAMP was analyzed further by examining the cAMP levels in nonadhered and adhered, TNF-{alpha}-treated moDC. Nonadhered moDC showed lower cAMP levels compared with adhered moDC. Furthermore, nonadhered moDC showed higher IL-12 content and allostimulatory ability compared with adhered moDC. The higher allostimulatory capacity was abolished in the presence of cAMP analogs and a PDE inhibitor. These results suggest that cAMP levels correlate with TNF-{alpha}-induced changes of moDC adhesion and allostimulatory capacity.

Key Words: IL-12 • signal transduction • second messengers


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Dendritic cells (DC) are professional antigen presenting cells that play a critical role in the induction of acquired immune responses [1 , 2 ]. DC precursors and progenitors exit the bone marrow and circulate via blood until they seed many tissues and nonlymphoid organs. DC exist in at least two stages of maturation. As immature DC in the tissues, they express low levels of major histocompatibility complex (MHC) and costimulatory molecules, and they are very effective in capturing and processing antigens. When DC encounter local inflammatory mediators, they become activated and undergo a maturation process. This process involves their mobilization from the periphery to the lymph node and spleen T cell areas, down-regulation of their antigen-capture capacity, as well as up-regulation of costimulatory molecules [3 4 5 6 ].

A wide array of inflammatory mediators contribute to DC maturation and migration. Lipopolysaccharides (LPS), interleukins (IL), interferons, colony-stimulating factors (CSF), tumor necrosis factor {alpha} (TNF-{alpha}), growth factors, and chemokines may contribute to DC migration [7 ] by inducing phenotype changes required for DC movement. Some of the phenotypic changes include expression of adhesion molecules that regulate interactions with the surrounding extracellular matrix and chemokine receptors [8 9 10 11 ]. Several adhesion molecules involved in DC emigration include CD11a/CD54, {alpha}6 integrin/CD49f, E-cadherin, and CD44 [12 13 14 15 ]. Other molecules such as matrix metalloproteinase-9 and P-glycoprotein multidrug resistance-1 may also be involved in this process [16 , 17 ]. However, early changes that DC undergo after an inflammatory stimulus, before starting the maturation process, still remain to be ascertained.

A relationship between TNF-{alpha} effects and the cyclic adenosine monophsophate (cAMP) system has been clearly demonstrated in other cells [18 , 19 ]. Therefore, we investigated the participation of cAMP in the response elicited by TNF-{alpha} on human monocyte-derived DC (moDC) adhesion and maturation.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Monoclonal antibodies (mAb)
mAb recognizing antigens present on DC were used for this study. These included mAb against human leukocyte antigen (HLA)-class I molecules [W6/32, immunoglobulin G (IgG)2a, American Type Culture Collection (ATCC), Manassas, VA], HLA-class II (HB55, IgG2a, ATCC), CD1a [NA134, IgG2a, 5th International Workshop on Human Leukocyte Differentiation Antigen (HLDA)], CD1b (WM25, IgG1, 5th International Workshop on HLDA), CD49d (HP2/1, IgG1, Immunotech, Marseilles, France), CD49e (SAM-1, IgG2b, Immunotech), CD86 (FUN-1, IgG1k, Pharmingen, San Diego, CA), CD83 (HB15e, IgG1, Pharmingen), CD18 (Ts1/18, IgG1, ATCC), TNF receptor type I (TNFR I; htr9, IgG1, 6th International Workshop on HLDA), and TNFR II (utr1, IgG1, 6th International Workshop on HLDA). mAb recognizing chemokine receptors CCR5 (2D7, IgG2a, Pharmingen) and CCR7 (3D12, kindly provided by Dr. Martin Lipp, Max-Delbruck-Centrum Berlin) were used.

Culture of moDC from peripheral blood
Human DC were generated from monocytes as described previously, with minor modifications [20 ]. Briefly, 100 ml blood was obtained from healthy donors. The peripheral blood mononuclear cells (PBMC) were isolated by centrifugation through a Ficoll-Hypaque (Pharmacia LKB Biotech, Piscataway, NJ) gradient as demonstrated [20 ]. To further isolate monocytes, the osmolarity of the PBMC medium was gradually increased in three steps from 290 to 360 mosmol by addition of 9% NaCl, as described by Boyum [21 ] and Recalde [22 ]. This protocol improved the monocyte purity and did not affect cell viability or function as reported previously [23 ]. After the incubation period, the PBMC were resuspended in hyperosmotic (360 mosmol) Ca++, Mg++-free Tyrode’s solution containing 0.2% ethylenediaminetetraacetate (EDTA), and 10% platelet poor plasma-Percoll (Pharmacia Fine Chemicals, Dorval, PQ) to achieve 56% Percoll concentration (based on 100%=isotonic Percoll). Five different Percoll cushions of 2.5 ml each were layered in a 15 ml conical polypropylene tube with 60% Percoll at the bottom, followed by 56%, 50% (containing the leukocytes), 46%, and 40%. Density gradient centrifugation was at 400 g (20 min at 22°C in a swinging bucket rotor). The purest monocyte fraction was recovered at the 46–40% Percoll interphase, yielding 8–10 x 106 monocytes from 100 ml starting blood with >90% purity, >95% viability by neutral red staining, and trypan blue exclusion. Monocytes (5x106 cells/ml) were cultured for 7 days in RPMI-1640, supplemented with 2 mM L-glutamine, 50 µg/ml gentamicin, 50 µM 2-mercaptoethanol, 800 U/ml granulocyte macrophage (GM)-CSF, 500 U/ml IL-4 (Sigma Chemical Co., St. Louis, MO), and 10% heat-inactivated, pooled AB human serum (HS) from three to four normal donors. More than four different batches of pooled HS were used through the experiments. moDC were identified by immunofluorescence staining with the mAb to HLA-DR, CD1b, and CD86. Before the experiments, the purity of the moDC was checked. The moDC preparations containing <3% contaminating cells were used. Whenever necessary, another Percoll gradient was performed to further isolate moDC from non-moDC at the end of the incubation time with GM-CSF and IL-4, i.e., 7 days. For the adhesion experiments, the moDC were resuspended at a final concentration of 5 x 105/ml in RPMI-1640, 0.5% HS albumin (HSA; Grifols Institute, Barcelona).

Adhesion of moDC to fibronectin (FN)
The 96-well flat-bottom plates (Costar, Cambridge, MA) were coated with purified FN from human plasma (Gibco, Grand Island, NY), 30 µg/ml, 60 min at 37°C, as described previously [20 ]. Protein-coated wells were washed with phosphate-buffered saline (PBS) and then incubated for 60 min at 37°C with RPMI-1640 medium containing 5 mg/ml HSA to block nonspecific binding sites before their use in cell-attachment assays. Cells were washed twice with RPMI-1% HSA and incubated with TNF-{alpha} (Sigma Chemical Co.), 10–1000 units/ml (0.2–2 ng/ml), or RPMI-1% HSA (control) during 30–60 min at room temperature (RT). In some experiments, moDC were treated for 30 min at 37°C with 1 mM of 8-bromo- or dibutyryl-cAMP (8Br-cAMP and Db-cAMP, respectively) or 1-methyl-3-isobutylxanthine (IBMX; 0.5 mM, 30 min at 37°C) alone or in combination with TNF-{alpha}. Afterwards, cells (5x104/well) were washed and allowed to adhere to protein-coated surfaces for 45 min at 37°C in 5% CO2. Nonadhered cells were removed by washing, and adhered cells were detached by using 0.3% trypsin-0.05% EDTA in PBS and washed with RPMI 1640-10% fetal bovine serum (FBS). The number of cells adhered and nonadhered was analyzed by flow cytometer "Cytoron" (Ortho Diagnostric Systems, Raritan, NJ) equipped with the software program Absolute as described previously [20 ]. According to the positivity of moDC for CD1a or CD3, two different gates on a forward-scatter versus side-scatter dot plot were defined: DC gate (R1=CD1a-positive, CD3-negative) and T lymphocyte gate (R2=CD1a-negative, CD3-positive). Only cells located in R1 were considered as moDC. The adhered and nonadhered moDC for each well (coated or uncoated with FN) were counted by flow cytometry. The results were expressed as the percentage of the total cells added to the wells (adhered and nonadhered) and recovered in each well. In some experiments, moDC were labeled with 51Cr sodium chromate (Amersham Biosciences, UK; 20 µCi/ml) by incubating for 30 min at 37°C. Then, 5 x 105 51Cr-moDC/ml were added above FN. After incubation, adhesion was stopped by washing to remove nonadhered moDC. The attached moDC were lysed by addition of 0.5% Triton X-100 and then analyzed for 51Cr. Results are expressed as the percentage of the total 51Cr-moDC added to wells and recovered in each fraction. All conditions were performed by triplicate.

Second challenge of nonadhered moDC to FN
moDC treated or untreated with TNF-{alpha} were tested for FN adhesion. After 45 min, nonadhered cells were recovered and challenged again for another adhesion to FN. Before the second adhesion assay to FN, nonadhered cells were treated with Db-cAMP or 8Br-cAMP (Sigma Chemical Co.; 1 mM, 30 min at 37°C) or IBMX (0.5 mM, 30 min at 37°C).

cAMP assessment
moDC cells were incubated for 30 min at 37°C in RPMI-1640 buffer in the presence or absence of 0.5 mM IBMX with or without TNF-{alpha}. After centrifuging for 10 min at 900 g, pellets were resuspended in distilled water and boiled for 2 min. cAMP levels were assessed as described previously [24 ]. Briefly, the homogenates were centrifuged at 5000 g for 5 min at 4°C. cAMP content was assessed in the supernatants by radioimmunoanalysis after acetylation. For this purpose, aliquots of samples or standards were acetylated with acetic anhydride/triethylamine. The acetylated products were incubated with [125I]-cAMP (15,000–20,000 dpm; specific activity, 140 mCi/mmol) and a rabbit antiserum, kindly supplied by the National Institute of Diabetes and Digestive and Kidney Disease (Torrance, CA), diluted 1:5000, and incubated overnight at 4°C. After adding 2 ml ethanol with 2% bovine serum albumin, the antigen-antibody complexes were precipitated by centrifugation at 2000 g for 30 min. The supernatants were separated by aspiration.

Flow cytometry analysis on nonadhered moDC
Suspensions of nonadhered, TNF-{alpha}-treated moDC recovered after the adhesion assays were incubated during 30 min at 37°C in the presence or absence of 1 mM 8Br-cAMP or Db-cAMP. Cells were incubated with pooled AB HS (1/10) for 30 min to block Fc receptors. Afterwards, moDC were incubated with the corresponding primary mAb at optimal concentrations (5–10 µg/ml) for 45 min. Finally, cells were treated with RPE-conjugated goat anti-mouse Ig (Dako, Roskilde, Denmark) for 45 min. All incubations and washings were performed at 4°C with RPMI 1640 supplemented with 5% FBS and 0.1% Na3N. Cells labeled successively with irrelevant isotype-matched primary mAb and RPE-conjugated goat anti-mouse Ig were used as negative controls. A FACStar plus (Becton Dickinson, Mountain View, CA) analyzed fluorescence intensity, and dead cells were excluded by gating with propidium iodide.

Intracellular IL-12 staining on adhered and nonadhered, TNF-{alpha}-treated moDC
moDC treated with TNF-{alpha} were tested for FN adhesion. After 45 min, adhered and nonadhered cells were recovered and incubated for 3 h at 37°C with Brefeldin A (10 µg/ml). After washing twice with PBS/sodium azide/FBS, cells were fixed with paraformaldehyde. Following, moDC were stained with anti-IL-12 R-PE kit (ImmunoQuality Products, Groningen, The Netherlands) in the presence of the permeabilizing agent as described by the manufacturer. The cells were then analyzed by flow cytometry.

Mixed leukocyte reaction (MLR)
moDC treated with TNF-{alpha} were tested for FN adhesion. After 45 min, adhered and nonadhered cells were recovered and tested for their allostimulatory ability. For this purpose, allogeneic or autologous PBMC were cultured in 96-well microplates with different concentrations (ranging from 5x103 to 2x104 cells/well) of adhered and nonadhered, TNF-{alpha}-treated moDC. Thymidine (TdR) incorporation was measured on day 5 by an 18 h pulse with [3H]TdR (1 µCi/well; specific activity, 5 mCi/mMol; Amersham Life Sciences). In some experiments, nonadhered and adhered, TNF-{alpha}-treated moDC were incubated further with cAMP analogs or IBMX before MLR assay.

Statistical analysis
Statistical analysis of results was performed by a Student’s t-test or by analysis of variance followed by post hoc Student Newman Kules analysis.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Relationship between cAMP content and moDC adhesion to FN
We evaluated the events elicited by TNF-{alpha} on moDC interaction with FN. moDC treated for 1 h with TNF-{alpha} were less adherent to FN (Fig. 1 , solid line) when compared with untreated moDC. Diminished adhesion was not changed whether or not TNF-{alpha} was removed before the adhesion assays (data not shown). This effect was statistically significant at concentrations of 10 and 100 U/ml TNF-{alpha} (Fig. 1 , solid line). When the incubation time was prolonged (up to 24 h) using TNF-{alpha} at 100 U/ml, the same extent of inhibition (25±7%, n=3) was observed. In all the cases, a possible cytotoxic effect was ruled out by staining with vital dyes (data not shown).



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  		Figure 1. TNF-{alpha} effect on moDC adhesion to FN and cAMP content. Monocytes differentiated with GM-CSF and IL-4 for 7 days were treated with TNF-{alpha} (10–1000 U/ml, 30 min at RT). Afterwards, 2.5–7.5 x 105 moDC were added above wells coated with FN or processed for cAMP assessment as described in Materials and Methods. Results are expressed as percentage of inhibition of moDC adhesion to FN ({circ}) and pmol cAMP for 106 moDC (•). cAMP content in untreated cells was 2.46 ± 0.06 pmol/106 cells. Data represent the mean value ± SEM of four experiments. *, P < 0.05; **, P < 0.01 compared with untreated moDC using post hoc Student Newman Kules analysis.

 
Figure 1 (dotted line) also shows that 1 h of TNF-{alpha} treatment decreased intracellular cAMP levels significantly in moDC. Similar to the adhesion assays, the effect of TNF-{alpha} on cAMP content was lower in the presence of higher concentrations of this cytokine.

To assess the subset of moDC capable of responding to TNF-{alpha}, we measured the expression of TNFR I and II on moDC. Figure 2 shows that only a subset of cells (>24%) expressed TNFR II. This value was variable among experiments, being the highest expression found at 53%. Moreover, four independent experiments indicated that TNFR I was almost undetectable on moDC. These results indicate that only the subset of moDC that expressed TNFR II was able to respond to TNF-{alpha}.



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  		Figure 2. TNFR I and II expression on moDC. moDC were stained with mAb anti-TNFR I or -TNFR II and analyzed by flow cytometry. Similar results were obtained in two additional experiments.

 
To determine the relationship between cAMP content and the binding ability of moDC, we examined the effect of IBMX (0.5 mM) and two cAMP analogs (8Br-cAMP, Db-cAMP) in adhesion assays to FN. IBMX significantly increased cAMP levels of untreated moDC (Fig. 3 , hatched bars), and it reduced the decrease of cAMP levels induced by TNF-{alpha} (Fig. 3 , hatched bars). IBMX per se did not affect the adhesion of untreated moDC, although it reversed the inhibitory effect of TNF-{alpha} on moDC adhesion to FN (Fig. 3 , open bars). The same pattern was observed with two cAMP analogs (data not shown).



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  		Figure 3. Effect of a PDE inhibitor on moDC adhesion to FN and cAMP content. TNF-{alpha}-treated and untreated moDC were incubated with IBMX (0.5 mM). Afterwards, 7.5 x 105 moDC were added above wells coated with FN or processed for cAMP assessment as described in Materials and Methods. Results are expressed as the percent of moDC adhered to FN and pmol cAMP for 106 moDC. Data represent the mean ± SEM of three experiments, each performed by triplicates. *, P < 0.05; **, P < 0.01 compared with untreated moDC; #, P < 0.05; ##, P < 0.01 compared with TNF-{alpha}-treated moDC using post hoc Student Newman Kules analysis.

 
Nonadhered cells (treated or not with TNF-{alpha}), which were harvested after the FN adhesion assay, showed different binding activity in a second challenge to FN. Figure 4 (open bars) shows that nonadhered, TNF-{alpha}-treated moDC attached less to FN than untreated moDC. In fact, when 8Br-cAMP (Fig. 4 , hatched bars) was added before the second adhesion assay, TNF-{alpha}-treated cells showed a binding activity similar to that of control cells. Similar results were obtained by using Db-cAMP (data not shown). The reversion of TNF-{alpha} effects on moDC also argues against a possible cytotoxic effect of TNF-{alpha}.



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  		Figure 4. Effect of cAMP analogs on nonadhered untreated and TNF-{alpha}-treated moDC. TNF-{alpha}-treated and untreated, "Non-adhered" moDC from FN adhesion assays were recovered and incubated further with 8Br-cAMP (1 mM, 30 min). Following, the cells were rechallenged by adding them above wells coated with FN. Data are expressed as the percentage of moDC adhered to FN and represent the mean ± SEM of three experiments, each performed by triplicates. *, P < 0.05 compared with untreated moDC; #, P < 0.05 compared with control, nonadhered TNF-{alpha} moDC using post hoc Student Newman Kules analysis. Similar results were obtained with Db-cAMP.

 
It is well known that CD49d and CD49e integrins are FN receptors [25 ]. We have previously described that moDC adhesion to FN is mediated mainly by CD49e [20 ]. Because 8Br-cAMP reversed the effect of TNF-{alpha} on FN adhesion, we studied its effects on the expression of FN receptors in nonadhered, TNF-{alpha}-treated moDC. Figure 5 shows that in spite of the differences in the adhesion ability, the expression levels of CD49d and CD49e were similar between both groups.



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  		Figure 5. CD49e/{alpha}5 and CD49d/{alpha}4 integrin expression on nonadhered, TNF-{alpha}-treated moDC. The nonadhered, TNF-{alpha}-treated moDC from FN adhesion assays were recovered and treated (empty profiles) or not (shaded profiles) with 8Br-cAMP or Db-cAMP (not shown). Afterwards, cells were stained with mAb anti-CD49e or CD49d and analyzed by flow cytometry. Similar results were obtained in two additional experiments.

 
Properties of adhered versus nonadhered, TNF-{alpha}-treated moDC
To further examine the relationship between moDC adhesion and cAMP content, nucleotide levels were assessed after adhesion assays, in adhered and nonadhered cells. For these experiments, we harvested the adhered and nonadhered cells pretreated with TNF-{alpha}. To allow comparison between adhered and nonadhered moDC, the latter received the same treatment as the former, using trypsin-EDTA before the experiments. Figure 6 shows that 106 nonadhered cells, recovered after 30 min of the adhesion assay, exhibited significantly lower levels of intracellular cAMP than the same number of adhered moDC.



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  		Figure 6. cAMP content on adhered and nonadhered, TNF-{alpha}-treated moDC. moDC were treated with TNF-{alpha} (10 U/ml, 30 min at RT) and added (7.5x105 moDC) above wells coated with FN. Adhesion was stopped after 45 min, and adhered and nonadhered cells were recovered and processed for cAMP assessment. Data are expressed as pmol cAMP for 106 moDC and represent the mean value ± SD of triplicates of one experiment representative of three. **, P < 0.01 compared with untreated moDC using Student’s paired t-test.

 
In addition, those moDC with low or high cAMP content showed a different immunostimulatory capacity. As shown in Figure 7 , nonadhered cells (low cAMP content) induced a high allogenic T cell proliferation. In contrast, adhered, TNF-{alpha}-treated moDC (with high cAMP content) were poor stimulators in MLR (Fig. 7) . The relationship between cAMP content and immunostimulatory capacity is shown in Figure 8 . By increasing cAMP content (with IBMX and 8Br-cAMP), the immunostimulatory capacity of nonadhered moDC decreased without affecting the poor capacity of adhered moDC (Fig. 8) .



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  		Figure 7. Allostimulatory capacity of adhered and nonadhered, TNF-{alpha}-treated moDC. moDC were treated with TNF-{alpha} (10 U/ml, 30 min at RT) and added (7.5x105 moDC) above wells coated with FN. Adhesion was stopped after 45 min, and adhered and nonadhered cells were recovered and used as stimulators for allogeneic T lymphocytes. T cells, 105 allogeneic, were added to irradiate adhered and nonadhered moDC. After 4 days, cells were pulsed with 1 µCi [3H]TdR per well for the last 16 h of culture and were harvested and counted in a beta counter. Results are mean counts per minute ± SD of triplicates of one experiment representative of three. *, P < 0.01 compared with untreated moDC using Student’s paired t-test.

 


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  		Figure 8. Effect of cAMP analogs and a PDE inhibitor on the allostimulatory capacity of adhered and nonadhered, TNF-{alpha}-treated moDC. moDC were treated with TNF-{alpha} (10 U/ml, 30 min at RT) and added (7.5x105 moDC) above wells coated with FN. Adhesion was stopped after 45 min, and adhered and nonadhered cells were recovered and treated or not with IBMX, 8Br-cAMP, or Db-cAMP (not shown). Following, the cells (104) were irradiated and used as stimulators for 105 allogeneic T lymphocytes. After 4 days, cells were pulsed with 1 µCi [3H]TdR per well for the last 16 h of culture and were harvested and counted in a beta counter. Results are mean counts per minute ± SD of triplicates of one experiment representative of three. **, P < 0.01; ***, P < 0.001 compared with control group using post hoc Student Newman Kules analysis.

 
Because immunostimulatory capacity is related to the maturation state of the cells, we next examined the expression of costimulatory molecules (CD86), chemokine receptors (CCR5, CCR7), CD83, HLA-DR, and CD18 on adhered and nonadhered, TNF-{alpha}-treated moDC. Figure 9a shows that following the adhesion assay, adhered and nonadhered, TNF-{alpha}-treated moDC did not express CD83 and CCR7. Nonadhered moDC had similar levels of HLA-DR and CD86, a slight increase in CD18, and a very slight decrease in CCR5 as compared with adhered moDC (Fig. 9a) . However, after 16 h of the adhesion assay, a subpopulation of the nonadhered, TNF-{alpha}-treated moDC expressed CD83. Moreover, an increase in CD86 and CD18 and a decrease in CCR5 were observed on nonadhered compared with adhered, TNF-{alpha}-treated moDC. In search for other mechanisms that contribute to the higher immunostimulatory capacity of nonadhered, TNF-{alpha}-treated moDC, we next assessed production of IL-12. Figure 10 shows that nonadhered moDC had higher intracellular IL-12 content than adhered moDC.



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  		Figure 9. MHC class II (DR), CD86, CD83, CCR5, CCR7, and CD18 expression on nonadhered (dotted-line histogram) and adhered (thick-line histogram), TNF-{alpha}-treated moDC. (a) moDC were treated with TNF-{alpha} (10 U/ml, 30 min at RT) and added (7.5x105 moDC) above wells coated with FN. Adhesion was stopped after 45 min, and adhered and nonadhered cells were recovered. Afterwards, cells were stained with mAb to MHC class II, CD86, CD83, CCR5, CCR7, and CD18 or control Ab (filled histograms) and analyzed by flow cytometry. Similar results were obtained in three additional experiments. (b) After the adhesion assay, adhered and nonadhered cells were incubated for 16 h. Then, cells were stained with mAb and were analyzed by flow cytometry as described above. Similar results were obtained in two additional experiments.

 


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  		Figure 10. Intracellular levels of IL-12 in adhered (dotted-line histograms) and nonadhered (thick-line histogram), TNF-{alpha}-treated moDC. moDC were treated with TNF-{alpha} (10 U/ml, 30 min at RT) and added (7.5x105 moDC) above wells coated with FN. Adhesion was stopped after 45 min, and adhered and nonadhered cells were recovered and incubated with Brefeldin A (10 µg/ml, 3 h, 37°C). Afterwards, cells were fixed and stained with anti-IL-12 R-phycoerythrin (RPE) or control Ab (filled histograms) and were analyzed by flow cytometry. Similar results were obtained in two additional experiments.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The foregoing results suggest that cAMP is a second messenger involved in early modulatory events of TNF-{alpha}-induced moDC adhesion and maturation. In the past several years, considerable progress has been made in defining some aspects of DC migration [8 9 10 11 ]. Although much attention has been devoted to identifying the molecules involved in the regulation of moDC adhesion and maturation process, knowledge of the intracellular events triggered by these signals is still incomplete. Two observations support the involvement of cAMP in the effect of TNF-{alpha} on moDC: TNF-{alpha}, with a very similar dose-dependency, decreased moDC adhesion to FN and cAMP intracellular content, and the effect of TNF-{alpha} on moDC adhesion and their immunostimulatory ability was significantly reduced in the presence of two cAMP analogs as well as a phosphodiesterase (PDE) inhibitor.

The effect of TNF-{alpha} on adhesion and cAMP levels was shown to be higher at lower concentrations. In agreement with our results, Stoitzner et al. [26 ] reported an inverted dose-dependency of the effect of TNF-{alpha} on DC migration. The TNF-{alpha} effect is transmitted via cross-linking the membrane-bound TNFR I (p55; CD120a) or TNFR II (p75; CD120b) [27 ]. Purified blood DC were shown to express TNFR II but undetectable levels of TNFR I [28 ]. Similarly, we found that a variable number (24–53%) of moDC also express TNFR II, which is down-modulated by TNF-{alpha} (unpublished observations). It is known that soluble TNFRs may function as TNF-{alpha} decoys [29 ] or may actually enhance TNF-{alpha} activity [30 ]. This phenomenon may account for the higher activity seen with low concentrations of TNF-{alpha} on moDC adhesion to FN and modulation of cAMP content. Although the effects of low concentration of TNF-{alpha} on moDC adhesion to FN might not be relevant in the context of an inflammatory response, they could be important in order to induce the low rate of DC migration existing in noninflamed tissues [11 ].

A link between TNF-{alpha} and the cAMP system has been postulated before in other cell types [18 , 19 ]. In endothelial cells, the cytokine increases PDE activity [18 ]. As the effect of TNF-{alpha} on cAMP intracellular levels and on moDC adhesion and immunostimulatory capacity was reverted in the presence of a PDE inhibitor, it seems likely that a similar mechanism operates in moDC. However, cAMP levels in the presence of TNF-{alpha} plus IBMX were significantly lower than with IBMX alone. This phenomenon can be explained by a noncomplete inhibition of PDE by IBMX.

If a decrease in cAMP levels accounts for the inhibitory effect of TNF-{alpha} on moDC adhesion, an increase in cell adhesion should be expected in the presence of a PDE inhibitor or cAMP analogs. This prediction was not evident in our experimental conditions. There is no ready explanation for this discrepancy. However, it is possible that basal levels of cAMP are enough to trigger activation of integrins and adhesion processes. Therefore, the increase in cAMP content that occurs on "resting moDC" will not result in an increased adhesion, unless integrins were previously inactivated. This idea is reinforced by the lack of change of integrin expression on moDC after "short" (30–60 min) treatment with TNF-{alpha} (unpublished observations) or cAMP analogs treatment (Fig. 4) and the increased adhesion of nonadhered, TNF-{alpha}-treated cells induced by cAMP analogs or a PDE inhibitor. It is worthwhile to mention that in apparent contradiction with our results, the expression of CD49d integrins may be induced on moDC after TNF-{alpha} treatment. However, this effect has been observed after 48–96 h of TNF-{alpha} addition [31 ].

The cAMP-dependent protein kinase (PKA) is activated as a result of interaction with cAMP [32 ]. The cAMP-PKA pathway can regulate cell adhesion, cytoskeletal structure, and focal contact formation [33 , 34 ]. Binding to integrins modulates cAMP-PKA pathway and stimulates an increase [35 ] or decrease in intracellular cAMP [36 ]. The relationship between cAMP levels and adhesion properties might depend on the cell type, state of activation [37 ], and compartmentalization of PKA at different subcellular locations. In monocyte-derived macrophages, cAMP analogs induce cell rounding and disassembly of structures that represent points of contact with substrate [38 ]. On the contrary, cAMP analogs attenuate the migration process of monocytes [39 ].

The duration of TNF-{alpha} treatment seems to be an important factor for integrin activation and IL-12 production. Immature DC treated with low concentration (0.2 ng/ml) of TNF-{alpha} for four days do not produce IL-12 [40 ]. However, we observed IL-12 production in nonadhered moDC after a 30-min treatment with similar TNF-{alpha} concentrations. This apparent contradiction may be related to the kinetics of cytokine production in maturing DC. In this sense, Langenkamp et al. [41 ] showed that DC produces IL-12 during a narrow time window and afterwards, becomes refractory to further stimulation. It is well known that DC maturation causes a dramatic up-regulation of MHC molecules I and II, costimulatory molecules, adhesion molecules, and the expression of CCR7 [11 , 42 ]. After 4 h of LPS stimulation, DC up-regulate MHC class II and CD86 [43 ]. Shortly after TNF-{alpha} treatment, IL-12 is produced even before any changes on those molecules. However, changes on DC markers are seen following 16 h of the adhesion assay. These observations are in agreement with the dynamics of DC response after exposure to pathogens [44 ].

The relationship between a decrease in cAMP content and the inhibition of adhesion elicited by TNF-{alpha} on moDC may have physiological implications. Resident, immature DC exhibit low PDE4 activity [45 ] and undetectable or low IL-12 content. TNF-{alpha} and proinflammatory cytokines released during an inflammatory process start a DC maturation that would involve an increase in PDE activity, which results in lower cAMP levels, IL-12 production [46 , 47 ], and their migration to the lymph node. Because IL-12 plays a central role in the immune system by skewing the immune response toward T helper 1 (Th1)-type responses, it is tempting to speculate that cytokines that decrease DC cAMP content would induce DC migration toward lymph nodes to elicite a Th1 immune response. Indeed, the modulatory effects of adenosine and cAMP on different cells of the immune response have been well characterized [48 ]. It is worthwhile to mention that at the time of submitting this manuscript, Kambayashi et al. [49 ] demonstrated that antigen presentation ability of murine DC was suppressed by camp-elevating agents via augmentation of IL-10 release.

For the first time, we described herein a relationship between cAMP content, adhesive properties of moDC, and early production of IL-12. We propose that the "early" events elicited by TNF-{alpha} on DC might use the cAMP pathway to modulate their physiology.


   ACKNOWLEDGEMENTS
 
This work has been funded by grants from CONICET PIP0905 to H. E. C., AGENCIA BID1202 OC-AR PICT04775 to R. E. R., "Beca Ramón Carrillo-Arturo Oñativia" to H. E. C. and R. E. R., and Universidad de Buenos Aires (TM16) to L. F. We thank Marcos Barbosa and Florencia Quiroga for their technical assistance. We also thank Max-Delbruck-Centrum and Dr. Martin Lipp for providing the mAb CCR7. We also appreciate the generous gift of [125I]-cAMP by Dr. Omar Pignataro. We thank Dr. G. Rabinovich for helpful discussions of the manuscript.

Received December 18, 2001; revised February 12, 2002; accepted February 13, 2002.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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