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(Journal of Leukocyte Biology. 2000;68:104-110.)
© 2000 by Society for Leukocyte Biology

Ligand activation of nerve growth factor receptor TrkA protects monocytes from apoptosis

Andrea la Sala*, Silvia Corinti*, Monica Federici*, H. Uri Saragovi{dagger} and Giampiero Girolomoni*

* Laboratory of Immunology, Istituto Dermopatico dell’Immacolata, IRCCS, Rome, Italy; and
{dagger} Department of Pharmacology and Therapeutics, McGill University, Montreal, Canada

Correspondence: Andrea la Sala, Laboratory of Immunology, Istituto Dermopatico dell’Immacolata, IRCCS, Via dei Monti di Creta, 104, 00167 Rome, Italy. E-mail: lasala{at}idi.it


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Nerve growth factor (NGF) receptors are expressed in different cell types outside the nervous system, and increasing evidence indicates that NGF can act as a regulatory molecule during inflammatory and immune responses. In this study, we show that triggering of the high-affinity NGF receptor TrkA with agonists protects monocytes from apoptosis induced by gliotoxin or UVB radiation. TrkA stimulation up-regulates the expression of the anti-apoptotic Bcl-2 family members, Bcl-2, Bcl-XL, and Bfl-1. On the other hand, TrkA stimulation does not change the expression of MHC, CD80, CD86, CD40, and CD54 molecules, nor the antigen-presenting function of monocytes. In addition, during in vitro monocyte to dendritic cell differentiation TrkA expression is progressively lost, suggesting that NGF selectively affects monocyte but not dendritic cell survival.

Key Words: neurotrophin • ultraviolet B radiation • dendritic cells • Bcl-2


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Nerve growth factor (NGF) is the best-characterized member of the neurotrophin family, which includes brain-derived neurotrophic factor, neurotrophin-3, and neurotrophin-4/5. Neurotrophins are critical for regulated development and survival of neuronal cells [1 , 2 ]. The biological effects of NGF are mediated by two classes of receptors [3 ]: the p75 glycoprotein, belonging to the superfamily of tumor necrosis factor {alpha} receptor [4 ], and TrkA, a transmembrane tyrosine kinase of 140 kDa. Stimulation of TrkA is necessary and sufficient to elicit a full biological response to NGF in different cell types [5 6 7 8 9 10 ].

In addition to the neurotrophic activity, NGF can exert different biological effects on various non-nervous cell types. In particular, expression of NGF receptors has been described on immunocompetent cells, such as monocytes [11 ], B lymphocytes [12 ], and T lymphocytes [13 14 15 ]. NGF promotes the differentiation of myeloid progenitor cells [16 , 17 ], induces proliferation and maturation of B lymphocytes [12 , 18 , 19 ], and stimulates the release of inflammatory mediators from basophils and mast cells [20 ]. NGF is also able to increase the expression of Bcl-2, thus conferring resistance to programmed cell death, in mast cells [21 ], B lymphocytes [15 ], and keratinocytes [22 , 23 ]. Moreover, increased levels of NGF have been reported in inflammatory and autoimmune diseases, suggesting that it may have a role in the pathophysiology of these disorders [24 25 26 ].

Less information is available on the expression and functional activity of NGF receptors on professional antigen-presenting cells (APCs) such as monocytes and dendritic cells (DCs). Human blood monocytes express exclusively the TrkA receptor but very little is known about its function [11 ], and no studies have investigated the expression or function of NGF receptors on DCs.

In this study, we show that stimulation of TrkA receptor protects monocytes from apoptosis induced by gliotoxin or UVB radiation and up-regulates Bcl-2 and Bcl-XL expression, but does not affect monocyte APC activity. In addition, TrkA expression is lost during in vitro differentiation of monocytes into DCs.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents and antibodies
NGF was purchased from R & D Systems (Minneapolis, MN). Lipopolysaccharide (LPS; from Escherichia coli, serotype 055:B5) was obtained from Sigma-Aldrich (Milan, Italy), and soluble CD40 ligand (CD40L) from Alexis Biochemicals (San Diego, CA). The anti-TrkA agonist mouse monoclonal antibody (mAb) 5C3 (IgG1) has been described previously [27 , 28 ]. Fluorescein isothiocyanate (FITC)-conjugated anti-HLA-DR (L243, IgG2a) and FITC-conjugated anti-CD14 (M{phi}P9, IgG2b), anti-CD3 (SK-7, IgG1), anti-CD2 (S5.2, IgG2a), anti-CD16 (GO22, IgG1), and anti-CD19 (4G7, IgG1) mAbs were purchased from Becton-Dickinson (San Jose, CA); FITC-conjugated anti-CD1a (HI149, IgG1), FITC-conjugated anti-CD86 (FUN-1, IgG1), anti-Bcl-2 (Bcl2/100, IgG1), and control mouse IgG1 (clone 107.3) from PharMingen (San Diego, CA); FITC-conjugated anti-CD80 (MAB104, IgG1) and anti-CD83 (HB15e, IgG1) from Immunotech (Marseilles, France); FITC-conjugated anti-CD40 (BB20, IgG1) from Ylem (Avezzano, Italy); FITC-conjugated anti-CD54 (84H10, IgG1), and anti-TrkA mAb 6G10 (IgG1) from Calbiochem (Cambridge, MA); anti-MHC class I (W6/32, IgG1) and FITC-conjugated swine anti-rabbit Ig from DAKO (Glostrup, Denmark); rabbit polyclonal anti-Bcl-XL (S-18) from Santa Cruz Biotechnology (Santa Cruz, CA); and FITC-conjugated goat anti-mouse Ig and rabbit Ig from Southern Biotechnology Associates (Birmingham, AL).

Monocyte and DC preparations
Peripheral blood monocytes were separated from blood of healthy donors, following a defined protocol [29 ]. Briefly, peripheral blood mononuclear cells isolated by standard density gradient centrifugation were separated on multistep Percoll gradients (Pharmacia, Uppsala, Sweden), and monocytes recovered from the light density fraction (42.5–50%; >90% CD14+). To generate DCs, monocyte cultures were added with 100 ng/mL granulocyte-macrophage colony-stimulating factor (GM-CSF; Mielogen®; Schering-Plough, Milano, Italy) and 200 U/mL interleukin (IL)-4 (Genzyme, Cambridge, MA). Medium was changed after 3 days. Incubation with 50 µg/mL LPS or 10 µg/mL soluble CD40L for 24 h was performed on day 6 of culture to induce DC maturation. Cells were cultured at 1 x 106 cells/mL in RPMI 1640 (GIBCO-BRL, Gaithersburg, MD) supplemented with 10% fetal calf serum (FCS; Hyclone Laboratories, Logan, UT), 1 mM sodium pyruvate, 0.1 mM nonessential amino acids, 2 mM L-glutamine, 25 mM HEPES, 100 U/mL penicillin, 100 µg/mL streptomycin (all GIBCO), and 0.05 mM 2-mercaptoethanol (Merck, Darmstadt, Germany) (complete medium) at 37°C with 5% CO2.

Flow cytometry analysis
For analysis of surface marker expression, cells were recovered at the indicated time points, washed with 2% FBS, 0.01% NaN3 PBS, and then stained with FITC-conjugated mAbs or unconjugated primary mAbs followed by FITC-conjugated goat anti-mouse Ig. For intracellular staining, cells were fixed with 2% paraformaldehyde in PBS (15 min at 4°C), washed, and permeabilized with 0.5% saponin and 1% FCS in PBS (20 min at 4°C) before incubation with mouse anti-Bcl-2 or rabbit anti-Bcl-XL Ab, followed by FITC-conjugated goat anti-mouse and FITC-conjugated swine anti-rabbit Ig, respectively. In control samples primary Ab was substituted with matched isotype control Ig. Cells (104 cells/sample) were analyzed in a FACScan with Cell Quest software (Becton Dickinson, Mountain View, CA).

Apoptosis
Monocyte cultures added or not with NGF (25–400 ng/mL) or 5C3 mAb (10 µg/mL = 67 nM) were left untreated, stimulated with 5 µM gliotoxin (Sigma), or exposed to ultraviolet B (UVB) radiation (20 mJ/cm2) using two unfiltered TL-12 fluorescent tubes (Philips, Hamburg, Germany; 250–400 nm with a peak at 313 nm). The UVB dose was measured with a radiometer equipped with a SCS 280 photodetector (International Light, Newburyport, MA). In control conditions, cells were stimulated with 10 µg/mL control IgG1 or 200 ng/mL NGF in the presence of 200 nM K252a (Calbiochem, Cambridge, MA). After 6 h incubation at 37% with 5% CO2, monocytes were harvested and washed with cold PBS. Cells were then stained with FITC-conjugated annexin V and propidium iodide using the Annexin V-FITC apoptosis detection kit from Genzyme (Cambridge, MA) as per the manufacturer’s protocol. A total of 10,000 events was analyzed by multiparameter flow cytometry immediately after staining.

RNA isolation, reverse transcription polymerase chain reaction (RT-PCR) analysis, and RNase protection assay (RPA)
Freshly isolated monocytes and differentiating DCs (days 1–7) were subjected to immunomagnetic separation with anti-CD2, anti-CD19, and anti-CD16 mAb followed by goat anti-mouse Ig-coated magnetic beads (Dynal, Oslo, Norway) to remove contaminating lymphocytes. This procedure gave >97% CD14+, CD3-, and CD19- cells on day 0, and >98% pure CD1a+, CD14-, CD3-, and CD19- DC preparations at day 7. PC12 cells were obtained from the American Type Culture Collection (Manassas, VA). Total RNA was extracted with a modified guanidine isothiocyanate-acid phenol protocol with the UltraspecTM RNA Isolation System (Biotecx, Houston, TX), as described [30 ].

For RT-PCR analysis, 1 µg RNA was reverse transcribed to cDNA using oligo-(d)T primers, and then the cDNA amplified by PCR for 25 (ß-actin) or 35 (TrkA) cycles (94°C for 1 min, 60°C for 1 min, and 72°C for 1 min) using the GeneAmp RNA PCR kit (Perkin-Elmer, Roche Molecular Systems, Branchburg, NJ) and the following TrkA-specific primers [11 ]. Sense primer, 5’-CCA TCG TGA AGA GTG GTC TC-3’; and antisense primer, 5-GGT GAC ATT GGC CAG GGT CA-3’ (amplified fragment size 456 bp). As an internal control for the amount of RNA used the following primers were used: ß-actin-specific primers, sense primer 5’-TGA CGG GGT CAC CCA CAC TGT GCC CAT CTA-3’; and antisense primer, 5’-CTA GAA GCA TTT GCG GTG GAC GAT GGA GGG-3’ (amplified fragment size: 631 bp). The PCR products were resolved on 1% agarose gel in the presence of ethidium bromide, and finally photographed with a UV transilluminator.

For RPA, the multi-probe template set hAPO-2, containing DNA templates for human Bcl-x L/S, Bfl-1, Bik, Bak, Bax, Bcl-2, Mcl1, L32, GAPDH, and the complete kit for this assay system were purchased from PharMingen and used per the manufacturer’s protocol. Briefly, the hAPO-2 DNA template was used to synthesize the [{alpha}-32P]UTP (3000 Ci/mmol, 10 mCi/mL; Amersham Italia, Milan, Italy)-labeled probes in the presence of a GACU pool, using a T7 RNA polymerase. Hybridization with 10 µg of each RNA sample was performed overnight, followed by digestion with RNase A and T1. The samples were treated with proteinase K/sodium dodecyl sulfate (SDS) mixture, extracted with Tris-saturated phenol plus chloroform/isoamyl alcohol (50:1), and finally precipitated in the presence of ammonium acetate. The samples and the 32P-labeled DNA molecular weight markers were loaded on an acrylamide-urea sequencing gel, and run at 50 W with 0.5x TBE. Gels were dried and exposed to Kodak films with intensifying screens.

Mixed leukocyte reaction (MLR)
T lymphocytes were purified from the heavy density fraction (50–60%) of Percoll gradients by two rounds of immunomagnetic depletion through the use of a mixture of anti-HLA-DR and anti-CD19 mAb-conjugated beads (Dynal). The purity of T cells was >95%, as assessed by flow cytometry using an anti-CD3 mAb. Monocytes (untreated or incubated for 16 h with NGF, 5C3 mAb, NGF plus K252a, or mouse IgG1) were washed, irradiated, and then cultured in 96-well microculture plates together with 2 x 105/well allogeneic T lymphocytes in complete medium containing 5% autologous plasma instead of 10% FCS. Co-cultures were pulsed at day 5 with 1 µCi/well [3H]thymidine (Amersham) for about 16 h at 37°C, and then harvested onto fiber-coated 96-well plates (Packard Instruments, Groningen, The Netherlands). Radioactivity was measured in a Top count (Packard Instruments). Results are given as mean cpm ± SD of triplicate cultures.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Monocytes, but not monocyte-derived DCs, express the TrkA receptor
TrkA expression was studied in monocytes freshly isolated from peripheral blood and in monocytes cultured in the presence of GM-CSF and IL-4, a procedure that induces differentiation into DCs [31 , 32 ]. CD14+ cells freshly isolated from peripheral blood expressed discrete amounts of TrkA receptor both at protein and mRNA levels as detected by flow cytometry and RT-PCR analysis, respectively (Fig. 1 ). During culture with GM-CSF and IL-4, TrkA mRNA was reduced after 24 h and was already not detectable after 3 days (Fig. 1B) . TrkA protein declined progressively to very low levels after 3 days and disappeared completely after 7 days, paralleling the loss of CD14 and the acquisition of CD1a, a DC-specific marker (Fig. 1A) . Stimulation of day 6 DCs with LPS for 24 h induced DC maturation as demonstrated by induction of CD83 expression, but was unable to restore TrkA expression. Similarly, incubation of DCs with soluble CD40L (1 µg/mL) could not induce TrkA, although CD40 triggering effectively promoted DC maturation. Consistent with the loss of TrkA expression, NGF did not affect the survival, membrane phenotype, or antigen-presenting function of monocyte-derived DCs (data not shown).



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  		Figure 1. CD14+ monocytes express TrkA, which is lost during monocyte to DC differentiation. Peripheral blood monocytes were either used immediately or cultured in the presence of GM-CSF and IL-4 to induce DC differentiation. LPS (50 µg/mL) was added on day 6 for 24 h to promote DC maturation. At the indicated time points cells were collected and examined by flow cytometry (A) or RT-PCR analysis (B). In panel A, the numbers indicate the mean fluorescence intensity subtracted from the fluorescence of isotype-matched control Ab (histograms with light lines). For RT-PCR analysis, cells were deprived of contaminating lymphocytes by negative immunomagnetic separation with anti-CD2, anti-CD19, and anti-CD16 mAbs followed by goat anti-mouse Ig-coated magnetic beads. RNA from PC12 cells (lane 6) served as positive control.

 
TrkA receptor protects monocytes from apoptosis
Because NGF has been reported to interfere with programmed cell death in different cell types including mast cells, keratinocytes, and B cells [12 , 21 , 22 ], we first examined the role of TrkA in the regulation of monocyte apoptosis. Expression of prophagocytic signals such as phosphatidylserine on the cell membrane is an early event during apoptosis [33 ]. Thus detection of phosphatidylserine exposure by FITC-labeled annexin V was used to study monocyte apoptosis in non-permeabilized cells. FITC-annexin V/propidium iodide double staining allowed us to distinguish early apoptotic (annexin V+/PI-) from necrotic (annexin V+/PI+) cells. After 6 h of exposure to gliotoxin, a fungal product inducing apoptosis on a variety of cell types, including monocytes [34 ], or physiological doses of UVB radiation, monocytes showed a significant increase in annexin V binding, but not propidium iodide staining. Incubation of monocytes with NGF markedly reduced the percentage of cells undergoing apoptosis (Fig. 2 and Fig. 3A ). This effect was TrkA mediated because apoptosis was restored by the addition of the alkaloid K252a, a specific inhibitor of the tyrosine kinase activity of Trk receptors [35 , 36 ]. Moreover K252a alone was not able to induce apoptosis, suggesting that it acted by interfering with NGF signaling and not exerting a direct toxic effect. In addition, protection from apoptosis was obtained by treating monocytes with the agonist anti-TrkA mAb 5C3, but not with control mouse IgG1. The anti-apoptotic activity of NGF was dose-dependent, with optimal effect at 200 ng/mL (Fig. 3B) .



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  		Figure 2. TrkA stimulation protects monocytes from gliotoxin- or UVB-induced apoptosis. Monocytes were left untreated, treated with 5 µM gliotoxin, or exposed to 20 mJ/cm2 UVB radiation to induce apoptosis. Five minutes before the addition of gliotoxin or UVB exposure, monocyte cultures were treated with NGF (200 ng/mL) or mAb 5C3 (10 µg/mL). As control, cells were also stimulated with NGF and the tyrosine kinase inhibitor K252a (200 nM) or isotype-matched control Ig (10 µg/mL). After 6-h incubation at 37°C with 5% CO2, monocytes were analyzed for propidium iodide staining and annexin V binding by double-color flow cytometry. Early apoptotic cells are characterized by high annexin V binding and low propidium iodide staining (lower right quadrants), whereas late apoptotic and necrotic cells show high propidium iodide and annexin V fluorescence (upper right quadrants). Numbers indicate percentage of cells in the quadrant.

 


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  		Figure 3. TrkA protects monocytes from apoptosis. (A) Monocytes were treated with 5 µM gliotoxin or 20 mJ/cm2 UVB radiation in the presence of 200 ng/mL NGF, NGF plus 200 nM K252a, 10 µg/mL 5C3 mAb or mouse IgG1, and analyzed for annexin V binding as described in Figure 2 . Results represent the mean (± SD) percentage of annexin V-positive cells from three independent experiments. (B) NGF dose-dependently limits monocyte apoptosis induced by UVB radiation or gliotoxin. Cells were exposed to UVB radiation (circles) or gliotoxin (squares) in the presence of increasing concentrations of NGF (filled circles, filled squares) or NGF plus K252a (open circles, open squares).

 
To investigate the mechanisms underlying the increased resistance to apoptosis of TrkA-stimulated monocytes, the expression of mRNA encoding for the Bcl-2 family proteins was studied by means of RPA with a multiprobe template set, which included both pro-apoptotic and anti-apoptotic Bcl-2 family members. Triggering of TrkA with NGF or 5C3 mAb enhanced mRNA expression of the anti-apoptotic factors Bcl-XL, Bcl-2, and Bfl-1, whereas no changes were observed for the pro-apoptotic factors Bak and Bax (Fig. 4 ). The increased amount of mRNA encoding for Bcl-2 and Bcl-XL was associated with a higher expression of intracellular Bcl-2 and Bcl-XL proteins (Fig. 5A ), the up-regulation of which had already started 6 h after NGF stimulation (Fig. 5B) . Enhanced expression of Bcl-XL, Bcl-2, and Bfl-1 mRNAs as well as Bcl-2 and Bcl-XL proteins induced by NGF was prevented by addition of K252a, and was not observed with control mouse IgG1. Finally, NGF was able to increase Bcl-2 and Bcl-XL protein levels also in monocytes exposed to gliotoxin or UVB radiation (Fig. 6 ).



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  		Figure 4. TrkA activation induces up-regulation of the anti-apoptotic Bcl-2 family members, Bcl-XL, Bfl-1, and Bcl-2 mRNAs. Monocytes were left untreated or stimulated with 200 ng/mL NGF, NGF and 200 nM K252a, 200 nM K252a, 10 µg/mL 5C3 mAb, or 10 µg/mL mouse IgG1. After 12 h, total RNA was extracted and RPA performed as described in Materials and Methods. Expression of mRNA for the housekeeping genes GAPDH and L32 is the control for the amount of RNA used.

 


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  		Figure 5. TrkA stimulation induces up-regulation of Bcl-2 and Bcl-XL proteins. (A) Monocytes were cultured in medium alone or in the presence of 200 ng/mL NGF, 10 µg/mL 5C3 mAb, NGF plus 200 nM K252a, or 10 µg/mL mouse IgG1 for 24 h, and then examined for Bcl-2 and Bcl-XL protein expression by flow cytometry. Saponin-permeabilized cells were incubated with anti-Bcl-2 or anti-Bcl-XL mAb (bold line) or irrelevant Ig (light line) followed by incubation with the appropriate secondary FITC-conjugated Ab. (B) Monocytes were cultured in medium alone (open symbols) or in the presence of 200 ng/mL NGF (closed symbols), and at different time points examined for Bcl-2 (squares) and Bcl-XL (circles) expression. Numbers indicate the mean fluorescence intensity subtracted from the fluorescence of isotype-matched control Ab.

 


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  		Figure 6. NGF enhances Bcl-2 and Bcl-XL expression also in gliotoxin- or UVB-treated monocytes. Cells were exposed or not to 5 µM gliotoxin or 20 mJ/cm2 UVB radiation in the presence of 200 ng/mL NGF for 24 h. Thereafter, Bcl-2 and Bcl-XL expression was evaluated by flow cytometry. Dashed line, isotype-matched control Ab; light line, untreated cells; bold line, gliotoxin- or UVB-treated monocytes; dotted line, monocytes exposed to gliotoxin or UVB and incubated with NGF.

 
TrkA triggering does not affect the antigen-presenting function of monocytes
In the next series of experiments we studied whether TrkA activation could affect the APC function of monocytes. Figure 7 shows that incubation of monocytes with NGF or 5C3 mAb for 16 h did not change their ability to activate allogeneic T cells in a primary MLR assay. Consistent with this finding, TrkA stimulation with NGF did not modify membrane expression of molecules relevant to T cell activation, including HLA-DR, MHC class I, CD80, CD86, CD54, and CD40. In contrast, LPS profoundly affected the membrane phenotype of monocytes (Fig. 8 ).



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  		Figure 7. NGF does not affect the antigen-presenting function of monocytes. Graded numbers of monocytes were left untreated or treated with NGF, 5C3 mAb, NGF plus K252a, or mouse IgG1 for 16 h were washed and then co-cultured with 2 x 105/well purified allogeneic T lymphocytes. [3H]thymidine incorporation was measured after 5 days. Results are given as mean cpm ± SD of triplicate cultures.

 


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  		Figure 8. NGF does not alter the membrane phenotype of monocytes. Monocytes were incubated with medium alone or cultured in the presence of 200 ng/mL NGF or 50 µg/mL LPS, and after 16 h examined for membrane expression of the indicated markers by flow cytometry. The numbers indicate the mean fluorescence intensity subtracted from the fluorescence of isotype-matched control Ab (histograms drawn with light lines).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Functional NGF receptors are expressed by different cell types such as B and T lymphocytes, mast cells, melanocytes, and keratinocytes [11 12 13 14 15 ]. Although NGF exerts its biological effects through both TrkA and p75 receptors, much evidence indicates that NGF can act on cells bearing TrkA molecule but lacking p75 receptor [5 6 7 8 9 10 ]. Previous studies have shown that freshly isolated human peripheral blood monocytes express TrkA but not p75 receptor [11 , 12 ], and that TrkA expression is lost during in vitro differentiation into macrophages [11 ]. Although NGF has been reported to trigger respiratory burst activity on human monocytes [11 ], the consequences of TrkA receptor stimulation on other monocyte functions have not been investigated.

In this study, we showed that triggering of TrkA with NGF or an agonist anti-TrkA mAb protects human monocytes from apoptosis induced by the immunosuppressant compound gliotoxin or physiological doses of UVB radiation. TrkA activation was indeed capable of selectively up-regulating mRNA and protein expression of the anti-apoptotic Bcl-2 family members, Bcl-2 and Bcl-XL, and mRNA expression of Bfl-1. These effects were specific because they could be prevented by K252a, a selective inhibitor of the Trk tyrosine kinase. Moreover, NGF was able to increase Bcl-2 and Bcl-XL protein levels also when monocytes were exposed to gliotoxin or UVB radiation. On the other hand, TrkA stimulation did not affect the expression of MHC, CD54, CD80, CD86, and CD40 molecules, and consequently did not change the capacity of monocytes to activate allogeneic T lymphocytes. An important attribute of monocytes is their capacity to differentiate into DCs when cultured in the presence of GM-CSF and IL-4 [30 ]. Incubation of monocytes with GM-CSF and IL-4 together with NGF did not alter the kinetics or the yield of differentiating DCs (data not shown). More interesting was the observation that DCs differentiated from monocytes lost TrkA expression, and hence became insensitive to NGF. Finally, LPS or soluble CD40L induced DC maturation but failed to restore TrkA expression.

NGF has been described to promote cell survival acting through its high-affinity receptor in different cell types outside the nervous system. Autocrine NGF can protect B lymphocytes, mast cells, and keratinocytes from apoptosis through up-regulation of Bcl-2 protein [22 , 23 ]. In addition, NGF secreted by keratinocytes enhances melanocyte survival through increased expression of Bcl-2 protein [37 ]. A key parameter that determines whether a cell will respond to an apoptotic signal is the ratio of death antagonist (Bcl-2, Bcl-XL, Bcl-w, Mcl-1, Bfl-1) to agonists (Bax, Bak, Bcl-Xs, Bad, Bid) belonging to the Bcl-2 family [38 ]. In this context, up-regulation of Bcl-2, Bcl-XL, and Bfl-1 induced by TrkA triggering can well account for the decrease in the number of monocytes undergoing apoptosis after exposure to gliotoxin or UVB radiation. Monocytes do not produce NGF [11, and our personal observation], and are thus dependent on NGF released by other cells for this effect. The resistance of NGF-stimulated monocytes to UVB-induced apoptosis can be relevant in the skin after UVB exposure. UVB radiation causes substantial perturbations of immune responses with suppression of cell-mediated immunity and induction of antigen-specific tolerance, mainly by affecting the skin APCs. In particular, UVB radiation alters the maturation and antigen-presenting functions of epidermal Langerhans cells, and induces their apoptotic cell death [39 , 40 ]. Together with the disappearance of Langerhans cells, UVB radiation induces, in both mouse and human skin, infiltration of monocytes, which are thought to be involved in the induction of immune tolerance [41 ]. These monocytes infiltrate first the dermis and soon after the epidermis and are thus themselves exposed to UV radiation. In this context, NGF released by keratinocytes or skin mast cells [42 , 43 ] may be important for protecting infiltrating monocytes from UVB-induced apoptosis. Of note, UVB radiation increases NGF expression by keratinocytes [44 ]. On the other hand, DCs differentiated from monocytes appear to be insensitive to the anti-apoptotic effects of NGF, a phenomenon that may contribute to DC apoptosis in UVB-exposed skin.

Elevated levels of NGF have been documented in the plasma as well as in the affected tissues of patients suffering from atopic disorders, such as bronchial asthma and rhinoconjunctivitis, or affected by autoimmune diseases including rheumatoid arthritis, lupus erythematosus, systemic scleroderma, multiple sclerosis, and psoriasis [24 , 45 ]. Enhanced survival of inflammatory cells, including monocytes, has been suggested to be an important factor in the establishment of chronic inflammation that characterize both atopic and autoimmune diseases [46 47 48 ]. We thus postulate that NGF may have a role in the persistence of inflammatory responses associated with these disorders by prolonging monocyte survival.


   ACKNOWLEDGEMENTS
 
This work was supported by grants from the Associazione Italiana per la Ricerca sul Cancro, the Istituto Superiore di Sanità (AIDS project), the European Community (Biomed 2 program), and the Ministero della Sanità.

Received December 11, 1999; revised February 28, 2000; accepted February 29, 2000.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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