* Laboratory of Immunology, Istituto Dermopatico dell’Immacolata, IRCCS, Rome, Italy; and
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
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Key Words: neurotrophin • ultraviolet B radiation • dendritic cells • Bcl-2
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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.
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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 [-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.
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 View larger version (41K): [in a new window] |
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.
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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).
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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.
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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).
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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.
Received December 11, 1999; revised February 28, 2000; accepted February 29, 2000.
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