Axel Lorentz, PhD, Department of Nutritional Medicine and Immunology, University of Hohenheim, Fruwirthstr. 12, D-70593 Stuttgart, Germany. Tel: +49 711 4592 4391; fax: +49 711 4592 4343; e-mail: firstname.lastname@example.org
Abstract Neurotrophins are potent regulators of neuronal cell survival and function. Nerve growth factor (NGF) was shown to reduce apoptosis in cord blood-derived mast cells. Here, we examined the effect of the neurotrophins NGF and neurotrophin (NT)-3 on survival and mediator release of human intestinal mast cells. Mast cells isolated from normal intestinal tissue were cultured in the presence of NGF, NT-3, or stem cell factor (SCF) alone or in the presence of SCF together with each neurotrophin. NGF or NT-3 alone did not promote mast cell survival. In contrast, mast cell recovery was increased twofold when mast cells were cultured with NT-3 in addition to SCF for 14 days compared with control. Mast cell recovery was further increased following a combined addition of NT-3, SCF and IL-4. NT-3 mediated mast cell growth was dependent on the primary receptor for NT-3 TrkC. NGF in combination with SCF or with SCF and IL-4 showed no effect on mast cell survival. Histamine release and histamine content per mast cell remained unchanged, whereas leukotriene C4 release decreased if mast cells were cultured with NGF or NT-3 in addition to SCF. In summary, NT-3 affects mature human mast cells by promoting mast cell survival, whereas NGF does not.
Neuroimmune interactions in the control of health and disease have received increasing attention. This is particularly true for the gastrointestinal tract where the enteric nervous system interact with local sensory receptors, the central nervous system and immune/inflammatory cells including mast cells.1 Intestinal mast cells are sentinels of the immune system located at the host–environment interface and often close to nerves. They act as inflammatory cells by releasing mediators, such as histamine, leukotriene C4 (LTC4) and prostaglandin D2 known to mediate smooth muscle contraction, vasodilatation and mucus secretion.2 Moreover, mast cells regulate activity and distribution of T cells, granulocytes and other cells by releasing these inflammatory mediators, as well as cytokines and growth factors.1–3
Neurotrophins, such as nerve growth factor (NGF) and neurotrophin-3 (NT-3), are known as regulators of neuronal functions. The effects of neurotrophins on immunocompetent cells are beginning to be explored. NGF was shown to promote in vitro growth and differentiation of murine and human mast cells, and to exert chemotactic effects on rat peritoneal mast cells.4–9 In addition, several studies identified NGF as a mast cell activator or priming agent directly inducing murine and human mast cell degranulation in the presence of phosphatidyl serine,10,11 and, as shown for murine mast cells, enhancing production and release of several cytokines and proinflammatory mediators.12–14 The biological activity of the neurotrophins is mediated via receptors of the Trk family. TrkA is known to bind NGF and, to a smaller extent, NT-3. TrkB is the receptor for brain-derived neurotrophic factor (BDNF), but is also able to bind NT-3. The primary receptor for NT-3 is TrkC.15
In this study, we examined the Trk receptor expression, and the effect of NGF and NT-3 on survival and mediator release of mature human intestinal mast cells. Our data demonstrate that NT-3, but not NGF, promotes the survival of human mast cells. Our findings might be of particular importance for our understanding of mast cell-associated diseases, such as allergic inflammation or inflammatory bowel disease.
Materials and methods
Isolation, purification, and culture of human intestinal mast cells
Human intestinal mast cells were isolated from surgical tissue specimens (macroscopically normal border sections), which were derived from patients who underwent bowel resection because of cancer. The local ethical committee gave us permission to conduct this study. The methods of mechanical and enzymatic tissue dispersion yielding single-cell preparations containing 4 ± 2% (mean ± SD) mast cells have already been described in detail elsewhere.16 After overnight incubation in culture medium (RPMI-1640 supplemented with 10% heat-inactivated fetal calf serum, 25 mmol L−1 HEPES, 2 mmol L−1 glutamine, 100 μg mL−1 streptomycin, 100 μg mL−1 gentamycin, 100 U mL−1 penicillin and 0.5 μg mL−1 amphotericin; all cell culture reagents were from Gibco Invitrogen, Paisley, UK), mast cells were enriched by positive selection of c-kit-expressing cells using magnetic cell separation (MACSTM system, Miltenyi Biotech, Bergisch-Gladbach, Germany) and the monoclonal antibody (mAb) YB5.B8 (Pharmingen, Hamburg, Germany) as described.17,18 The fraction containing the c-kit-positive cells (mast cell purity 60 ± 35 %) was cultured at a density of 2 × 105 MC per ml in medium which was supplemented with 50 ng mL−1 of recombinant human stem cell factor (SCF; Amgen, Thousand Oaks, CA), IL-4 (2 ng mL−1, Novartis, Vienna, Austria), NGF (50 ng mL−1, R&D Systems, Minneapolis, MN, USA), NT-3 (50 ng mL−1, PeproTech), or with a combination of these cytokines. To block TrkC-NT-3 interaction, neutralizing anti-human-TrkC-Ak (R&D Systems) or matching isotype control IgG1 (R&D Systems) were used. Half of the culture medium was carefully exchanged weekly, and cytokines and neurotrophins were re-supplemented at the same final concentrations. During the culture period mast cell purity increased to 98–100%. Mast cell purity and recovery (expressed as percent of mast cell numbers at the start of the culture) were determined by cell counting using trypan blue staining (Sigma Chemicals, St Louis, MO, USA), and differentiation of the cells on cytospin smears stained with May–Grünwald/Giemsa (Merck, Darmstadt, Germany). To confirm the mast cell recovery data, histamine content per well was measured by commercial RIA (Coulter-Immunotech, Krefeld, Germany) after washing the cells and subsequent cell lysis. Histamine recovery was calculated as percent of histamine content at the start of culture.
Mediator release assays
After a culture period of 14 days, mast cells were counted, washed in Hepes buffer, and seeded at a density of 0.5–1 × 105 cells per mL in culture medium without antibiotics or cytokines. Mast cells were stimulated by IgE receptor cross-linking using the mAb 22E7 (100 ng mL−1, 90 min incubation time, Hoffmann-La Roche, Nutley, NJ, USA) directed against a non-IgE-binding epitope of the high-affinity IgER α-chain. Histamine release into supernatants was measured by RIA (Coulter-Immunotech) and expressed according to the following formula: (histamine released after IgE receptor cross-linking − histamine released spontaneously in response to buffer control)/total cellular histamine content as determined after cell lysis. LTC4 release was measured by RIA as previously described.19 The release of LTC4 into the supernatant was expressed as a specific mediator release obtained by subtraction of spontaneous mediator release in response to buffer control.
RNA isolation and RT-PCR
Total RNA was prepared from 1 × 105 human intestinal cells containing 98–100% mast cells, and semi-quantitative RT-PCR was performed as described.17,18 The following specific sense and antisense primers were used for the cDNAs of: Glyceraldehyde 3-phosphate dehydrogenase (GAPDH): 5′-ACCACAGTCCATGCCATCAC-3′; 5′-TCCACCACCCTGTTGCTGTA-3′, product size: 452 bp; TrkA: 5′-GGCTCCTCGGGACTGCGATG-3′; 5′-CAGGAGAGAGACTCCAGAGCG-3′, product size: 273 bp; TrkB: 5′-AGCAACCTGCAGCACATCAA-3′; 5′-CTTCCTCCACAGTGAGGTTA-3′, product size: 289 bp; TrkC: 5′-GTGTCTGCAGCAAGACTGAG-3′; 5′-CTTCCTCCACAGTGAGGTTA-3′, product size: 316 bp. The PCR products were separated on 1% agarose gel containing ethidium bromide (500 ng mL−1) and then photographed.
Immunocytochemistry was performed using a mAb against the human nuclear cell proliferation-associated antigen Ki-67 (Clone MIB-1, dilution 1:2, Dianova, Hamburg, Germany), and appropriate isotype control mAbs (mouse IgG1; Southern Biotechnology, Birminghman, AL, USA) as primary antibodies (overnight incubation at 4 °C) and the streptavidin–biotin detection system (Histostain-Plus kit; Zymed, San Francisco, CA, USA). Percentage of apoptotic or necrotic mast cells was assessed using the terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL) in situ cell death detection assay for immunocytochemistry (Boehringer Mannheim, Mannheim, Germany) according to the manufacturer's instructions.
To obtain whole cell extracts, 0.5 × 106, mast cells were lysed in extraction buffer containing 25 mmol L−1 Tris–HCl, pH 7.5, 0.5 mmol L−1 EDTA, 0.5 mmol L−1 EGTA, 0.05% Triton X-100, 10 mM β-mercaptoethanol, supplemented with the protease inhibitor cocktail CompleteTM Mini (Roche Diagnostics, Mannheim, Germany). Protein concentration was determined using the Bio-Rad protein assay (Bio-Rad, Hercules, CA, USA). Cell extracts (10–20 μg protein each) were separated on a 12% SDS-polyacrylamide gel and blotted onto a nitrocellulose membrane (Schleicher and Schuell, Einbeck, Germany) in 0.1% SDS, 20% methanol, 400 mmol L−1 glycine, 50 mmol L−1 Tris–HCL, pH 8.3 at 4 °C for 4 h at 40 V by electroblotting using the Trans-BlotTM Cell system (Bio-Rad). Membranes were blocked with 5% skim milk in PBS containing 0.1% tween overnight. Membranes were probed with anti-TrkA (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-TrkB, TrkC (R&D Systems), and anti-extracellular signal-regulated kinase (ERK = mitogen-activated protein kinase, MAPK)-2, and antiphospho-ERK (MAPK)-1/2 mAbs (Alexis, Lausen, Switzerland). The membranes probed with antiphospho-ERK-1/2 mAb were stripped following probing using Restore TM Western Blot Stripping Buffer (Pierce, Rockford, IL, USA) and probed again with anti-ERK2 mAb. The antigen–antibody complexes were visualized using electrochemiluminescence detection system as described by the manufacturer (NENTM Life science, Boston, MA, USA).
Significance of differences was assessed using the Wilcoxon signed rank test. A value of P < 0.05 was considered to be statistically significant.
Expression of Trk receptors in human intestinal mast cells
Mast cells purified from intestinal tissue and cultured in the presence of SCF or SCF and IL-4, respectively, were either stimulated by IgE receptor cross-linking using the antibody 22E7 or treated with buffer control. Fig. 1A shows the expression of mRNA encoding for the Trk receptors, TrkA and TrkC. We did not detect the expression of mRNA encoding for TrkB (not shown). Moreover, the culture conditions or activation of the cells by IgE receptor cross-linking had no influence on mRNA expression for the Trk receptors. To demonstrate protein expression of the Trk receptors on human intestinal mast cells, mast cell lysates were analysed by Western blot. Confirming the data on mRNA expression, we found the expression of TrkA and TrkC, but not the expression of TrkB (Fig. 1B).
NT-3 promotes growth of cultured human intestinal mast cells
TrkA is known to bind NGF and, to a lesser extent, NT-3. TrkC is the primary receptor for NT-3.15 NGF or NT-3 added to the culture medium had no visible effect on human intestinal mast cell survival or proliferation during culture. Confirming previous data on the crucial role of SCF, in the presence or absence of NGF or NT-3, all mast cells died within 4–9 days unless SCF was added to the culture medium (Fig. 2A).16,17 Noteworthy, we found that the combined addition of SCF and NT-3 to the culture medium for 14 days caused a significant increase of the mast cell recovery (start of the culture was set at 100%) in comparison with the addition of SCF alone from 130 ± 57% to 206 ± 56% (mean ± SD). These data indicate a synergistic effect of SCF and NT-3 on mast cell growth (Fig. 2B). We have shown, previously, that IL-4 is a very potent growth factor for human intestinal mast cells acting also in synergism with SCF.17 Thus, we addressed the question, whether NT-3 further enhances mast cell numbers if added to culture medium in combination with SCF and IL-4. Fig. 2B shows that adding NT-3 to the culture medium supplemented with SCF and IL-4 indeed further enhances mast cell recovery from 232 ± 99% to 311 ± 118% (mean ± SD). Surprisingly, in contrast to NT-3, a combined addition of NGF to the culture medium caused no significant increase of mast cell numbers, neither in combination with SCF alone, nor in combination with SCF and IL-4 (Fig. 2C).
Fig. 3A shows mast cells after culture with SCF alone or with SCF and NT-3 for 14 days. Using light microscopy, we could not detect any differences in cell sizes, metachromasia or the numbers of granules. The growth-promoting activity of NT-3 appeared in a dose-dependent fashion with a maximal effect at 50 ng mL−1 (Fig. 3B). To address the question whether TrkC transduces the NT-3 caused mast cell growth, they were treated with a blocking antibody directed against TrkC during culture with SCF or SCF and NT-3 respectively. Treatment of the cells with 30 μg mL−1 anti-TrkC antibody resulted in a significant inhibition of the NT-3 caused increase in mast cell numbers (Fig. 3C). The data show that the NT-3-mediated mast cell growth depends on TrkC demonstrating TrkC as the relevant NT-3 receptor also on human intestinal mast cells. To elucidate whether the NT-3-mediated increase in mast cell numbers was a consequence of decreased apoptosis or an increased cell proliferation, we performed the TUNEL assay, which is known to identify DNA fragmentation indicating apoptotic and necrotic cells. We found that NT-3 decreases the number of TUNEL positive cells which was most distinctive in the presence of suboptimal concentrations of SCF (range 1–10 ng mL−1) hardly sufficient to provide mast cell survival (Fig. 4A, B). To analyse mast cell proliferation in response to NT-3, immunocytochemistry was performed using an antibody directed against the human nuclear cell proliferation-associated antigen Ki-67. We found that NT-3 caused an increase in Ki-67 positive cells, which was most pronounced in the presence of 10–50 ng mL−1 SCF (Fig. 4A). MAPK ERK1/2 is known to be involved in cell proliferation.20,21 To test activation of the ERK1/2 in response to NT-3, mast cells were treated 1 week after the last feeding with 50 ng mL−1 SCF, 50 ng mL−1 NGF or 50 ng mL−1 NT-3, respectively, for 15 min. In accordance with our previous data,22 phosphorylation of ERK1/2 was clearly found in response to SCF stimulation. ERK1/2 phosphorylation was not found in response to NGF and also not detectable in response to NT-3 (Fig. 4C).
NT-3 and NGF change the release of LTC4 in human intestinal mast cells
We reported previously that IL-4 and IL-3 enhance not only mast cell growth, but also histamine release and LTC4 synthesis following IgE receptor cross-linking.17,23 Noteworthy, addition of NT-3 or NGF to the cultures supplemented with SCF did not affect the release of histamine, but even down-regulates the release of LTC4 from mast cells following stimulation with mAb 22E7 (Fig. 5). NT-3 caused a decrease of the LTC4 release from 5.5 ± 1.9 to 3.6 ± 2.0 ng of LTC4/106 cells. NGF caused a decrease of the LTC4 release from 5.5 ± 1.9 to 4.0 ± 2.2 ng of LTC4/106. NT-3 or NGF by themselves did neither affect the release of histamine or LTC4 nor the histamine content per cell (not shown).
Our study shows expression of TrkA and TrkC, the high-affinity receptors for NGF and NT-3, in mature human intestinal mast cells. Expression of TrkA and TrkC was also found in preparations of human umbilical cord blood-derived mast cells (CBMC). In contrast, the human leukaemia mast cell line HMC-1 as well as highly purified populations of human lung mast cells expressed TrkB in addition to TrkA and TrkC.24 Although mature mucosal mast cells derived from the gut should be more related to mucosal lung mast cells than to cord blood-derived mast cells, we did not detect TrkB expression on intestinal mast cells as described for CBMC. TrkC was recently found on immature mast cells from neonatal mouse skin.25 The data from Metz et al. suggest a role for NT-3 in the maturation of mast cells, such as TrkC-mediated stimulation of the differentiation of pre-existing, less mature mast cells and/or by enhancing the migration of circulating mast cell precursors into the skin. The lack of the low-affinity neurotrophin receptor p75 on human intestinal mast cells is consistent with findings showing the absence of p75 on mast cells from neonatal mouse skin, on mature rat mast cells and on HMC-1.6,24–26 This observation is remarkable concerning the increased mast cell recovery in response to NT-3 demonstrated in this study, because p75 is thought to mediate pro-apoptotic effects of neurotrophins.27,28
A number of neurotrophins, neuropeptides and neurotransmitters has been investigated for their possible role in regulating mast cell growth and function. Among them, NGF gathered the widest attention, as increased MC numbers in tissues of neonatal rats were found following NGF administration.29 In accordance with this finding, NGF was also shown to promote in vitro growth and differentiation of murine and human mast cells.4–9 In contrast, we have demonstrated above that NT-3, promote survival of mature mast cells derived from human gut, but not NGF. NGF, in synergy with SCF, has been described to promote the survival of peritoneal rat mast cells as well as cord blood-derived human mast cells and to play a role in mast cell maturation and differentiation.4,5,8,12 We have not seen anti-apoptotic effects of NGF on human intestinal mast cells as reported for CBMCs.8 These differences between intestinal and cord blood-derived mast cells may be due to a tissue-specific mast cell phenotype. It is well known that mast cells derived from different species and different tissues were found to be heterogeneous not only in terms of morphology and immunohistochemistry but also in functional criteria.30 The only NGF effect on human intestinal mast cells found was the reduction of LTC4 synthesis and release in response to activation by FcɛRI cross-linking. This result was also unexpected because in human basophils NGF strongly enhances LTC4 release in response to various stimuli.19 Interestingly, we found a similar reduction of LTC4 release following treatment with NT-3. The reduction of LTC4 release may be a sign of a protective role of neurotrophins in inflammation of the gut as it has been already postulated elsewhere.31 But, overall, our data suggest that NGF does not play an important role in the relationship between mast cells and intestinal inflammatory diseases.
In contrast to NGF, our data demonstrate that NT-3, in synergy with SCF, clearly enhances human mast cell recovery. This is particularly remarkable, because NT-3 by itself had no visible effect on human intestinal mast cells. The situation changes importantly when mast cells are treated with NT-3 in the presence of SCF. Clarification of the responsible mechanisms is of especial interest for our understanding of the biology of mature human mast cells. NT-3 is known as an important factor in the development of the nervous system.32 NT-3 affects melanocytes, chemotaxis and nitric oxide production of macrophages as well as migration of Schwann cells.33–36 Moreover, NT-3 has been, along with NGF, BDNF and NT-4, described to be a survival and activation factor for eosinophils in patients with allergic bronchial asthma.37 In this study, we found that NT-3 mediates enhancement of mature human mast cell growth in a dose-dependent fashion. Treatment of the cells using a blocking anti-TrkC antibody prior to treatment with NT-3 resulted in a significant, but not total, inhibition of the up-regulated mast cell growth. Thus, NT-3 caused increase of mast cell numbers depends mainly on TrkC, but may also be mediated by TrkA, which acts as a low-affinity receptor for NT-3.38
Increase of mast cell numbers could be a consequence of an increased proliferation or a decreased apoptosis. We found both enhancement of proliferation and a reduction of apoptosis in human intestinal mast cells in response to NT-3. ERK-mitogen-activated protein kinase is known to be involved in cell proliferation.20,21 Activation of ERK was seen in HMC-1 cells in response to NGF stimulation.24 As expected, we did not detect ERK phosphorylation in intestinal mast cells in response to NGF stimulation, but ERK activation was also not found or only hardly detectable, respectively, in response to NT-3. Possibly, this is due to an inappropriate time frame of NT-3 stimulation. Recently, we found higher levels of activated ERK in mast cells after culture with IL-4 in addition to SCF compared with cells cultured with SCF alone.22 The effect of IL-4 on mast cell proliferation is clearly more pronounced than the effect of NT-3. Nevertheless, the detectable enhanced ERK phosphorylation during mast cell culture in the presence of IL-4 is comparatively low. Here, we were not able to detect differences in ERK activation between mast cells cultured with SCF alone or with SCF and NT-3 maybe because of an insufficient sensitivity of the Western blot analysis (not shown). On the other hand, NT-3 may act through anti-apoptotic genes like Bcl-2. In CBMC apoptosis was found to be regulated through Bcl-2 and Bcl-XL.39 NGF was capable of inducing bcl-2 expression in rat mast cells.12 Further studies are necessary to clarify the mechanisms of mast cell regulation by NT-3.
In summary, we did not detect significant effects of NGF on the survival of mature human mast cells derived from gut tissue, as reported earlier for human cord blood-derived mast cells. In contrast, our data demonstrate that NT-3 affects human intestinal mast cells by promoting cell survival. This observation further shows a mechanism for the crosstalk between mast cells and the enteric nervous system in the gut.
The work was supported by the Deutsche Forschungsgemeinschaft (SFB621-A8 and BI 424/2-1)