Evidence for a restricted rather than generalized stimulatory response of skin-derived human mast...

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Journal of Neuroimmunolo

Evidence for a restricted rather than generalized stimulatory response

of skin-derived human mast cells to substance P

Sven Guhl1, Hae-Hyuk Lee1, Magda Babina1, Beate M. Henz1, Torsten ZuberbierT,1

Department of Dermatology and Allergy, Charite, Campus Mitte, Schumannstr. 20/21, D-10117 Berlin, Germany

Received 26 November 2004; accepted 28 February 2005

Abstract

To resolve the controversy regarding substance P (SP) mediated stimulation of mast cells (MC), we demonstrate that SP triggers histamine

release from purified human skin MC (sMC), but contrast to stimulation via FcqRI, does not effect the production of TNF-a or IL-8.

Conversely, both anti-IgE and SP are suppressive in terms of IL-6. By quantitative RT-PCR, the amount of templates at baseline (per 25 ng

total RNA) is 2178 (IL-6), 2,665 (IL-8) and 94 (TNF-a), and remains unaltered by SP. Contrast to sMC, LAD2 MC respond to SP with

stronger histamine release and robust TNF-a production in an only partially neurokinin-1R mediated manner, while histamine release of sMC

is chiefly mediated by this receptor. We conclude that human sMC are responsive to SP in a selective manner by eliciting degranulation

without the induction of cytokines and that SP-triggered cytokine production varies among MC subtypes, likely through differences in

signaling mechanisms.

D 2005 Elsevier B.V. All rights reserved.

Keywords: Mast cells; Substance P; Histamine; Cytokines; Neurokinin receptors

1. Introduction

Mast cells (MC) are specialized myeloid cells involved in

allergic reactions and innate immunity towards intruding

pathogens, based in part on the presence of various

receptors that can signal activation or danger, combined

with the ability to produce numerous mediators, including

preformed biogenic amines, rapidly de novo generated lipid-

derived factors, as well as cytokines and growth factors

(Metcalfe et al., 1997; Artuc et al., 1999; Maurer et al.,

2003). Unlike the previous view that MC activation

proceeded through a simple on and off mechanism resulting

in the simultaneous generation of most or all mediators, it

has become obvious over the years and through various

studies that MC distinguish between different stimulatory

0165-5728/$ - see front matter D 2005 Elsevier B.V. All rights reserved.

doi:10.1016/j.jneuroim.2005.02.015

T Corresponding author. Tel.: +49 30 450 518135; fax: +49 30 450

518919.

E-mail address: [email protected] (T. Zuberbier).1 Member of GA2LEN (Global Allergy and Asthma European Network).

signals from the outside by responding with the production

of a specific and restricted spectrum of soluble mediators.

Substance P (SP) represents a potent neuropeptide acting

primarily via neurokinin-1 and -2 receptors (NK1R and

NK2R). It is abundant both in the periphery and in the central

nervous system, and the release of SP from nerves is induced

by danger signals and by stress (Mantyh, 2002). SP is,

however, produced by additional cells and has even been

localized to MC in the skin (Toyoda et al., 2000).

Importantly, SP is believed to evoke immunoinflammatory

responses and to participate in disease conditions such as

allergic asthma (Joos, 2001), and the close proximity

between MC and nerves has early on stimulated significant

research into the communication between these two cell

types. In fact, it was demonstrated by in vivo and in vitro

studies that the triggering of nerves in different organs leads

to inflammation through MC degranulation and that the

neuronal stimulation of MC relies, to a significant part, on SP

(Suzuki et al., 1999; Singh et al., 1999; Saban et al., 2002).

However, the studies also revealed that MC responsiveness

to SP stimulation may differ substantially among different

gy 163 (2005) 92–101

S. Guhl et al. / Journal of Neuroimmunology 163 (2005) 92–101 93

species and MC subsets. For example, murine MC in the skin

and peritoneal cavity respond to SP by histamine release,

while BMMC generated in the presence of IL-3 only become

responsive to SP treatment upon further pretreatment with

IL-4+SCF (Karimi et al., 2000). In addition, rat MC

subtypes also differ considerably in their response to SP

(Amano et al., 1997; Azzolina et al., 2003; Cocchiara et al.,

1995; Bradesi et al., 2001; Cocchiara et al., 1999). In

humans, skin MC that represent typical MCTC type MC (MC

that express both tryptase and chymase) have been regarded

as SP responders, but this has not been studied in detail with

purified MC, and from the available studies, there even

appears to be variation among individual subjects, especially

with regard to the effects apart from histamine release

(Columbo et al., 1996; Okayama et al., 1998; Okabe et al.,

2001). Human pulmonary MC have also been reported to

release histamine on SP stimulation (Heaney et al., 1995),

whereas highly purified MC from human intestinal tissue

have been shown to be resistant to SP stimulation and to lack

SP receptors (Bischoff et al., 2004).

The recent optimization of MC purification protocols to

obtain homogeneous cell populations in large quantity from

human skin has now allowed us to address the question of

the exact response pattern of purified skin MC to SP in

comparison with immunological stimulation via FcqRI.In this study, we show that skin MC can be triggered by

SP to rapidly release histamine. However, steady-state

transcript levels of pro-inflammatory cytokines remain

unaltered following SP administration in skin MC, and this

is reflected by ELISA of their respective protein products.

IL-6 concentrations, on the other hand, even decrease

significantly in the presence of SP. Conversely, the MC

sarcoma cell line LAD2 responds to SP by more

pronounced histamine release and the production of large

quantities of TNF-a. Whereas the skin MC response to SP is

mediated predominantly by NK1R, NK1R independent

activation seems to account for the bulk histamine release

of LAD2 cells. The data imply that skin MC respond to SP

stimulation by the release of preformed mediators but that

this type of stimulation does not affect the transcription of

cytokine genes in the prototype connective tissue MC. In

addition, the study provides direct evidence that the

response pattern of MC to SP is strongly cell subtype

dependent through differential contributions of at least two

separate signaling mechanisms.

2. Materials and methods

2.1. Mast cells

Human MC were isolated from adult breast skin, as

described recently (Babina et al., 2004). In brief, skin was

cut into strips and treated with dispase (Boehringer-

Mannheim, Mannheim, Germany) at 0.5 mg/ml and 4 8Covernight. After removal of the epidermis, the dermis was

chopped in small pieces and digested with collagenase at 10

mg/ml (type 4, Worthington, Lakewood, NJ), hyaluronidase

at 5 mg/ml (type 1S, Sigma), DNAse I at 10 Ag/ml (both

from Roche, Basel, Switzerland), and 5 mM MgSO4 for 1 h

at 37 8C. After a second digestion step of 1 h at 37 8C, cellswere separated from the remaining tissue by three steps of

filtration. MC purification from these dispersates was

achieved by positive selection using anti-c-kit mAb

YB5.B8 (kindly provided by Dr. L. K. Ashman), goat-

anti-mouse-Ig coated magnetic beads (Miltenyi Biotec,

Bergisch Gladbach, Germany), and an Auto-MACS sepa-

ration device. MC purity in these preparations typically

exceeded 95%, as assessed by acidic toluidine blue staining

(0.1% in 0.5 N HCl).

The LAD2 MC sarcoma cell line was recently estab-

lished from a patient with MC sarcoma (Kirshenbaum et al.,

2003) and kindly provided by Dr. D. D. Metcalfe.

2.2. Histamine release

Histamine release assays were performed as described

elsewhere (Guhl et al., 2005). In brief, cell suspensions were

divided into aliquots, washed twice with PAG-CM (PIPES

albumin glucose buffer containing Ca2+ and Mg2+),

resuspended at 4�105 cells/ml and challenged for 30 min

at 37 8C with SP at different concentrations or mouse-anti-

human IgE (dilution 1 :20,000), or kept in buffer only for

spontaneous release. Supernatants were stored at �20 8Cuntil measurement. Total cellular histamine content was

assessed upon cell lysis with 1% perchloric acid. Histamine

amounts were determined by an automated fluorescence

method (Siraganian, 1975), using an autoanalyzer (Borg-

wald Technik, Hamburg, Germany). To delineate the

involvement of the NK1R, skin MC and LAD2 cells were

preincubated with the specific NK1R antagonist L732,138

(Sigma, Taufkirchen, Germany) at 20 AM or vehicle control

(ethanol) for 1 h prior to the addition of SP (30 AM) or

buffer (for blank release).

2.3. ELISA

MC were plated in culture dishes at 1�106/ml in RPMI

medium (supplemented with 10% heat-inactivated fetal calf

serum, 4 mM l-glutamine, and antibiotics; all from

Seromed, Berlin, Germany) and kept at 37 8C for 24 h,

either untreated, or stimulated with SP at the concentrations

indicated, mouse-anti-human IgE (dilution 1 :20,000), or a

combination of both stimuli. After incubation, supernatants

were removed, remaining cells and debris pelleted, and

aliquotted cell free supernatants kept at �80 8C until

assaying for cytokine concentration. Remaining cells were

lysed at 1�106/ml in lysis buffer (1% Triton X-100, 50 mM

Tris–HCl, pH 8.0, 150 mM NaCl, 100 Ag/ml PMSF, 1 Ag/ml

aprotinin, and 1 Ag/ml leupeptin) for 30 min on ice,

centrifuged at 20,000 �g and the supernatants thereof were

also used for the quantitation of cytokines by ELISA.

S. Guhl et al. / Journal of Neuroimmunology 163 (2005) 92–10194

Concentrations of tumor necrosis factor-a (TNF-a) were

determined by a high-sensitivity enzyme-linked immuno-

sorbent assay, while standard kits were utilized for IL-6 and

IL-8 ( all from R and D Systems, Wiesbaden, Germany), as

detailed by the supplier.

2.4. Quantitative reverse transcription-PCR (qRT-PCR)

qRT-PCR was performed, essentially as described

(Babina et al., 2005). Briefly, total RNA was isolated using

the RNeasy Total RNA Kit, digested with RNAse free

DNAse (Qiagen, Hilden, Germany), and quantitated by the

RiboGreen RNA Quantitation Kit (Molecular Probes,

Leiden, The Netherlands). Total RNA (1 Ag per 20 Alreaction volume) was reverse-transcribed with a first strand

synthesis kit (Roche Applied Science, Mannheim, Ger-

many), using random priming, as detailed by the manufac-

turer. For standard curve preparation, specific fragments

were produced from cDNA by PCR, electrophoresed, the

corresponding bands excised from the gels, and purified

by the bhigh pure PCR product purification kitQ (Roche

Applied Science). DNAwas quantitated with the PicoGreen

dsDNA Quantitation Kit (Molecular Probes) and mixed with

100 Ag/ml sheared Herring sperm DNA (Gibco BRL)

(Babina et al., 2005). PCR was carried out with the LC Fast

Start DNAMaster SYBR Green kit (Roche Applied Science)

using cDNA from untreated and SP treated MC (correspond-

ing to 25 ng total RNA/assay), in a 20 Al final volume, 4 mM

MgCl2 and 0.4 AM of each primer (final concentration).

The primer pairs were as follows: 5V-ATGTAGCCGCCC-CACACAGA and 5V-CATCCATCTTTTTCAGCCAT for

IL-6; 5V-ATGACTTCCAAGCTGGCCGTGGCT and 5V-TCTCAGCCCTCTTCAAAAACTTCTC for IL-8; 5V-TCTCGAACCCCGAGTGACAA and 5V-TCAGCCACTG-GAGCTGCC for TNF-a. PCR was performed 40 cycles at

95 8C for 20 s, specific annealing temperature (62 8C for IL-6

and IL-8; 65 8C for TNF-a) for 4–5 s and 72 8C for 8 s. For

the quantitation of NK1R and NK2R, cDNA from skin

MC and LAD2 cells was used along with the following

primers: 5V-CAGAGACCATGCCCAGCAGA and 5V-ACCTTGCGCTTGGCAGAGA for NK1R; 5V-GAGA-CAAGGGCTGGGTGTCA and 5V-ATCATGAAGG-

CAGGGCAGGA for NK2R. Transcript copies were

normalized to the housekeeping gene G6PDH, as described

(Babina et al., 2005).

Amplification specificity was checked using melting

curve and gel analysis. Results were analyzed with the

LightCycler Software (Roche Applied Science) using the

second derivative maximum method to set CT. Quantifica-

tion of cDNA samples was performed automatically with

reference to the standard curve.

2.5. Flow-cytometry

Purified MC were washed twice in Ca2+/Mg2+-free PBS,

blocked with human AB-serum (Biotest, Dreieich, Ger-

many), reacted with saturating concentrations (10–20 Ag/ml)

of either rabbit anti-NK1R (ab 466, Abcam, Cambridge, UK)

or with goat anti-NK2R (C-21, Santa Cruz, Heidelberg,

Germany) for 30 min at 4 8C, washed and stained with R-PEor FITC-conjugated F(abV)2 fragment of secondary Ab (goat

anti-rabbit or rabbit anti-goat) for 20 min at 4 8C. After twowashes, cells were analyzed by an EPICS XL flow cytometer

(Coulter Electronics, Krefeld, Germany). For negative

control, cells were stained with goat IgG or rabbit IgG

(Jackson Immuno Research, Dianova, Hamburg, Germany).

3. Results

3.1. Substance P triggers histamine release from purified

human skin MC in a dose-dependent manner

First, we investigated the ability of SP to trigger

histamine release from human skin MC. As shown in Fig.

1A, SP at 30 AM induced histamine release in the range of

22.8% (versus 6.7% spontaneous release), similar to that

induced by immunological stimulation through cross-bridg-

ing of IgE receptors (29.8%; the difference between SP and

anti-IgE mediated stimulation was not statistically signifi-

cant). In addition, SP-triggered histamine release was a

dose-dependent and saturable event, reaching a maximum at

around 30 AM (Fig. 1B). In conclusion, SP is able to trigger

histamine release from pure skin MC preparations.

3.2. SP does not increase the release of pro-inflammatory

cytokines from human skin MC

There is some debate as to whether SP can induce events

in MC other than degranulation. To test this, we treated

purified skin MC preparations with SP at 0.3 up to 30 AM,

or anti-IgE in direct comparison, or a combination of the

two stimuli (SP at 30 AM plus anti-IgE). Supernatants were

collected after 24 h and analyzed by ELISA for TNF-a, IL-

6, and IL-8 contents, cytokines known to be produced by

skin MC (Babina et al., 2004). SP (as well as anti-IgE)

significantly decreased (rather than increased) the amount of

IL-6 that had accumulated in the supernatant (Fig. 2, top).

On the other hand, the concentration of IL-8 remained

unaffected in the presence of SP, while there was a slight

increase in the presence of anti-IgE (Fig. 2, middle), in

accordance with previous data (Babina et al., 2004). TNF-a,

that is highly inducible via the IgE receptor pathway, was

strongly increased by anti-IgE also in this series of experi-

ments, but remained unaffected by SP (Fig. 2, bottom).

Likewise, SP had no effect on the production of any

cytokine when added in combination with anti-IgE (Fig. 2,

last bar). In addition, positive effects from SP on cytokine

levels were not detected when assaying after shorter times of

incubation (4, 8, 12 h, data not shown). The low, yet

detectable intracellular amounts of cytokines that are

extractable from MC after 24 h (Babina et al., 2004), again

0

5

10

15

20

25

30

35

blank anti-IgE substance P

His

tam

ine

rele

ase

(% o

f to

tal)

***

***

0

2

4

6

8

10

12

14

16

18

0 3,75 7,5 15 30 60

SP (µM)

Net

his

tam

ine

rele

ase

*** ***

**

**

A

B

Fig. 1. Histamine release from human skin MC induced by SP in

comparison to anti-IgE. Cells were isolated from human breast skin, and

treated with buffer alone, SP (30 AM) or anti-IgE (1 :20,000) for 30 min

at 37 8C (A) or with different doses of SP (B), as described in the

Materials and methods. Histamine release was assessed by a histamine

analyzer. (A) Gross histamine release (% of complete); (B) net histamine

release (gross histamine release minus blank release). Results are expressed

as meanFSEM of 12 (A) or 7 (B) independent experiments. **p b0.01;

***p b0.001.

0

10

20

30

40

50

60

70

baseline anti-IgE SP 0.3µM

SP 3 µM SP 30 µM anti-IgE +SP 30 µM

IL-6

(p

g/m

l)

**

**

0

1000

2000

3000

4000

5000

6000

7000


SP 3 µM SP 30 µM anti-IgE +SP 30 µM

IL-8

(p

g/m

l)

**

60

80

100

120

140

TN

F-α

(pg

/ml)

** **


remained unaltered in the presence of SP (data not

illustrated), suggesting that the lack of effect was not due

to a defect in cytokine release. Most importantly, while the

absolute amounts of cytokines varied among skin specimens

0

20

40


SP 3 µM SP 30 µM anti-IgE+SP 30 µM

Fig. 2. SP does not induce the release of proinflammatory cytokines from

human skin MC but decreases IL-6 levels. Skin MC were treated with the

different stimuli for 24 h at 37 8C. Cell-free supernatants were collected andanalyzed for IL-6, IL-8, and TNF-a content. Results are the meanFSEM of

8 separate MC preparations. **p b0.01; significantly lower from baseline

control, with either cytokine, the difference between SP 30 AM and SP 30

AM+anti IgE is not significant.

0

100

200

300

400

500

600

700

800

900

baseline SP 30 µM

TN

F-α

(pg

/ml)

**

Fig. 4. SP triggers a vigorous TNF-a response in LAD2 MC. LAD2 cells

were treated with SP at 30 AM for 24 h or kept in medium alone (baseline).

TNF-a levels in supernatants were assessed by ELISA. The data are the

meanFSEM of 4 separate tests.


and thus individuals, the lack of increase by SP was found

with all separate samples investigated for the production of

each cytokine (n =8–12), thereby largely excluding donor-

specific differences in the response to SP that had been

described in the past with regard to LTB4 production by

human MC (Okabe et al., 2001).

3.3. SP does not affect the steady-state levels of cytokine

transcripts in human skin MC

To gain a better quantitative measure of cytokine

transcripts in skin MC and to assess by an additional

method if the transcription of cytokine genes is targeted by

SP, we employed a real-time quantitative PCR technique

and quantitated template numbers in skin MC. In prelimi-

nary studies, experimental conditions for the three primer

pairs, including primer concentrations, annealing temper-

atures and MgCl2 concentrations, were optimized first,

yielding highly pure amplification products. As depicted in

Fig. 3, skin MC expressed significant amounts of IL-6 and

IL-8 transcripts at baseline, while copy numbers for TNF-a

were rather low. This difference corresponds well with the

data for spontaneous release of the three cytokines (Fig. 2

and Babina et al., 2004) and with the fact that TNF-a is

tightly controlled. MC treatment with SP for 8 h had no

effect on any of these transcripts. Thus, with the exception

of IL-6 whose concentration was reduced in the supernatant

upon SP triggering but remained unaffected at the mRNA

level (Figs. 2 and 3), there was a perfect correlation between

the mRNA and the protein level. Taken together, highly

purified skin MC cultured in medium alone express

0

500

1000

1500

2000

2500

3000

3500

ControlIL-6

SP ControlIL-8

SP ControlTNF-α

SP

Tra

nsc

rip

t c

op

ies

pe

r 25

ng

to

tal

RN

A

Fig. 3. Quantitation of cytokine transcripts by quantitative real-time PCR in

human skin MC at baseline and following SP treatment. Skin MC were

treated with or without SP (30 AM for 8 h at 37 8C), and RNA extracted

from each group. cDNA synthesis and real-time PCR were carried out as

described under Materials and methods. The amount of template copies was

assessed relative to standard curves. Results are the meanFSEM of 4

separate MC preparations.

transcripts for both IL-6 and IL-8, while levels of TNF-a

mRNA are low. SP has thus no impact on the concentration

of these transcripts.

3.4. The TNF-a response to SP is MC subtype dependent

To clarify if other MC subtypes may react towards SP

with a similar response pattern as skin MC, we employed

0

10

20

30

40

50

60

70

80

LAD2 skin MC

Net

his

tam

ine

rele

ase

(%)

vehicle control

L732,138

*

*

Fig. 5. Differential involvement of NK1R in the histamine response of

human skin MC versus LAD2 cells. Skin MC and LAD2 cells were

incubated with either NK1R antagonist L732,138 (at 20 AM) or vehicle

control for 1 h, and then stimulated with SP at (30 AM) or buffer for 30 min

at 37 8C. Net histamine release was then determined as described in Fig 1.

The data are the meanFSEM of 5 (LAD2) and 6 (skin MC) independent

assays. *p b0.05.

0

20

40

60

80

100

120

140

160

NK1R

Tra

nsc

rip

t co

pie

s

skin MC

LAD2

*

0

200

400

600

800

1000

1200

NK2R

Tra

nsc

rip

t co

pie

s

skin MC

LAD2

*

A

B

32E

vent

s0

10o 101 102 103 104

32E

vent

s0

10o 101 102

FL2/PE103 104

Fig. 6. Expression of NK1R by human skin and LAD2 MC. (A) Skin MC were investigated for NK1 expression by flow-cytometry. Representative (2 out of 20

cell preparations) histogram overlays showing NK1R on skin MC, one expressing higher, one lower levels. White curve: Control antibody, gray curve: anti-

NK1R antibody. (B) Quantitation of NK1R and NK2R transcripts in skin MC and in LAD2 cells by qRT-PCR. Specific transcripts were normalized to 30,000

copies of G6PDH, as described Babina et al. (2005), and are the meanFSEM of 12 separate cDNA preparations. *p b0.05.



the MC sarcoma cell line LAD2 for comparison. These

cells represent a fairly differentiated MC subtype and

resemble human connective tissue type MC in many

aspects, including the low, yet detectable expression of

MC chymase (Kirshenbaum et al., 2003). When stimulated

by SP, very high levels of TNF-a were surprisingly

generated in these cells, with a 400–500fold induction

over baseline (Fig. 4). The finding for TNF-a thus provides

clear evidence that the response pattern of MC to SP differs

substantially among MC subtypes, even among those that

share many characteristics.

3.5. NK1R involvement in SP-mediated histamine release

differs between skin MC and LAD2 cells

The strongly divergent result for TNF-a led us to assess

the impact of an NK1 receptor antagonist on histamine

release from the two MC subsets. As shown in Fig. 5, LAD2

cells displayed a much higher histamine response than skin

MC on SP mediated stimulation with nearly 70% of the

cellular histamine content being liberated, which is several

times higher than the IgE/anti-IgE inducible process of

LAD2 cells (around 20–30%, not depicted). This is in

contrast to skin MC where the two activation protocols (i.e.

SP and anti-IgE) result in very similar levels of histamine

release (Fig. 1). Even more interesting, while the histamine

response of skin MC could be blocked by more than 65%

with the NK1R antagonist L732,138, the analogous process

of LAD2 cells was only inhibited by ~30%, so that the

amount of histamine released in the presence of L732,138

was still almost 50% of the total cellular content (Fig. 5).

Thus, the vigorous response of LAD2 cells to SP is

mediated to a minor part by NK1R, while NK1R is the

predominant SP signal transducing entity of skin MC.

3.6. NK receptor expression by human skin and LAD2 MC

Based on the above results, we finally tested if the two

MC subsets differed in the expression pattern of NK1 and

NK2 receptors. By flow-cytometry, NK1R was present with

an average of 24.4F4.7% positively staining cells in highly

purified skin MC (n=20). One striking feature was the

presence of a clearly distinct subpopulation of cells that

expressed NK1R at high levels, while a second subpopu-

lation expressed low levels only or was entirely negative

(Fig. 6A). The relative distribution of these populations was

donor-dependent, but the pattern of reactivity was constant.

On the other hand, NK2R expression was low or absent on

flow-cytometric evaluation (not shown). When LAD2 cells

were tested by flow-cytometry, a lower expression of NK1R

was detectable, while NK2R was absent (not depicted). To

quantitate the differences between skin MC and LAD2 cells

directly, transcripts for both receptor subtypes were com-

pared by qRT-PCR (Fig. 6B). Both NK1R and NK2R

specific transcripts were detectable in skin MC, while much

lower levels of both transcripts were found in LAD2 cells.

Together with the data obtained with the NK1R antagonist

(Fig. 5), these data imply that the contribution of NK1R to

the response towards SP differs between LAD2 and skin

MC, and that this difference is, at least in part, the

consequence of reduced NK1R expression levels in the

former cell type.

4. Discussion

Mast cells (MC) are specialized myeloid cells involved in

allergic reactions and innate immunity towards intruding

pathogens (Metcalfe et al., 1997; Galli et al., 1999; Henz et

al., 2001; Malaviya and Georges, 2002). The cells express a

variety of receptors by which they can be activated, but the

outcome of the activation process will differ in dependence

of the receptor subtype engaged by its ligand. MC can be

found in close proximity to nerves (Wiesner-Menzel et al.,

1981), and this has stimulated a large body of research into

the mediators and receptors that might mediate MC–nerve-

interactions. By the use of in vitro and in vivo rodent

models, one prominent mediator turned out to be SP, but the

extent of MC responsiveness towards SP and the partic-

ipation of specific NK receptors in the response have

remained controversial, especially in humans. Human tissue

MC are rare, and their purification is costly, reasons why

these cells have remained less well characterized than their

rodent counterparts. To help resolve the existing controversy

with regard to SP effects, we decided to study the

contribution of SP to skin MC activation, the prototype

connective-tissue MC, and to compare these data with a

further MC model.

We show that MC highly purified from skin specimens

are responsive to SP by which they are induced to secrete

histamine in a way similar to immune stimulation via FcqRI.The effects of SP are known to be mediated predom-

inantly by neurokinin receptors NK1R and NK2R, with

NK2R on smooth muscle and airways mediating part of the

bronchoconstrictor effect of tachykinins, whereas most of

the proinflammatory and immunoregulatory effects of SP

are likely to be transduced by the NK1R subtype (Joos,

2001). In addition, only the NK1R is coupled to the calcium

phospholipid pathway (Guillemain et al., 1992), and a

calcium signal is indispensable for MC histamine release

(Metcalfe et al., 1997). Thus, NK1R appeared the primary

candidate to trigger the histamine response. To address this

issue, we studied the expression of NK1R and NK2R. By

quantitative RT-PCR, skin MC expressed transcripts for

both receptors, but since we detected only NK1R at the

protein level, while the expression of NK2R was low or

absent (Fig. 6A and data not shown), the former receptor

subtype was in fact most likely to mediate the response to

SP. The use of the NK1R antagonist L732,138 eventually

proved that this was indeed the case, where almost 70% of

the histamine release of skin MC could be antagonized by

the compound. Taken together, in contrast to mucosal MC


that lack NK receptors and appear resistant to SP at baseline

(Bischoff et al., 2004), skin MC display NK1R and

responsiveness to SP without the requirement for any

pretreatment, thus distinguishing further these two major

MC subcategories in humans. In comparison to skin MC,

the LAD2 cell line exerts a more robust histamine response

upon SP stimulation (Fig. 5). This is obviously not the result

of enhanced NK1R levels in theses cells. On the contrary,

both NK1R and NK2R levels are substantially lower in

LAD2 cells than in skin MC (Fig. 6). In addition, only

around 30% of the histamine response of LAD2 cells can be

antagonized by L732,138, while the still vigorous release of

~50% of total histamine in the in the presence of the

antagonist implies that SP operates through additional routes

in the MC line. Since a calcium signal is absolutely required

for MC histamine release, other NK receptor subtypes are

unlikely involved in signal propagation in these cells

(Guillemain et al., 1992). Rather, SP seems to exert its

effects mainly in a receptor independent manner in LAD2

cells. In fact, SP is well known to activate G proteins

directly without the necessity for cell surface receptors

(Repke and Bienert, 1987; Maggi, 1997).

In contrast to histamine, there is no evidence that

cytokine genes are directly targeted by signals transduced

by SP in skin MC. And this could be verified at i. the level

of cytokine release after various time points, ii. the level of

cell-associated cytokines, and iii. the level of cytokine

specific mRNAs. In addition, the lack of response is based

on a large amount of independent MC preparations, thereby

largely excluding donor-dependency. In direct comparison,

FcqRI triggering was fully potent to induce TNF-a

production, but again SP had no impact on this response.

The comparison of these two pathways extends the concept

of divergent intracellular pathways being induced by the

two stimuli to skin MC (Columbo et al., 1996).

The only cytokine that was significantly affected by SP

turned out to be IL-6. The negative effect of both

stimulation pathways, i.e. anti-IgE and SP, on IL-6 amounts

in the supernatant (but not on the corresponding transcript),

implies a post-transcriptional mechanism of action that

should be common to both activation pathways. In fact, IL-

6 was recently reported to be a specific target of MC

tryptase by which it is proteolytically cleaved (Mallen-St

Clair et al., 2004). It is highly probable that the reduced

IL-6 levels reflect the degradation of IL-6 by tryptase that

is released together with histamine from MC granules upon

stimulation.

There is much controversy as to the ability of MC to

generate mediators de novo following SP stimulation

(Ansel et al., 1993; Azzolina et al., 2003; Cocchiara et

al., 1995, 1999; Okabe et al., 2001; Bischoff et al., 2004).

One group reported that skin MC express higher amounts

of TNF-a mRNA and protein, but not of any other

cytokine, following SP stimulation (Okayama et al., 1998),

while another study reported rapid release of presumably

preformed TNF-a and IL-8 from enriched skin MC

populations (Gibbs et al., 2001). The reasons for these

contradictory results may include the level of MC purity

achieved in the previous settings and thus indirect effects

from other cells, differences in the purification protocols,

and most importantly, the in vitro pretreatment of the cells

prior to their testing for SP mediated effects. In contrast,

we used the cells without any in vitro manipulation in

order to be as close as possible to the in vivo situation. In

addition, we used highly pure MC preparations with

virtually no residual contaminants, largely excluding

indirect effects of SP on MC that could be mediated by

other cells. That the MC subtype is in fact decisive was

finally proven by our finding that the MC line LAD2 that

displays many features of mature human MC (Kirshen-

baum et al., 2003), responds to SP by vigorous TNF-a

production (Fig. 4). Together with the pronounced histamine

response of LAD2 cells towards SP, the result obtained for

TNF-a may imply that a more sustained activation of G

proteins (followed by other signal transducers, e.g. p38

MAPK and JNK (Azzolina et al., 2002) is required to trigger

TNF-a production, while a lower level of activation may be

sufficient to induce histamine release (albeit at lower level),

as seen in skin MC. From the combined results, it is

conceivable to postulate that SP operates via at least two

distinct mechanisms, one that employs the NK1R and

another that is independent of NK1R. While the latter makes

up for the largest portion of the LAD2 response, its

contribution to skin MC stimulation (if any) is only minor.

Based on the available literature, the second pathway seems

to proceed through receptor independent activation of G

proteins (Repke and Bienert, 1987; Maggi, 1997), although

future efforts will be required to formerly prove this for

LAD2 cells. The focus of the present study, however, was to

delineate the response pattern of skin MC, in which the

receptor independent pathway does not seem to play a major

role. It needs to be emphasized that although distinct

mechanisms may operate in different MC subtypes (that

would or would not result in the production of cytokines),

there is no indication that cytokines are induced in more

physiological contexts, i.e. in normal skin MC without in

vitro pretreatments, thus resembling conditions of a natural

tissue environment. The strongly divergent data obtained for

the two MC subtypes employed in the present study also

demonstrate that caution is required when results acquired

with one single MC type are transferred to the entire lineage

or to normal human MC. In fact, together with the data

published recently on mucosal MC (Bischoff et al., 2004),

MC in human tissues (in contrast to cell lines or animal

models) appear much less susceptible to SP stimulation,

possibly by downregulating (a) critical component(s) of the

SP signal transduction machinery and/or by changes in

membrane architecture, resulting in a strongly diminished

NK1R independent stimulation. The identification of the(se)

factor(s) will have to await future work. The present study,

however, clearly demonstrates that MC subtypes are

strongly divergent in terms of SP responsiveness, and that


human skin MC are much less susceptible to SP triggering

than has been suspected so far.

In conclusion, we have shown here that purified human

skin MC show a limited, yet detectable response towards

SP: They express NK1R by which they can be stimulated

to release preformed histamine stored in MC granules,

confirming previous reports (Columbo et al., 1996; Okabe

et al., 2001; Okayama et al., 1994). However, SP does

obviously not trigger cytokine transcription in highly

purified skin MC nor does it lead to increased cytokine

levels in MC supernatants. SP may, on the contrary,

reduce the bioavailability of cytokines (i.e. IL-6) in the

vicinity of MC through the release of degrading enzymes,

such as tryptase. We conclude that in human skin, nerve-

derived SP may stimulate MC to participate in acute

responses but seems unlikely to contribute significantly to

late-phase responses, such as those found in most allergic

settings, since these latter rely to a significant part on MC-

derived cytokines. On comparison with the results

obtained for LAD2 MC, we speculate that human skin

MC downregulate components required for an NK1R

independent pathway of SP signal transduction during

terminal differentiation.

Acknowledgments

This work was supported by a grant from the German

Federal Ministry of Education and Research for the Clinical

Study Group Allergy (CMBF, Klin. Forschergruppe Aller-

gologie, TPV II, 01GC0002). We thank Anke Herrmann and

Renate Franke for expert technical assistance with quanti-

tative RT-PCR.

We are also grateful to Dr. Metcalfe (NIH, Bethesda) for

providing the LAD2 cell line.

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