Variations of neuronal nitric oxide synthase in systemic sclerosis skin

ARTHRITIS & RHEUMATISMVol. 54, No. 1, January 2006, pp 202–213DOI 10.1002/art.21543© 2006, American College of Rheumatology

Variations of Neuronal Nitric Oxide Synthase inSystemic Sclerosis Skin

Lidia Ibba-Manneschi,1 Sirkku Niissalo,2 Anna Franca Milia,1 Yannick Allanore,3

Angela Del Rosso,1 Alessandra Pacini,1 Mirko Manetti,1 Annarita Toscano,1 Paola Cipriani,4

Vasiliki Liakouli,4 Roberto Giacomelli,4 Andre Kahan,3 Yrjo T. Konttinen,2

and Marco Matucci-Cerinic1

Objective. In systemic sclerosis (SSc), derange-ment of the peripheral nervous system is linked tovascular tone dysfunction. Nitric oxide (NO) producedby neuronal nitric oxide synthase (nNOS, NOS-I) mightplay a dynamic role in the control of vascular tone. Thisstudy was performed to verify, by immunohistochemicaland biochemical analyses, the presence and expressionof nNOS and protein gene product 9.5 (PGP 9.5) in SScskin, in different subsets and various phases of thedisease.

Methods. Biopsy samples of clinically involvedskin from 32 SSc patients (12 with limited cutaneousSSc [lcSSc] and 20 with the diffuse form [dcSSc]) andskin samples from 6 healthy controls were either immu-nostained with anti–PGP 9.5 and anti-nNOS antibodiesor analyzed by semiquantitative reverse transcription–polymerase chain reaction and Western blotting.

Results. Immunohistochemical and biochemicaldata showed a decrease in PGP 9.5 and nNOS innerva-

tion and in their messenger RNA (mRNA) levels inlcSSc and dcSSc skin. In the edematous phase of SSc, alight alteration in cutaneous innervation was initiatedand slowly progressed into the sclerotic phase, becom-ing most evident in the atrophic phase. Levels of nNOSmRNA were significantly lower between the edematousphase and the sclerotic phase in both dcSSc and lcSScskin, which was attributable to the earlier occurrence ofmore severe pathologic alterations.

Conclusion. Total cutaneous innervation andnNOS innervation slowly disappear in the skin of SScpatients. Expression of nNOS depends on the severity oftissue damage in SSc, and increased synthesis of NOalso contributes to this process. It remains to be deter-mined whether the changes in cutaneous innervationare due to the disease itself or whether these changescontribute to the pathogenesis and evolution of SSc.

Systemic sclerosis (SSc) is a connective tissuedisorder of unknown etiology that is characterized in itsearly phases by microvascular changes and later isfollowed by typical fibrotic changes in the skin andinternal organs. Microvascular derangement is one ofthe main factors implicated in the pathogenesis of SSc(1). The process of microvascular derangement is char-acterized by endothelial injury, enhanced expression ofadhesion molecules interacting with lymphocytes, pres-ence of perivascular inflammatory cell infiltrates, oblit-erative microvascular lesions, and increasing vessel wallthickness (2).

The endothelial injury that occurs in SSc impairsthe endothelial-dependent control of vascular tone andblood flow. However, functional changes in the peri-pheral nervous system (PNS), such as a reduction insensory and parasympathetic nerves and an increase in�2-receptor activity, have been described (3). A signifi-

Supported by Helsinki University Central Hospital (evo-grantTYH 2235), Finska Lakaresallskapet, Suomen HammaslaakariseuraApollonia, and the Ministero dell’Universita e della Ricerca (MIUR40%).

1Lidia Ibba-Manneschi, MD, Anna Franca Milia, PhD, An-gela Del Rosso, MD, Alessandra Pacini, PhD, Mirko Manetti, BSc,Annarita Toscano, BSc, Marco Matucci-Cerinic, MD: University ofFlorence, Florence, Italy; 2Sirkku Niissalo, DDS, Yrjo T. Konttinen,MD: Helsinki University Central Hospital, Helsinki, Finland; 3Yan-nick Allanore, MD, Andre Kahan, MD: Cochin Hospital, AP-HP,Paris V University, Paris, France; 4Paola Cipriani, MD, VasilikiLiakouli, MD, Roberto Giacomelli, MD: University of L’Aquila,L’Aquila, Italy.

Drs. Ibba-Manneschi and Niissalo contributed equally to thiswork.

Address correspondence and reprint requests to Lidia Ibba-Manneschi, MD, Associate Professor of Anatomy, Department ofAnatomy, Histology and Forensic Medicine, Viale G. B. Morgagni, 85,Florence 50134, Italy. E-mail: [email protected].

Submitted for publication June 1, 2005; accepted in revisedform October 6, 2005.

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cant generalized reduction of calcitonin gene–relatedpeptide (CGRP) and vasoactive intestinal polypeptide(VIP), present in sensory and parasympathetic fibers,respectively, was described in the digital skin of SScpatients (4). A relevant decrease in protein gene product9.5 (PGP 9.5), a pan-neuronal marker (5), was alsoobserved (4). This evidence suggests that neuropeptide-containing nerves contribute to the pathologic processesof SSc digital skin and to vasomotor dysfunction (3,4,6).Both in the diffuse cutaneous subset and in the limitedcutaneous subset of SSc (dcSSc and lcSSc, respectively),ultrastructural changes have been observed in cutaneousmyelinated and unmyelinated nerves, comprising disin-tegration of myelinic sheaths, irregular thickening ofbasal membranes, edema of the axoplasm, and, lessoften, a reduction in the number of neurofibrils andmicrotubules (7). In some cases, nerve fibers wereobserved to be compactly encased by a considerableamount of collagen fibrils (7).

An ultrastructural study of PNS alterations, ac-cording to subsets and phases of SSc (early and ad-vanced), showed that ultrastructure modifications to thePNS were linked to the progression and severity of skininvolvement (8). Alterations evolved from the early tothe advanced phase, mainly in the diffuse disease subset;indeed, the severe PNS lesions found in advanced lcSScwere already widely diffuse in early dcSSc, and themicrovascular damage in early lcSSc skin preceded thePNS modifications (8). Moreover, ultrastructural dam-age was observed in innervation of the gastrointestinal(GI) tract (9,10) and sural nerve (11).

A large body of evidence has demonstrated thatnitric oxide (NO) plays a key role in these physiologicand pathologic events. NO is a short-lived reactivemolecule with biologic signaling function (12). It may actas a messenger or as a modulator, playing a role indefense of the immune system (12), and it can becharacterized both as a neurotransmitter and as a hor-mone (12). NO is produced by neuronal NO synthase(nNOS, NOS-I), inducible NOS (iNOS, NOS-II), andendothelial NOS (eNOS, NOS-III) (13). Localization ofNOS in the skin is now well described, and the majorskin cell types express NOS and appear capable ofreleasing NO (14,15). In healthy human skin, NO playsa pivotal role in regulatory processes but is also involvedin many pathophysiologic events (14).

In SSc, eNOS and iNOS have been widely studied(16–20), but no study has focused on the nNOS that isconstitutively expressed in the central nervous system(CNS) and PNS, and in other cell types (13,21). Thisisoform contributes to neural physiologic functions, such

as neurotransmitter release, neural development andregeneration, synaptic plasticity, and regulation of geneexpression (22). Neuronal NOS is sometimes colocalizedwith acetylcholine and VIP in the perivascular parasym-pathetic fibers, wherein they modulate vascular tone andcontrol blood flow (23).

NO is synthesized on demand and diffuses to itsintracellular targets (12). In low doses, NO has vasopro-tective actions, such as inhibition of leukocyte adhesion,mast cell degranulation, neutrophil diapedesis, endothe-lial permeability, and associated microvascular leakage,and a long-term inhibitory effect on migration andproliferation of smooth muscle and intimal cells. Exces-sive and impaired synthesis of NO, such as occurs ininflammation, may be self-destructive in autoimmunedisorders, in conditions of immune rejection, and ingraft-versus-host disease (12). These data indicate thatNO may have either positive or negative effects at themicrovascular level in SSc skin (24).

An overexpression of nNOS has been identifiedin several neurologic disorders that are characterized byneural injury (21,22). Up-regulation of nNOS messengerRNA (mRNA) is considered to be a generic response ofthe neuronal cells to different mechanisms of stress,caused by thermal, chemical, or other biologic agents orby pathologic lesions, such as nerve injuries, local isch-emia, and hypoxia (21,25). However, recent reportsindicate that chronic hypoxia can induce a decline innNOS levels in the cerebral circulation of adult and fetalsheep (26). Moreover, a decrease in nNOS expressionand an up-regulation of iNOS were found after long-term ischemia in an animal model of atherogenic erec-tile dysfunction (27).

This evidence led us to investigate whether nNOSexpression was modified in human SSc skin, in thedifferent subsets and various phases of the disease, inorder to understand if a loss of nNOS could be involvedin the pathogenesis of SSc.

PATIENTS AND METHODS

Patients. Thirty-two consecutive patients with SSc at-tending the rheumatology outpatient clinic of the HospitalCochin in Paris, France (8 patients), the rheumatology outpa-tient clinic of the University of Florence, Italy (8 patients), andthe internal medicine outpatient clinic of the University ofL’Aquila, Italy (16 patients) were enrolled and gave theirwritten informed consent to participate in the study. Theprotocol was approved by the ethics committees of all hospi-tals. SSc was classified in the lcSSc subset (12 patients) ordcSSc subset (20 patients) according to the criteria describedby LeRoy et al (28). Patients with lcSSc or dcSSc were furtherclassified according to disease duration (29), and according to

NEURONAL NOS IN SYSTEMIC SCLEROSIS 203

clinical skin involvement in the edematous phase (4 with lcSScand 5 with dcSSc), sclerotic phase (5 with lcSSc and 7 withdcSSc), or atrophic phase (3 with lcSSc and 8 with dcSSc). SScpatients were assessed in accordance with the recently pro-posed guidelines (30). Demographic and clinical characteris-tics of the SSc patients and healthy control subjects are listedin Table 1.

Biopsy samples. Full-thickness biopsy samples (3 � 0.5cm) were obtained from the clinically involved skin of one-third of the distal forearm of each subject. In this area wedefined clinically involved skin as that with a skin thicknessvalue of �2, according to the Rodnan modified skin thicknessscore (31). Control skin samples were obtained from 6 healthysubjects who underwent forearm surgery for traumatic lesions.Controls were matched to the patients by age and sex. All skinbiopsy samples were processed for immunohistochemistry.Eleven of 12 samples from patients with lcSSc (3 in theedematous, 5 in the sclerotic, and 3 in the atrophic phase), 13of 20 samples from patients with dcSSc (5 in the edematous, 5in the sclerotic, and 3 in the atrophic phase), and 3 of 6 controlbiopsy samples were divided into 2 specimens and processedfor either immunohistochemistry or biomolecular analysis.

For biomolecular analysis, the specimens were imme-diately immersed in liquid nitrogen and then stored at �80°Cuntil used. All SSc patients underwent a 15-day drug-washoutperiod before the biopsy was performed. During this period,only proton pump inhibitors and clebopride were allowed.Patients who could not discontinue their pharmacologic treat-ment due to the presence of pulmonary hypertension, severecutaneous ulcers, active pulmonary alveolitis, renal failure,severe heart disease, or GI involvement were not enrolled inthe study.

Immunohistochemistry. The skin samples were fixedin Zamboni’s solution overnight at 4°C, followed by a wash for

3 days in 15% sucrose in 0.1 phosphate buffered saline (PBS)(pH 7.4) containing 0.01% sodium azide. Two fragments wereobtained from each specimen. The first fragment was dehy-drated and embedded in paraffin. Six micrometer–thick sec-tions were stained with hematoxylin and eosin and observedunder a light microscope to evaluate histopathologic changes.The second fragment was snap-frozen in OCT compound andstored at �20°C until processed. Twenty micrometer–thickcryostat sections were mounted on 3-aminopropyltri-ethoxysilane–coated slides (Sigma, St. Louis, MO) and post-fixed in Zamboni’s solution for 10 minutes. Endogenousperoxidase was inhibited by soaking the sections in 0.3% H2O2in methanol for 30 minutes at �22°C.

The sections were incubated sequentially in a humidchamber with 1) primary rabbit anti-human antibodies (forPGP 9.5, 1:6,000 dilution [Cambridge Research Biochemicals,Cambridge, UK], and for nNOS, 1:1,000 dilution [Santa CruzBiotechnology, Santa Cruz, CA]) diluted in 0.1M PBS contain-ing 0.1% (weight/volume) bovine serum albumin (BSA; Sigma)overnight at �4°C, 2) biotinylated goat anti-rabbit IgG in PBSwith 0.1% (w/v) BSA for 60 minutes at �22°C, and 3)avidin–biotin–peroxidase complex (diluted 1:200 in PBS; Vec-tor, Burlingame, CA) for 60 minutes at �22°C. After each stepthe sections were rinsed 3 times in PBS. Neuronal NOS andPGBP 9.5 were labeled with 3,3�-diaminobenzidine tetrahydro-chloride (DAB) and DAB nickel, respectively. DAB- and DABnickel–labeled sections were mounted with Gel-Mount(Biomeda, Foster City, CA). Normal rabbit IgG was used atthe same concentration to replace the primary antibodies, as anegative staining control. The stained sections were observedunder a Nikon Eclipse E400 microscope (Nikon, Tokyo,Japan).

Some cryosections (n � 36) labeled with anti-nNOSantibody were counterstained with Mayer’s hemallumen to

Table 1. Demographic and clinical characteristics of the SSc patients and controls*

SSc patients

Healthy controls(n � 6)

All SSc(n � 32)

lcSSc(n � 12)

dcSSc(n � 20)

Age, mean � SD years 50 � 14 53 � 14 48 � 17 54 � 6.5Disease phase, no.

Edematous 9 4 5Sclerotic 12 5 7Atrophic 11 3 8

Sex, no.Male 7 2 5 1Female 25 10 15 5

Disease duration, mean � SD years 8.5 � 5.4 8.1 � 3.9 8 � 6.4Skin score, mean � SD 15.4 � 7.2 8.6 � 5.1 22.1 � 4.0Fingertip ulcers, no. 7 2 5Autoantibodies, no.

ANA� 30 12 18Scl-70� 11 3 8ACA� 11 8 3

DLCO, mean � SD % predicted 75.38 � 25.71 86.14 � 26.14 69.64 � 24.13Heart involvement, no. 4 0 4Esophageal involvement, no. 13 3 10

* SSc � systemic sclerosis; lcSSc � limited cutaneous SSc; dcSSc � diffuse cutaneous SSc; ANA � antinuclearantibodies; ACA � anticentromere antibodies; DLCO � carbon monoxide diffusion capacity.

204 IBBA-MANNESCHI ET AL

detect endothelial cells. Three fields (40� objective magnifi-cation) from 2 random sections of 3 skin biopsy samples ineach disease phase were analyzed by a blinded observer (MM).We performed a semiquantitative analysis of the nerve fibersand blood vessels in the examined fields, with consideration ofthe distinct areas such as the epidermis and dermal–epidermaljunction, papillary dermis, and reticular dermis, excluding thecutaneus appendages. Histologic grading was carried out toevaluate the presence or absence of nerve fibers and vessels,with results expressed as positive or negative.

Western blot analysis. Biopsy samples were homoge-nized with 300 �l of ice-cold buffer (50 mM Tris HCl, pH 7.5,150 mM NaCl, 1 mM EDTA, 1% Triton, 0.25% sodiumdodecyl sulfate) supplemented with a cocktail of proteaseinhibitors (Complete; Boehringer Ingelheim, Milan, Italy).Total extracts were cleared by centrifugation for 30 minutes at4°C at 15,000 revolutions per minute (10,000g) and assayed forprotein content using Bradford’s method. Fifty micrograms ofproteins was electrophoresed through a 10% polyacrylamidegel, before being transferred to nitrocellulose membranes. Thefilters were stained with 10% Ponceau S solution for 2 minutesto verify equal loading and transfer efficiency. Blots wereblocked overnight with 5% nonfat dry milk and then incubatedwith anti-nNOS polyclonal antibody (Santa Cruz Biotechnol-ogy) and anti–PGP 9.5 polyclonal antibody (Chemicon, Te-mecula, CA) in PBS–0.1% Tween 20 at 4°C overnight. Afterwashing with PBS–0.1% Tween 20, filters were incubated with1:1,000 peroxidase-conjugated anti-rabbit Ig for 1 hour atroom temperature, and finally analyzed using the Opti 4CNsystem (Bio-Rad, Hercules, CA) according to the manufactur-er’s instructions.

Immunoreactive bands were quantified using NIHImage Analysis software. Data are expressed as the mean �SD of triplicate determinations in a representative experimentof 2 independent experiments with similar results (P � 0.05, byanalysis of variance [ANOVA] and Tukey’s w-test).

Semiquantitative reverse transcription–polymerasechain reaction (RT-PCR) analysis. For RT-PCR analysis, totalRNA was isolated from the forearm skin of controls and SScpatients, using TriReagent (Sigma) according to the manufac-turer’s instructions. The final total RNA pellet was suspendedin 30 �l of diethyl-pyrocarbonate–treated water, and 2 �l wasused for spectrophotometric determination of RNA concen-tration at 260 nm and RNA quality (260:280 nm ratio). Anequal amount of total RNA (1 �g) from each skin sample wasreverse transcribed and amplified in a total volume of 50 �lusing the SuperScript One-Step RT-PCR System with Plati-num Taq DNA (Invitrogen, Milan, Italy).

Neuronal NOS and PGP 9.5 primer sequences weredesigned on the basis of the published sequences (GenBankaccession no. NM_00620 for nNOS and NM_004181 forPGP 9.5). The forward and reverse primers for nNOS were5�-GGCCCATATTAATCCCTCGT-3� and 5�-ACATGAG-GGCTCTGCTCACT-3�, and for PGP 9.5 were 5�-CAG-CATGAGAACTTCAGGAAA-3� and 5�-GCCATCCAC-GTTGTTAAACAG-3�. The expected product size was 211 bpfor nNOS and 339 bp for PGP 9.5. All RT-PCRs werenormalized with �-actin (GenBank accession no. NM_001101;forward primer 5�-GGACTTCGAGCAAGAGATGG-3�, re-verse primer 5�-AGCACTGTGTTGGCGTACAG-3�, ex-pected product size 234 bp) and the optimal PCR cycles within

the linear amplification ranges were determined (data notshown). Neuronal NOS, PGP 9.5, and �-actin were reversetranscribed by performing one cycle of 55°C for 30 minutes and94°C for 2 minutes. PCRs were conducted immediately aftercomplementary DNA synthesis, under the following condi-tions: 40 cycles of 94°C for 15 seconds (denaturation), 54°C for30 seconds (annealing), 68°C for 1 minute (extension), fol-lowed by a final extension of 68°C for 5 minutes.

The size of the predicted products was visualized with1.0% agarose gel electrophoresis and ethidium bromide stain-ing. The amounts of RT-PCR products were determined bydensitometric analysis using NIH Image Analysis software.Within the linear range of amplification, at least 3 values fromeach amplification product were normalized to the startingRNA volumes and referred to the corresponding �-actinvalues (P � 0.05, by ANOVA and Tukey’s w-test).

RESULTS

Morphologic findings. Control skin. PGP 9.5. AllPNS cutaneous fibers in the healthy control skin wereimmunopositive for PGP 9.5. Nerve fibers appeared asdotted strands and continuous fibers running in thedermis, passing through the dermal–epidermal junction,and branching into thin fibers toward the skin surface(Figure 1A). PGP 9.5–immunoreactive fibers were visi-ble around the microvessels. Moreover, hair follicles(Figure 1B) and sweat glands (Figure 1C) were inner-vated by an extensive network of PGP 9.5–immunoreactive fibers.

Neuronal NOS. The nNOS-immunoreactive fi-bers in control skin were similar in appearance anddistribution to the PGP 9.5–immunoreactive fibers in thedermis, dermal–epidermal junction, and epidermis, aswell as around microvessels and skin appendages (Fig-ures 1D and E). Some nNOS-immunoreactive mast cellswere also observed in the dermis (Figure 1E).

Limited cutaneous SSc. Cutaneous changes in thelcSSc samples had a patchy distribution, characterized byareas with pathologic features that were close, withabrupt transition, to healthy-looking areas and were notdifferent from those in control biopsy skin (Figure 2A).In the edematous phase, the alterations were repre-sented by a relevant dermal inflammatory infiltrate,mainly consisting of neutrophils, monocytes, and somelymphocytes, as well as edema around the microvessels.In the sclerotic and atrophic phases in lcSSc skin,flattening of papillae and fibrosis were evident. Thesecutaneous changes were clearly focally distributed.

The impairment of blood vessels commenced inthe edematous phase, often with partial occlusion of thelumen of small vessels by hypertrophic endothelial cells.


In the sclerotic and atrophic phases, most of the microves-sels disappeared and the remaining ones were occluded.

PGP 9.5. In the edematous phase in lcSSc skin,PGP 9.5 innervation was similar to that in control skin.In the sclerotic and atrophic phases, affected areas, closeto the healthy-looking areas, showed sparse, discontin-uous PGP 9.5–immunoreactive nerve fibers, and onlysome of these nerve fibers reached the dermal–epidermal junction (Figures 2A and B). Nerve fiberbundles with dotted features were detectable in thedermis (Figure 2A). The network of nerve fibers aroundthe hair follicles, sebaceous glands, and sweat glands was

extensive in the edematous and sclerotic phases, becom-ing discontinuous in the atrophic phase. The healthy-looking areas showed innervation similar to that incontrol skin.

Neuronal NOS. In affected areas of lcSSc skinduring the edematous and sclerotic phases, the dermaldistribution of nNOS-immunoreactive nerve fibers wassimilar to that in control skin. Numerous nNOS-immunoreactive degranulating mast cells were observedclose to the vessels, particularly in the edematous phase(Figure 2C). In the atrophic phase, very few nNOS-immunoreactive nerve fibers were seen intraepider-

Figure 1. Innervation of healthy control skin as assessed by protein gene product 9.5 (PGP 9.5)and neuronal nitric oxide synthase (nNOS) immunostaining. For PGP 9.5, nerve fibers are visibleat the dermis, dermal–epidermal junction, and epidermis level (arrows) (A). Note the Meissner’scorpuscle in the dermis papilla (A). Nerve fibers are also evident around hair follicles (arrows) (B)and are forming a network around the sweat glands (arrows) (C). For nNOS, nerve fibers areevident at the dermis, dermal–epidermal junction, and epidermis level (arrows) (D) and aroundthe glandular duct (long arrows) (E). In the dermis, nNOS-immunoreactive mast cells are present(short arrow) (E). (Original magnification � 40 in A and E; � 10 in B and C; � 25 in D.)


mally, at the level of the epidermal basal cell layer or justbeneath the dermal–epidermal junction (Figure 2D),but they were still detectable in the dermis. Innervationof nNOS was discontinuous around the hair follicles,sweat glands, and sebaceous glands in the atrophic phase(Figure 2E). In the healthy-looking areas, nNOS inner-vation was similar to that in control skin.

Diffuse cutaneous SSc. In dcSSc skin, pathologicchanges were diffuse and homogeneously distributed. Aprominent accumulation of melanin pigment in the basallayer of the epidermis was found in most of the samples.

In the dermis in the edematous phase, a mononuclearinflammatory cell infiltrate and some degranulated mastcells, accompanied by edema, were observed. Most ofthe microvessels were occluded in their lumen by hyper-trophic endothelial cells and showed delaminated basalmembranes. The fibrotic process was already beginningin the edematous phase, characterized by abnormal andirregular deposition of collagen bundles in the papillarydermis. In the sclerotic phase, the thickness of both theepidermis and the papillae was reduced. Hair follicles,sebaceous glands, and sweat glands were sparse in the

Figure 2. Innervation of limited cutaneous systemic sclerosis (lcSSc) skin as assessed by PGP 9.5 and nNOS immunostaining. For PGP 9.5 in skinin the atrophic phase, a section with a healthy-looking area can be identified (on the left in A) close, with an abrupt transition, to an affected area(on the right in A). PGP 9.5–immunoreactive fiber distribution in the healthy-looking area is similar to that in control skin, and limited at thedermal–epidermal junction in the affected area (up arrows in A). Note a positive nerve fiber bundle in the dermis (right-pointing arrow in A). PGP9.5–immunoreactive fibers are evident near the dermal–epidermal junction (arrow in B). For nNOS in skin in the edematous phase,nNOS-immunoreactive degranulating mast cells are evident in the dermis (arrows in C). In skin in the atrophic phase, nNOS-immunoreactive fibersare sparse and discontinuous and far from the dermal–epidermal junction (arrows in D). A discontinuous network of nNOS-immunoreactive fibersis detectable around the gland units (arrows in E). (Original magnification � 10 in A; � 40 in B and C; � 20 in D; � 25 in E.) See Figure 1 for otherdefinitions.


papillary dermis and microvessels were rare, whereasmost blood vessels, localized in the deep dermis, werestill present in the atrophic phase. Cutaneous nervefibers were scant in the whole dermis and mostly local-ized around the remaining skin appendages.

PGP 9.5. In edematous and sclerotic dcSSc skin,PGP 9.5 innervation in the dermis was scanty, but thefibers appeared with dotted features similar to thoseseen in control samples (Figure 3A). In the atrophicphase, only a few, swollen PGP 9.5–immunoreactivenerve fibers were present in the dermis (Figure 3B).

These fibers rarely reached or crossed through thedermal–epidermal junction. Scanty PGP 9.5–immunoreactive fibers were observed around the vesselsand the few remaining sweat and sebaceous glands. Inthe reticular dermis, hardly any nerves were detectable,and these were edematous and weakly stained for PGP9.5.

Neuronal NOS. In edematous and sclerotic dcSScskin, nNOS-immunoreactive fibers were scarce and al-ways far from the dermal–epidermal junction (Figure3C). In the atrophic phase, rare nNOS-immunoreactive

Figure 3. Innervation of diffuse cutaneous systemic sclerosis (dcSSc) skin as assessed by PGP 9.5and nNOS immunostaining. For PGP 9.5 in skin in the sclerotic phase, PGP 9.5–immunoreactivefibers are rare (A), and most of them are visible in the dermis. In skin in the atrophic phase, raredisrupted fibers can reach the dermal–epidermal junction (arrows in B). For nNOS, rarenNOS-immunoreactive fibers are visible in the papillary dermis (long arrow in C). Note a fewnNOS-immunoreactive mast cells in the dermis (short arrows in C). A network of nNOS-immunoreactive fibers is evident around the gland units (arrows in D). Nerve fiber bundles withsome positive fibers are also detectable (arrows in E). (Original magnification � 10 in A; � 20 inB; � 25 in C and E; � 40 in D.) See Figure 1 for other definitions.


nerve fibers with a disrupted and swollen appearancewere observed in the papillary dermis (Figure 3C). SomenNOS-immunoreactive fibers were still found aroundthe few remaining sweat and sebaceous glands andvessels (Figure 3D). Immunopositive nerves, with a fewnNOS-positive fibers, were still observed in the reticulardermis (Figure 3E). Histologic scoring of the nNOSnerve fibers and microvessels is summarized in Table 2.

Biochemical findings. To evaluate whether thepathologic changes to the nervous system in SSc skinwere associated with alterations in the level of PGP 9.5and nNOS mRNA and proteins, semiquantitative RT-PCR and Western blot analyses were carried out in thecutaneous samples from patients and controls.

Western blot analysis. The molecular detection ofthese proteins, using the anti–PGP 9.5 and anti-nNOSpolyclonal antibodies, revealed a single band of theexpected size. The densitometric quantification of thebands, performed using NIH Image Analysis software,revealed that there was a significant decrease, comparedwith control skin, in the expression of both PGP 9.5 andnNOS in the edematous and sclerotic phases, reachingthe lowest values in the atrophic phase both in lcSSc skinand in dcSSc skin (Figure 4A). In dcSSc skin, levels ofboth PGP 9.5 and nNOS were found to be significantlylower than in lcSSc skin (Figure 4A).

The reduction in both SSc subsets in the atrophicphase is significant when compared with the otherphases, whereas no significant difference was observedbetween the edematous and sclerotic phases. Figure 4Bshows nNOS and PGP 9.5 protein levels that decreasedin both subsets of SSc, maintaining the same ratio.

Semiquantitative RT-PCR analysis. PGP 9.5 andnNOS mRNA levels were significantly reduced in theSSc samples compared with the control samples (Figure4C). Specifically, semiquantitative RT-PCR showed de-

creased levels of mRNA for both proteins in the edem-atous, sclerotic, and atrophic phases. No relevant differ-ence between the edematous phase and the scleroticphase was detected in either SSc subset. The reductionin the atrophic phase in both subsets was significantwhen compared with that in the control skin, and withthat in the edematous phase and in the sclerotic phase.Levels of mRNA for nNOS were significantly higher inthe edematous and sclerotic phases of lcSSc with respectto the same phases of dcSSc (Figure 4C).

DISCUSSION

NO of neural origin, produced by nNOS, partic-ipates in vascular tone and control of blood flow. Inhealthy skin, nNOS has been detected in mast cells andkeratinocytes (15), as well as in the PNS and in other celltypes (13,21). Our findings in healthy skin are consistentwith a constitutional expression of nNOS by PNS nerves(32). As far as we know, this is the first report onnNOS-immunoreactive nerve fibers in SSc skin. More-over, we compared the nNOS-immunoreactive nervefibers with the total cutaneous innervation, using thepan-neuronal marker PGP 9.5, and focused our atten-tion on disease subsets in different phases of the disease.

It is well known that stress caused by thermal,chemical, and biologic agents, as well as tissue ischemiaand cellular hypoxia, up-regulate nNOS expression inneural cells (21). However, recent reports indicate thatchronic hypoxia can induce a decline in nNOS levels incerebral vascular innervation in adult and fetal sheep(26). Moreover, a decrease in nNOS expression and anup-regulation of iNOS were found after long-term isch-emia in an animal model of atherogenic erectile dysfunc-tion (27).

It is also well known that SSc is characterized by

Table 2. Scoring of nNOS nerve fibers and microvessels in skin biopsy samples*

Controls

lcSSc dcSSc

Edematous Sclerotic Atrophic Edematous Sclerotic Atrophic

Nerve fibersEpidermis �� /� � � � �Dermal–epidermal junction �� /� � �/� � �Papillary dermis �� /� �/�Reticular dermis �� /� �/�

Blood vesselsDermal–epidermal junction �� /� �� Papillary dermis �� /� �� /�Reticular dermis ��

* Scores are defined as follows: �� plenty of fibers/vessels; �� moderate presence of fibers/vessels; � � few fibers/vessels; �/� � very fewfibers/vessels; � � hardly any fibers/vessels. nNOS � neuronal nitric oxide synthase (see Table 1 for other definitions).


Figure 4. Biochemical analyses of protein gene product 9.5 (PGP 9.5) and neuronal nitric oxide synthase (nNOS) in skin biopsy samples fromsystemic sclerosis (SSc) patients. A, Western blot analysis for the expression of PGP 9.5 and nNOS proteins in skin from patients with limitedcutaneous SSc (lcSSc) (3 edematous [lane e], 5 sclerotic [lane s], and 3 atrophic [lane a] phase) and from patients with diffuse cutaneous SSc (dcSSc)(5 edematous [lane e], 5 sclerotic [lane s], 3 atrophic [lane a] phase). Lane C, Control skin. Histograms show PGP 9.5 and nNOS protein levelsquantitated by densitometric analysis; bars represent the mean and SD of the intensity of the bands from triplicate determinations in a representativeexperiment of 2 independent experiments with similar results. P � 0.05, PGP 9.5 and nNOS in lcSSc and dcSSc groups versus controls, and SSc skinin the atrophic versus edematous and sclerotic phases. B, Comparison between PGP 9.5 and nNOS protein expression levels. PGP 9.5 and nNOSproteins were decreased in both SSc subsets, maintaining an unaltered ratio between them. O.D. � optical density. C, Semiquantitative reversetranscription–polymerase chain reaction (RT-PCR) analysis of PGP 9.5 and nNOS mRNA expression levels. Agarose gels show RT-PCR productsof PGP 9.5, nNOS, and �-actin mRNA expression levels in the lcSSc group (3 edematous [lane e], 5 sclerotic [lane s], 3 atrophic [lane a] phase) andthe dcSSc group (5 edematous [lane e], 5 sclerotic [lane s], 3 atrophic [lane a] phase). Lane C, Control skin. Data are representative of at least 3separate experiments. Histograms show PGP 9.5 and nNOS mRNA levels quantitated by densitometric analysis; each bar represents the mean andSEM of 3 values of PGP 9.5 or nNOS amplification products normalized to the starting total RNA volumes and referred to the corresponding �-actinvalues. P � 0.05, PGP 9.5 and nNOS in lcSSc and dcSSc groups versus controls, and SSc skin in the atrophic versus edematous and sclerotic phases.


continuous episodes of ischemia–reperfusion, clinicallyevident as Raynaud’s phenomenon. Ischemia generateslarge amounts of reactive oxygen species (33–35), and ithas been hypothesized that these reactive oxygen speciescould be involved in the release of cryptic, immunogenicautoantigens (36) and in the pathogenesis of SSc (37). InlcSSc, which is characterized by a prolonged period ofischemia–reperfusion, reactive oxygen species might sig-nificantly affect the perivascular nerve fibers, in partic-ular nitrergic fibers, with an initial up-regulation fol-lowed by a significant down-regulation of nNOS, due toan exhaustion of nerve fibers or direct damage of thefibers. In dcSSc, this hypothesis is unlikely, because thedisease may be contemporaneous to the onset ofRaynaud’s phenomenon. This point suggests that indcSSc, the observed damage to the PNS is independentof the vascular involvement and linked to another un-known factor. In contrast to what we expected, nNOSwas not up-regulated in the edematous phase, but, inboth SSc subsets, down-regulation occurred progres-sively from the edematous phase through the atrophicphase.

Our findings confirm that the PNS undergoesalterations, as shown in previous studies demonstrating asignificant reduction in parasympathetic, VIP-ergic, andsensory, CGRP-containing peripheral nerves in SSc skin(4). In our samples, we observed a decrease in thecutaneous innervation of PGP 9.5–immunoreactive fi-bers, as already shown by Terenghi et al (4), and aparallel decrease in the nNOS fibers, which progres-sively worsened with the evolution of the disease in bothsubsets. These data are consistent with our previousultrastructural findings in SSc skin, in which structuraldamage with consequent degeneration and destructionof nerve fibers has been shown (8). This evidence,together with a down-regulation of PGP 9.5 and nNOS,might explain the progressive reduction in PGP 9.5– andnNOS-immunoreactive fibers.

Based on our morphologic findings, neither thetotal cutaneous innervation nor the nNOS innervationdiffered between the edematous and sclerotic phases, asconfirmed by the biochemical data that indicated nodifference in the expression levels of the enzymes. Thisis probably attributable to the continuous presence ofthe pathologic features from the edematous phasethrough to the sclerotic phase. In the SSc edematousphase, a light alteration of cutaneous PGP 9.5– andnNOS-immunoreactive nerve fibers was initiated andslowly progressed to the sclerotic phase, becoming mostevident in the atrophic phase of the disease.

As previously demonstrated (8), the onset of the

alterations in cutaneous innervation was similar in itsorder but different in its timing between the dcSSc skinand lcSSc skin; the progression of cutaneous changeswas earlier and more severe in dcSSc skin than in lcSScskin (see Table 2). Nerve fibers, as well as blood vessels,seemed to disappear as the disease progressed from theouter to the inner skin layers. Whereas the loss of thenerve fibers was already evident in sclerotic lcSSc andedematous dcSSc skin, the blood vessels seemed to bespared. It has to be considered, however, that thenumber of nerve fibers and vessels cannot be easilycorrelated, and furthermore, this ratio does not providea view of the structural and functional situation. Withstandard histology, ultrastructural modifications ob-served in the vessels earlier than in the nerve fibers (8)are not detectable.

In the dcSSc skin, nNOS mRNA levels as well asits protein levels were significantly lower compared withthose in the lcSSc skin, whereas PGP 9.5 decreased onlyon the protein level. This may be attributable to thesynthesis of nNOS from mast cells, which are activatedin lcSSc.

During the main phases of clinically active SSc,iNOS is activated, resulting in increased levels of NO(17,18). In SSc, a controversy exists regarding whethercirculating NO levels are elevated or decreased (17–20).Most authors have reported increased serum NO levels,

Figure 5. Schematic representation of the suggested mechanismleading to nerve fiber damage in systemic sclerosis. Reactive oxygenspecies (ROS) are the main products of the ischemia–reperfusionsyndrome, which can act directly or indirectly both on endothelium andon nerve fibers. The increased levels of nitric oxide (NO) frominducible nitric oxide synthase (iNOS), induced by the inflammatorymilieu, together with the increased ROS, lead to peroxynitrate pro-duction. This, in turn, can play a role in the damage of the cutaneousperipheral nervous system. nNOS � neuronal nitric oxide synthase;S.M.C. � smooth muscle cells.


especially in the early stage of dcSSc (20). The effects ofNO in SSc could be both beneficial (as a vasodilator)and harmful (as a cytotoxic effector) with regard to theclinical manifestations (20,24).

In SSc, free radicals produced by inflammatorycells may react with NO, forming peroxynitrate, a potentoxidant that mediates cellular and tissue injury (23,24).Thus, NO can mediate free radical–related tissue injuryand cell death through necrosis and apoptotic mecha-nisms (24,38). Moreover, in the CNS, ischemic condi-tions excessively increase NO levels, by activating nNOS,and this is toxic to the surrounding neurons (38), sug-gesting the involvement of elevated levels of NO in thedamage to the PNS and in the reduced presence of PNSfibers in SSc, particularly in the atrophic phase. Thismight also account for the reduced expression of nNOSand PGP 9.5.

In the SSc edematous and sclerotic phases, thefinding of nNOS-immunoreactive, perivascular-activated mast cells suggests that these cells can poten-tially contribute to the toxic damage of nerve fibers byreleasing NO. In the edematous phase of dcSSc, com-pared with lcSSc, the inflammatory process was lessrelevant and differed in the types of involved cells and inthe amount of degranulating mast cells. This finding,together with the further observation of patchy skininvolvement in lcSSc, could explain the differences ob-served in the expression of nNOS between the 2 subsetsof the disease.

In conclusion, we provide evidence that thenNOS enzyme is down-regulated in SSc skin and de-creases from the edematous to the atrophic phase inboth subsets of the disease, in conjunction with worsen-ing of the disease. The total cutaneous innervation andthe nNOS innervation slowly disappear in the skin of SScpatients, probably due to the toxic effect of increasedproduction of NO, the effect of which is likely to bederived from iNOS as the main source (Figure 5).

It is clear that the first event, primary or second-ary, that leads to damage of the PNS in SSc has not yetbeen identified. Several times it has been suggested thatthe PNS and the endothelium are tightly correlated sothat they may influence each other’s function. Thepresent study cannot solve the problem of “who comesfirst,” but clearly our findings demonstrate the progres-sion of PNS damage from the early phase to the mostadvanced phase of the disease.

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Variations of neuronal nitric oxide synthase in systemic sclerosis skin

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