Increased pulmonary epithelial permeability in systemic sclerosis is associated with enhanced cutaneous nerve growth factor expression

European Journal of Internal Medicine 11 (2000) 156–160 www.elsevier.com / locate / ejim

Original article

Increased pulmonary epithelial permeability in systemic sclerosis is associated with enhanced cutaneous nerve growth factor expression M. Piga a , G. Passiu b , P. Carta c , L. Satta a , V. Cherchi d , R. Pala b , G. Sanna b , A. Cauli b , e e b, M.A. Tuveri , L. Aloe , A. Mathieu * a Chair of Nuclear Medicine, Department of Medical Sciences, University of Cagliari, Cagliari, Italy Second Chair of Rheumatology, Center for Systemic Rheumatic Diseases, Department of Medical Sciences, University of Cagliari, Via San Giorgio 12, 09100 Cagliari, Italy c Chair of Occupational Medicine and Respiratory Pathophysiology, Department of Occupational Medicine, University of Cagliari, Cagliari, Italy d Center for Diagnostics Imaging, Department of Medical Sciences, University of Cagliari, Cagliari, Italy e CNR Institute of Neurobiology, Rome, Italy b

Received 8 November 1999; received in revised form 22 February 2000; accepted 29 February 2000

Abstract Background: Nerve growth factor (NGF), a neurotrophic factor that indirectly induces fibroblast proliferation and collagen production, has been found to be increased in the affected dermis of patients with systemic sclerosis (SSc). To investigate the possibility of a relationship between cutaneous NGF production and pulmonary damage in SSc, we studied seven non-smoking scleroderma patients. Methods: Abnormalities in lung structure were assessed by radiological lung examination, and pulmonary epithelial permeability (PEP) was determined by ventilation lung scintigraphy. All patients underwent skin punch biopsy with NGF immunohistological staining. Results: A statistically significant correlation was found between the PEP values and the cutaneous NGF staining scores, which were markedly increased in all of the patients examined, irrespective of the age, disease duration, or radiologically defined lung abnormalities. Conclusion: These results support the hypothesis that functional and anatomical changes in SSc target organs may be determined by a local tissue hyperproduction of NGF.  2000 Elsevier Science B.V. All rights reserved. Keywords: Pulmonary epithelial permeability; Nerve growth factor; Systemic sclerosis; Pulmonary fibrosis; Connective tissue diseases

1. Introduction Systemic sclerosis (SSc) is the clinical expression of a progressive, fibrosing disorder. Pulmonary involvement is a frequent occurrence in this disease [1,2]. Histological lung damage includes fibrotic replacement of the normal components of interstitial tissue within the alveolar walls and small arterial vessel obliteration due to endothelial changes. Pulmonary function tests and radiological chest examinations have revealed the presence of interstitial lung disease in approximately 70% of SSc patients [3,4]. The *Corresponding author. Tel.: 139-070-660-211; fax: 139-070-660213. E-mail address: [email protected] (A. Mathieu)

main functional changes characterizing the chronic interstitial lung involvement in this disease are the reduction in forced vital capacity (FVC), the decrease in CO diffusion lung capacity (DLCO) observed in the majority of patients [3,4] with very few exceptions [5–7], and the increased pulmonary epithelial permeability (PEP), as documented by an increase in 99mTechnetium-DTPA pulmonary clearance. The abnormality of this last function is expressed by a reduction in DTPA half-time (DTPA T 1 / 2 ) lung residence [8,9] and is constantly present in the series examined [10–15]. As for the DLCO decrease, the DTPA T 1 / 2 reduction is believed to be dependent on interstitial edema or fibrosis [16]. Recently, several lines of evidence have been found to support the view that mast cells and nerve growth factor

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M. Piga et al. / European Journal of Internal Medicine 11 (2000) 156 – 160

(NGF) may play a role in the pathogenesis of the skin changes in SSc. NGF, a neurotrophic factor produced by nerve fibers in human tissues, including skin and lung, is capable of inducing MC histamine release [17,18] and consequent fibroblast proliferation and collagen production [19]. The expression of NGF in the skin has been found to be increased in SSc patients compared to healthy controls [20]. The aim of this preliminary study was to investigate the possibility of a relationship between disease-related functional or morphological pulmonary changes and NGF expression in the affected skin of SSc patients, assuming that the pathogenetic mechanism inducing the histological abnormality is common to all organs involved and considering that the effect of NGF is not restricted to the site of production [17].

2. Materials and methods

2.1. Patients Seven non-smoking patients (six females, one male, mean age 57.4 years, range 36–72 years) affected by diffuse SSc and fulfilling the preliminary criteria of the American College of Rheumatology for the classification of SSc [21] were enrolled in this study after giving informed consent. The study procedures had been reviewed by the Ethics Committee of the Faculty of Medicine of the University of Cagliari. In five patients the disease duration (DD) was less than 3 years. Four of the seven patients had never been treated, while three had received D-penicillamine as the sole treatment for at least 2 years. Clinical evaluation, appropriate hematological and serological tests, chest X-ray examination, cutaneous punch biopsy, and ventilation lung scintigraphy were performed in all cases. Six of the seven patients were also subjected to high-resolution computed tomography (HRCT) evaluation of the lungs and to respiratory function tests. Ten age-matched, non-smoking subjects undergoing ventilation lung scintigraphy for clinical evaluation of chronic obstructive pulmonary disease were examined as controls for DTPA T 1 / 2 values that are unaffected by this condition. Control cutaneous punch biopsies in the forearm were obtained from two healthy volunteers for NGF staining and distribution.

2.2. Respiratory function tests All patients underwent evaluation of conventional parameters including forced vital capacity (FVC), forced expiratory volume in 1 s (FEV1), its ratio (FEV1 / FVC), and single-breath diffusion lung capacity for carbon monoxide (DLCO), according to the criteria of the American

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Thoracic Society [22]. The results were calculated as a percentage of the reference values supplied by the European Coal and Steel Community [23].

2.3. Ventilation lung scintigraphy PEP was determined by means of ventilation lung scintigraphy performed using 740 MBq 99mTechnetiumDTPA placed in the nebulized commercially available system (Venticis II, CIS, France). Aerosols with a mass diameter of 0.8 mm were generated with an air inflow of 10 l / min. The subjects inhaled the radioaerosol for 4 min. The patients were then placed in the sitting position in front of a g-camera (Elscint, Haifa, Israel) and lung fields were imaged in posterior projection. After the radioaerosol inhalation, data were acquired for 30 min (20 s / frame). Two regions of interest were selected over each lung. The exponential line was determined by regression analysis and pulmonary half-time (T 1 / 2 expressed as minutes) of the inhaled DTPA was assessed using the formula T 1 / 2 5 0.693 /k, where k is the slope of the line. DTPA T 1 / 2 represents the rate of the DTPA pulmonary clearance which, in turn, inversely expresses the PEP measurement.

2.4. High-resolution computed tomography This technique has been applied in several progressive lung diseases, including pulmonary involvement in SSc, to detect fine morphological changes in lung architecture. In our study, the category of the changes was respectively classified and scored according to Remy-Jardin et al. [24]. Group 1 included patients with normal findings at CT, group 2 was composed of patients with abnormal CT findings but without honeycombing, and group 3 was composed of patients with abnormal CT findings including honeycombing lung tissue.

2.5. Skin NGF identification Biopsy samples of affected skin (usually of the extensor forearm) were taken from each SSc patient and from two healthy individuals for cutaneous histological examination. Local anesthesia was administered without direct infiltration of the area to be biopsied by the injection of lidocaine 1% around the biopsy site. The full-thickness skin sample was obtained with a disposable biopsy punch (4 mm diameter) and processed for histological and immunohistochemical studies of NGF distribution, as reported elsewhere [20]. Briefly, skin samples were immediately fixed in 2% paraformaldehyde / 0.4% p-benzoquinone / 50 mM phosphate buffer pH 7.4 for 6 h at room temperature. For the study of NGF distribution, 5 mm sections were incubated overnight with either affinity-purified polyclonal NGF antibodies diluted 1:600–1:3000 (initial concentration 1.0

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mg / ml) or monoclonal NGF antibodies raised against mouse NGF at the Institute of Neurobiology, Rome, Italy, diluted 1:30 (initial concentration 1.0 mg / ml). Sections were then stained with immunoperoxidase and specificity checked by: (1) a pre-absorption of the first antibody with an excess of NGF, (2) the staining procedure without the primary antibody, and (3) an incubation of sections with preimmune rabbit serum. As described in a previous report [25], a blind estimate of the NGF staining was made, scoring its intensity 11 to 31.

expected value), and two had normal values (102 and 96% of the expected value). A statistically significant relationship was observed between the DTPA T 1 / 2 values and the dermal NGF staining score (r 5 0.84, P , 0.01), which was increased in five SSc patients (score 1 in two patients, score 2 in three, and score 3 in the remaining two patients) compared to that in the healthy controls (score 1 in each normal control; Fig. 1A–C). No significant correlation was found between DTPA T 1 / 2 and DLCO values (r 5 0.46), HRTC score (r 5 2

2.6. Statistical analysis Statistical analysis of the data was carried out with a two-sided Student’s t-test and the calculation of linear regression, as appropriate.

3. Results The HRCT, DLCO, and DTPA T 1 / 2 values and the expression of NGF in the skin are shown in Table 1. The mean value of DTPA T 1 / 2 determined in SSc patients (50611 min) was significantly lower than that found in the control group (105614 min; P , 0.001). In all seven patients the individual DTPA T 1 / 2 (range 36–70 min) was below the lowest value observed in the control group. The decrease in DTPA T 1 / 2 values observed in our patient series ranged from 33 to 65% (mean 52%) of the mean DTPA T 1 / 2 values found in the control group. This decrease appeared to be irrespective of the age or disease duration (DD) of the patients. Of the six patients who underwent DLCO, two had significantly reduced values (49 and 55% of the expected value), two had borderline values (82 and 83% of the Table 1 DTPA T 1 / 2 and DLCO values with cutaneous NGF scores in SSc patients with lung involvement a Patient

Age

DD

TC

DLCO

DTPA T 1 / 2

NGF

P.A. P.M. L.G. C.G. M.A. C.L. M.M.A.

64 72 53 36 63 54 60

3 2 5 2 8 2 1

2 3 3 1 3 3 ND

82 102 83 96 49 55 ND

70 55 53 48 44 45 36

1 1 2 2 2 3 3

a DD, disease duration (in years); TC, score of morphological changes in the high-resolution lung tomography (group 1, normal findings at CT; group 2, abnormal CT findings without honeycombing; group 3, abnormal CT findings including honeycombing lung tissue); DLCO, values of lung diffusion capacity of CO (as percent of expected values); DTPA T 1 / 2 , value of DTPA pulmonary half-time or clearance rate (in minutes); NGF, dermal content in nerve growth factor (as immunoperoxidase staining score) (1, low level of staining intensity; 2, intermediate level; 3, high level); ND, not determined.

Fig. 1. Typical immunostaining of skin section from two patients (A and B) with systemic sclerosis (SSc), showing nerve growth factor (NGF)positive cells (arrows) and (C) skin of control (original magnification 360).

M. Piga et al. / European Journal of Internal Medicine 11 (2000) 156 – 160

0.21) and DD (r 5 2 0.04), or between NGF score and the above-mentioned parameters (vs. DLCO r 5 2 0.63; vs. HRTC r 5 0.16; vs. DD r 5 2 0.17). Age had no influence on the DTPA T 1 / 2 values (r 5 0.25) or on the NGF staining score (r 5 2 0.39). Similarly, no statistically significant correlation was observed for DLCO and HRTC (r 5 2 0.45).

4. Discussion Despite the small size of our patient sample, the results of the pulmonary function tests are consistent with those of previous reports indicating an increase in PEP (as expressed by the decrease in DTPA T 1 / 2 ) in SSc patients. In our study, this increase was closely correlated to enhanced NGF immunoreactivity in the dermis. This finding suggests that the increased expression of NGF in tissue may be one of the factors responsible for the characteristic changes in the sclerosis process and, thus, play a role in the pathogenesis of SSc. Pulmonary function tests are non-invasive diagnostic tools capable of revealing the development of lung involvement earlier than radiological chest assessment. Of these tests, DLCO and DTPA T 1 / 2 measurement are considered the most appropriate tools for the detection of interstitial functional changes, at least during the initial stages of the disorder. DLCO is affected by interstitial fibrosis and related alterations that yield a reduced diffusion of CO through the alveolar–capillary barrier [3]. Nonetheless, there is some evidence that DLCO may be increased in a minority of patients [5–7]. However, significant DLCO impairment usually develops at a late stage of the disease, when the interstitial fibrosis is more evident and widespread. In this study, the average disease duration was short, which may explain why the functional defect was restricted to only two patients. Conversely, in interstitial lung disease (associated with heavy smoking, long-term use of free-base cocaine, highdose bleomycine, sarcoidosis, systemic sclerosis), the DTPA clearance is always accelerated [10–15]. The permeability of the alveolar–capillary barrier depends on the physiologic ability of the DTPA molecule, a hydrophilic solute, to migrate from the alveolar chamber across the alveolar epithelium and basal membrane toward the capillary endothelium and the interepithelial porus channels. Edema or fibrosis will lead to the separation and disarrangement of epithelial cells and, consequently, to an increase in the diameters of the porus channels. This, in turn, leads to an increase in PEP. The same PEP abnormality occurs in disorders leading to interstitial edema, such as acute respiratory distress syndrome [26], or when chronic epithelial damage develops, as in chronic smoking [10]. Early changes involving pulmonary endothelial and epithelial surfaces have been reported in SSc patients [27].

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The lack of a significant correlation between DTPA T 1 / 2 values or DLCO findings and HRTC scores may be explained by the different significance of these tests: the first two express functional changes, while the third expresses morphostructural alterations. Further explanation derives from the fact that these test categories do not account to the same extent for the differences in disease stage and, thus, in pathological changes among patients. These differences are probably also responsible for the lack of correlation between NGF and HRTC scores in our patients. Recently, the presence of anti-endothelial cell antibodies in SSc patients was found to be associated with alveolo–capillary impairment measured by 99m Tc-DTPA radioaerosol, but not with the morphological expression of pulmonary fibrosis as detected by HRTC [28]. The limited number of cases does not allow us to make an evaluation of the apparent trend towards an inverse correlation between NGF scores and DLCO values. Conversely, the considerable overlap between the alterations in skin and lung tissue in patients with the diffuse form of SSc may explain the significant correlation observed between the increased expression of NGF in the skin and the hyperpermeability of the pulmonary epithelium. Our findings suggest a role for NGF in the pathogenesis of SSc and in the genesis of lung alterations, but at this stage it is not possible to draw definite conclusions. Experimental investigations or more direct studies on lung tissue are required to better understand the pathogenesis of abnormal permeability of pulmonary epithelium and the functional role of NGF in SSc.

Acknowledgements This work was supported by grants from the National Ministry of University and Scientific and Technological Research (No. 2208 UNI.CA) to M. Piga, from the Consorzio NIRECO to L. Aloe, and from the Regional Health Authority to the Center of Systemic Rheumatic Diseases of the University of Cagliari and the National Ministry of University and Scientific and Technological Research to A. Mathieu.

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