Clinical immunology
The influence of sera from interstitial lung disease patients on human mononuclear cells production of VEGF and bFGF

Introduction
Angiogenesis is a complex process depending on many stimulating and inhibiting factors. Under conditions of homeostasis, the rate of capillary endothelial cell turn-over is typically measured in months or years because in postnatal life, anti-angiogenic factors such as endostatin, angiostatin, thrombospondin, inhibitors of metalloproteinases (MMPs) and platelet factor 4 dominate [1]. Neovascularization can be induced by many indirect and direct proangiogenic factors in response to diverse pathophysiologic stimuli. Vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) are the most important direct pro-angiogenic factors [2]. The basic fibroblast growth factor was discovered as the first pro-angiogenic molecule [3]. This angiogenic mitogen can stimulate production of MMPs by endothelial cells [4]. Vascular endothelial growth factor is a specific endothelial cell mitogen that regulates endothelial cell differentiation, angiogenesis and maintenance of existing vessels [5]. It has been shown that VEGF and its receptor system is the fundamental regulator in the cell signalling angiogenesis. The levels of this growth factor in tumour cells can be significantly enhanced by hypoxia [6]. Hypoxia and inflammation are main causes of neovascularization in non-cancerous diseases [7]. Recently, the important role of angiogenesis in interstitial lung diseases (ILD) has been emphasized [8]. Previously, we have demonstrated, in vivo, with the Sidky and Auerbach animal model that sera from ILD patients constitute source of mediators participating in angiogenesis but the nature of these mediators is unknown [9-11]. However, correlation between angiogenic activity of sera from patients with ILD and serum tumor necrosis factor α (TNF-α) level was demonstrated [12-13]. The open question is if in exami­ned model direct proangiogenic factors such as VEGF and bFGF are produced.
The aim of the study was to assess, in vitro, VEGF and bFGF release by mononuclear cells of healthy donors pre-incubated with sera from ILD patients such as sarcoidosis, extrinsic allergic alveolitis and systemic sclerosis with pulmonary manifestations.


Material and methods


Patients
The protocol was approved by a local Ethics Committee and informed consent was obtained from every subject. In the study, sera from 5 patients with sarcoidosis, 5 patients with extrinsic allergic alveolitis (EAA), 5 patients with systemic sclerosis (SSC) and 5 healthy volunteers were used. The diagnosis of sarcoidosis was based on clinical and radio­logical symptoms and confirmed by histological criteria according to ATS/ERS/WASOG standards [14]. The diagnosis of EAA was based on clinical, radiological, functional, serological, BAL and histological findings according to Lacasse et al. criteria [15]. All patients fulfilled the diagnostic criteria of American Rheumatism Association for SSC [16]. All cases with SSC demonstrated pulmonary manifestation by high resolution computed tomography of the thorax. For control purpose we used sera from 5 healthy volunteers recruited from our medical staff. The scheme of the study is presented in Figure 1.


Isolation of normal human peripheral blood mononuclear cells
Normal human peripheral blood mononuclear cells (MNC) derived from buffy-coat cells of healthy donors (Warsaw Central Blood Bank) were prepared using a gradient technique according to the Boyum method [17]. The samples were diluted 1 : 1 with a phosphate buffered saline (PBS), layered over Histopaque 1077 (Sigma) and centrifuged at room temperature for 10 min at 500 G. The MNC were suspended in culture medium RPMI 1640 (Biomed, Lublin) in a concentration of 5 × 106 cells/ml.


Preincubation of MNC with sera
MNC were suspended in RMPI with supplementation of 25% of pooled sera from 5 patients with sarcoidosis or pooled sera from 5 EAA patients, or from 5 SSC patients or from 5 healthy donors in concentration 107 cells/ml and incubated one hour at 37°C in a saturation of 5% CO2.


Culture of pre-incubated MNC
After pre-incubation with pooled sera from patients or healthy donors, MNC were washed with PBS and suspended in RMPI 1640 in concentration 2 × 106 cells/ml, enriched with inactivated in 56°C 10% fetal bovine serum (FBS), L-glutamine 2 mM/ml and antibiotics: penicillin (1000 U/ml ) and streptomycin (10 µg/ml). MNC cultures were established in microplates (four repetitions of each MNC sample) and cultivated for 24, 48 and 72 hours in humidified atmosphere at 37°C, 5% CO2 (ASSAB, Sweden). After culture, supernatants and cell sediments were separated and frozen at –70°C.


Measurement of VEGF and bFGF concentration
Thawed cells or supernatant from culture was supplemented with 1% fetal calf serum for stabilization of VEGF or bFGF. Before measuring the cytokines concentration, cells were homogenized with an ultrasonic disrupter VirSonic (Virtis) for 2 minutes at a frequency of 22.5 KHz. Human VEGF and bFGF in cell supernatant and cells homogenate were evaluated by sandwich enzyme-linked immunosorbent assay using commercially available kits (R&D Systems, Inc. Minneapolis MN, USA) according to the manufacturer’s instructions. Optical density was measured at 450 nm using spectrophotometric reader Elx800 (Biotec Instruments, Inc. USA). Cytokine concentration was expressed as pg/ml.


Statistical analysis
The data were presented as mean ± SD and p < 0.05 was regarded as statistical significance. Test t was used for statistical analysis (Statistica 6 for Windows).


Results
The results are graphically presented on the Figures 2-5.
Vascular endothelial growth factor concentration in cell supernatants of MNC cultures collected after 72 hours was significantly (p < 0.001) higher compared to concentration after 24 hours (respectively 1445 ±223 pg/ml vs. 441 ±48 pg/ml for MNC pre-incubated with sera from sarcoidosis patients, 1531 ±336 pg/ml vs. 564 ±72 pg/ml for MNC pre-incubated with sera from EAA patients, 1382 ±342 pg/ml vs. 325 ±84 pg/ml for MNC pre-incubated with sera from SSC patients, 772 ±103 pg/ml vs. 0 pg/ml for MNC pre-incubated with sera from healthy volunteers, and 179 ±26 pg/ml vs. 0 pg/ml for MNC pre-incubated with PBS). VEGF level in supernatant of 72 hours cultures of MNC pre-incubated with sera from ILD in all cases was significantly higher than VEGF content of corresponding cell homogenate (respectively 1531 ±336 pg/ml vs. 29 ±3.1 pg/ml after pre-incubation of MNC with sera from EAA patients, 1382 ±342 pg/ml vs. 25 ±2.1 pg/ml for SSC patients, 1445 ±223 pg/ml vs. 21 ±1.6 pg/ml for sarcoidosis patients, 772 ±103 pg/ml vs. 26 ±1.2 pg/ml for healthy donors, and 179 ±26 pg/ml vs. 5 ±1.2 pg/ml for PBS control).
Concentration of bFGF was undetectable in supernatant as well as in homogenate of MNC pre-incubated with sera from patients, healthy donors or PBS in all examined groups.


Discussion
Our results demonstrate, that MNC pre-incubated with sera from some ILD patients produce during 3-day in vitro culture increasing amounts of VEGF. Increase of VEGF level in the epithelial lining fluid on 4th day patients with ARDS was showed [19]. However, this increase was associated with resolution of lung injury.
Significantly higher level of VEGF in supernatant from cultures of MNC than in cell homogenate from these cultures was demonstrated. Vascular endothelial growth factor and its receptors are the most important factors regulating process of angiogenesis [20, 21]. Vascular endothelial growth factor stimulates migration and proliferation of endothelial cells, increases vascular permeability and modulates coagulation system [5]. This factor stimulates NO release by endothelium cells, provokes dilatation of vessels [22] and protects endothelium against apoptosis [23]. Vascular endothelial growth factor is produced by tumour cells, macrophages and other immunological cells [24]. It is abundantly expressed in the normal lung, where may be released by numerous cell types found around the airspaces, including alveolar type 2 cells, alveolar macrophages and polymorphonuclear neutrophils. Many different factors stimulate production of VEGF, but hypoxia is one of the most important. Hypoxia-inducible factor (HIF) provokes transcription of gene for VEGF [25]. Hyperoxia decreases the expression of VEGF and its receptors in rat lungs [26]. The role of VEGF in early phases of angiogenesis was demonstrated [27]. Our results confirm early participation of VEGF in angiogenesis. However, we did not observe significant differences between VEGF level in supernatant from cultures of MNC pre-incubated with sera from different ILD patients. Previously, we demonstrated that sera from sarcoidosis and EAA patients significantly stimulated and from Systemic sclerosis inhibited neovascularization measu­red by Sidky and Auerbach assay [11, 12]. Systemic sclerosis was characterized by hypovascularity subunit expression [28]. However, concentration of VEGF was increasing in the serum of SSC patients in the earliest stages of the disease [29]. Mackiewicz [30] suggests that the imbalanced expression of VEGF and its vascular receptors, in part due to insufficient local production of VEGF, which was low compared to VEGFR expression, is responsible for angiogenesis fail in SSC. The data confirm that mechanism of angiogenesis is complicated and depends on many factors.
The second angiogenic factor, bFGF, was not released by MNC pre-incubated with sera from ILD patients and probably was not important in the induction of early steps of MNC-induced angiogenesis in Sidky-Auerbach model (in vivo). Basic fibroblast growth factor joins in the process of neovascularization latter than VEGF and plasmin mobilizes bFGF from extracellulary matrix [30]. In presented assay of MNC produced VEGF, however, lack in vitro of extracellulary matrix and clotting factors such as plasmin can explain lack of bFGF in this study. The further studies to explore the role of angiogenesis in interstitial lung diseases are needed.


Conclusion
The results suggest that VEGF released by MNC pre-incubated with ILD patients sera may be partly responsible for the results obtained previously in vivo in the Sidky and Auerbach assay.

Acknowledgements
This work was supported by the statutory budget of Institute of Tuberculosis and Lung Diseases in Warsaw.
No conflict of interest was declared with relation to this work.


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