Clinical immunology
Angiogenic activity of sera from interstitial lung diseases patients in relation to IL-6, IL-8, IL-12 and TNFα serum level

Introduction

Angiogenesis is a process involving the formation of new vessels from the pre-existing vasculature. It occurs in many physiological and pathological conditions [1]. Neovascularisation is essential for an embryonic development, mainly facilitating embryo implantation and placenta formation
[2-4]. In adult life it occurs during endometrium regeneration and corpus luteum formation [5]. In physiological reactions angiogenesis contributes in wound healing [6] and sportsmen’s myohypertrophy [7]. The process plays an important role in the pathogenesis of numerous diseases especially neoplasia [8], Crohn’s diseases and psoriasis [9] etc. It is also closely associated with inflammation in several diseases including chronic inflammatory disorders (e.g. rheumatoid arthritis) [9]. It occurs with frequency in other conditions e.g. in diabetic retinopathy [10] and obesity [11]. Although neovascularisation is a principal vascular response in chronic hypoxia and chronic inflammation, the role of angiogenesis in chronic pulmonary inflammatory disorders such as sarcoidosis or hypersensitivity pneumonitis is not clear. However, microvascular changes were observed in lung specimens in ILD [12].
The angiogenetic process is controlled through the balance of two groups of modulators: stimulators and inhibitors. Many proangiogenic factors such as interleukin-1 (IL-1), interleukin-6 (IL-6), interleukin-8 (IL-8), basic fibroblast growth factor (bFGF), platelet derived growth factor (PDGF), tumor necrosis factor alpha (TNFα), prostaglandin E2 (PGE2), and vascular endothelial growth factor (VEGF) also demonstrate proinflammatory activity [13-15]. However, certain antiangiogenic factors as interleukin-10 (IL-10), interleukin-12 (IL-12), interferon alpha (INF), interferon gamma (INFγ), transforming growth factor beta (TGFβ), 1,25OH-VitD3, thrombospondin and angiostatin inhibit inflammation [13, 16]. The interstitial pneumonia is characterized by inflammation, cell proliferation and excessive extracellular matrix deposition. Several cytokines, including those involved in angiogenesis, have been implicated in ILD pathogenesis [17, 18].
The aim of the study was to asses the effect of sera from ILD patients on angiogenesis induced by MNC in relation to IL-6, IL-8, IL-12, and serum TNFα level.

Material and methods

The study population consisted of 68 ILD patients who had not received the immunosuppressive treatment. According to the final diagnosis patients were divided into 5 groups: (i) 26 patients with SAR, (ii) 13 patients with AFL, (iii)13 with IPF, (iv) 8 patients with HIS, and (v) 8 patients with SCL with pulmonary manifestations. The diagnosis was based on clinical, radiological, immunological and histological criteria. All blood samples were obtained with consent of fully informed patients. Sera from 14 healthy volunteers were used as a control (group). All samples were stored at –80°C.
Serum cytokines were measured by sandwich enzyme-linked immunosorbent assay using commercially available kits (QuantikineTM human IL-6 and IL-8, R&D Systems, Inc. Minneapolis MN, USA, IL12+p40 and TNFα BIOSOURCE Europe SA) according to the manufacture’s instructions.
Normal human peripheral blood mononuclear cells (MNC) derived from buffy-coat cells of healthy donors were prepared using a Histopaque 1077 (Sigma) and gradient technique at a room temperature for 20 min at 450 g, according to Boyum method [19]. MNC viability was assessed by trypan blue exclusion and found to be ł98%.
Angiogenesis was quantified by the Sidky and Auerbach test (leukocyte induced angiogenesis assay) [20] in modification [21]. MNC were incubated in phosphate buffered saline (PBS) mixed with serum from patients and from healthy volunteers (25% of serum and 75% of PBS). As a control, MNC were preincubated in PBS only. Following an hour of incubation at 37°C with saturation of 5% CO2, MNC were suspended in Parker liquid (5x10⁶ cells/ml). Inbred 8 weeks old female BALB/c mice served as the recipients of normal MNC preincubated with sera or PBS. The mice, anaesthetised with 3.6% chloral hydrate, were injected intradermally with 5x10⁵ cells in Parker liquid supplemented with 0.01% trypan blue. Three mice constituted a group per each patient. Every mouse received 6 injections (3 into each side). Three days following the injection of MNC angiogenic reaction was quantified on the inner surface of the skin of each mouse. New blood vessels localized to the injection sites, trypan blue negative and contrasting with the pre-existing vasculature by a virtue of their tortuosity and divarications were counted under
a dissection microscope (Nicon, magnification of 6x). The results are introduced as the mean number of new blood vessels grown in one group (18 injection points per patient). They are expressed as mean ±SD (p<0.05 is regarded as
a statistical significance). Student’s and Pearson’s tests were used in statistical analysis (Statistica 6 for Windows).

Results

Sera from AFL, SAR and IPF patients significantly stimulated angiogenic activity of normal MNC compared with the sera from the healthy subjects measured by the mean number of new vessels following the injection of MNC (p<0.001) (table 1). The most important proangiogenic effect was observed following preincubation of MNC with sera from AFL patients (17.8±1.3). Weaker stimulation was induced by MNC preincubated with sera from SAR (16.2±0.97) and IPF patients (15.3±1.4) (figure 1). However, sera from the healthy donors significantly (p<0.001) stimulated the angiogenic activity of MNC (13.3±0.8) as compared with MNC preincubated with sera from HIS (11.1±0.5) and SCL patients (10.1±0.3). The differences between the mean numbers of new vessels in all groups were significant (table 1).
IL-12 serum level from 17 patients with SAR, 13 with AFL, 13 with IPF, 8 with SCL, 8 with HIS, and 10 healthy donors was measured. The serum level of IL-12 in SAR patients was significantly elevated (302±180 pg/ml, p<0.001) as compared with the control group (64±69 pg/ml) as well as with SCL patients (66±110 pg/ml, p<0.01) and AFL patients (72±89 pg/ml, p<0.001) (figure 2). An elevated IL-12 serum level was also observed in the group of IPF patients (246±285 pg/ml) but the differences in
IL-12 level in other groups were not significant. In the sera of HIS patients IL-12 serum level was 147±211 pg/ml. No significant correlation has been established between IL-12 serum level and the number of new vessels formed, following injections of MNC preincubated with the sera from ILD patients and from healthy controls (figure 3).
Serum TNFα level was measured in sera from 10 patients with SAR, 10 with IPF, 10 with AFL, 8 with SCL, 6 with HIS, and from 8 healthy donors. The serum level of TNFα was significantly elevated in HIS patients (28±3.9 pg/ml) as compared not only with the healthy controls (20.7±3.6 pg/ml, p<0.005, figure 4), but also with SCL patients (22.7±2.9 pg/ml, p<0.01) and IPF patients (22.4±3.9 pg/ml, p<0.05). Also, in the SAR group the TNFα serum level (27±5 pg/ml) was significantly elevated compared with the healthy controls (p<0.01) and IPF patients (p<0.05). The difference between serum TNFα level from patients with SAR and SCL was not significant (p=0.052), and similarly, between the serum TNFα level from AFL patients (25.1±5.1 pg/ml) and the healthy donors (p=0.06). The TNFα level in the sera from IPF and SCL patients was similar to its level in the sera from the control group.
A significant correlation between the serum TNFα level and the number of new vessels created after injection of MNC preincubated with the sera of the studied patients was observed (r=0.29, p<0.05, figure 5a). In contrast, in the group of 46 patients with SAR, IPF, AFL, SCL and healthy controls (without HIS patients) the correlation between the serum TNFα level and the number of new vessels appeared stronger (Pearson coefficient r=0.39, p<0.01, figure 5b).
The IL-6 serum level was measured in sera from
43 patients (25 with SAR, 10 with AFL, and 8 with IPF). The highest IL-6 level was observed in the sera from IPF patients (33.7±7.9 pg/ml) and the lowest in the sera from AFL patients (26.8±6.7 pg/ml), but the differences between these groups were not significant (p<0.056, figure 6). In
the SAR patients the mean serum level of IL-6 was 30.8±13.5 pg/ml. We have not found any correlation between the IL-6 serum level and the number of new vessels formed following the injection of MNC preincubated with the sera from ILD patients (figure 7a).
The IL-8 serum level was measured in sera from
42 patients (25 with SAR, 9 with AFL, and 8 with IPF). The highest IL-8 level was observed in sera from
AFL patients (872±129 pg/ml), lower in those from IPF patients (741±120 pg/l) and the lowest in SAR patients (699±234 pg/l). The differences between the IL-8 serum level from AFL patients and SAR patients were significant (p<0.05, figure 8). We have not found any correlation between the IL-8 serum level and the number of new vessels created after injection of MNC preincubated with the sera from ILD patients (figure 7b). However, the IL6 and IL8 serum levels correlated with each other (r=0.46, p<0.01, figure 9).

Discussion

The role of neovascularisation in pathogenesis of ILD is not clear. Microvascular changes have been observed in specimens of lung parenchyma in sarcoidosis and IPF
[12, 22]. Keane et al. demonstrated an increase of the angiogenic activity in the lung tissue of IPF and experimental fibrosis [23]. An increased angiogenesis-inducing ability of activated alveolar macrophages was found in bronchoalveolar lavage (BAL) specimens from sarcoidosis patients [24]. Moreover, proinflammatory and antiinflammatory chemokines modulating angiogenesis were found in sera from ILD patients [18]. Generally, the role of angiogenesis in pathogenesis of ILD requires further research. Our results showed that the sera from ILD patients constitute a source of mediators modulating angiogenesis. Serum effect varies depending on the disease. The sera from AFL, IPF and SAR patients demonstrate proangiogenic activity. The most pronounced proangiogenic effect is exerted by the sera from AFL patients. Interestingly, no information on angiogenesis in hypersensitivity pneumonitis has been found. In contrast to AFL, IPF and SAR patients, sera from SCL patients with pulmonary manifestations and from HIS patients exert an inhibitory effect on angiogenesis as compared with the healthy controls and the control with PBS. Previously, Majewski
et al. demonstrated the sera from patients with acrosclerosis and diffuse scleroderma modify angiogenic capability of normal MNC. The effect decreased or disappeared in severe and chronic cases [25]. We have not come across any research papers on angiogenesis in histiocytosis.
The results suggest that the angiogenesis may play a role in the pathogenesis of chronic inflammation and fibrosis in ILD. Chronic inflammation is accompanied by neovasularisation [9, 15]. An inflammatory state can promote angiogenesis, and in turn, angiogenesis can facilitate chronic inflammation. Macrophages and lymphocytes which constitute a cellular infiltrate secrete both angiogenic and inflammatory mediators such as the examined IL-6, IL-8, and TNFα [9]. Most of the agents produced during the inflammation process are direct or indirect promoters of angiogenesis. They act by altering the balance between angiogenesis inhibition and stimulation promoting the production of some more directly acting inducers such as bFGF or VEGF which influence endothelial cells proliferation and migration [15].
IL-6 is a multifunctional cytokine presenting biological activities. They involve: activation of B and T cells, production of acute-phase reactants and production of immunoglobulins [26]. In vivo IL-6 production is dominated by cells of macrophage/monocytes lineage [26]. IL-6 induces endothelial cells activation and vascular smooth muscle cells proliferation and migration [27]. Although this cytokine has been implicated in angiogenesis, in vitro it has demonstrated both stimulatory and inhibitory effects on the endothelial cell growth [9, 28]. IL-6 has been reported to be involved not only in the development of inflammatory disorders such as sarcoidosis and rheumatoid arthritis but also in diabetes mellitus and osteoporosis [26, 29-31].
IL-8 is secreted by activated monocytes, neutrophiles, endothelial cells, fibroblasts, and lung epithelial cells
[32, 33]. Various studies have implicated IL-8 as pro-
-inflammatory mediator stimulating neutrophil accumulation, chemotaxis and degranulation [33, 34]. However, it is not only chemotactic but also mitogenic for endothelial cells [9]. Aberrant increases in IL-8 caused by psoriatic keratinocytes have been said to be crucial in their ability to produce an angiogenic response [35]. IL-8 is implicated in the pathogenesis of lung damage and remodelling, especially in IPF [36]. This cytokine regulates angiogenesis, a key component of the fibrotic response in IPF [23].
An increase of angiogenic activity has been attributed to an imbalance between proangiogenic chemokines (as IL-8) and antiangiogenic CXC chemokines (IP-10) [23]. The correlation between IL-6 and IL-8 serum level in IPF, AFL and sarcoidosis patients is particularly interesting. It confirms the role of the cytokines in pathogenesis of ILD.
IL-12 plays a crucial role in controlling the development of Th1 immune response and stimulates the proliferation and lytic activity of the activated lung T cells and natural killer cells [37]. It is a heterodimeric molecule composed of the p35 and p40 subunits. The data have indicated IL-12 involvement in the development of the lung granulomas typical for sarcoidosis and tuberculosis [38]. In our experiments IL-12 serum level in SAR patients was significantly higher as compared with other ILD groups containing a dominant fibrotic component. IL-12 strongly inhibits tumoral neovascularisation [39]. Induction of INFg by IL-12 appears to play a decisive role in the antiangiogenic effects of IL-12 [40]. However, we did not establish any negative correlation between IL-12 serum level and the number of new vessels created after injection of MNC preincubated with the sera from ILD patients.
TNFα is a proinflammatory, pleiotropic and heparin-binding cytokine secreted mainly by monocytes and macrophages [41]. The cytokine stimulates the synthesis of IL-1, GMCSF and ICAM-1 by endothelial cells. It is well established that there exists a strong link between the overexpression of TNFα and the development of IPF [42]. The importance of TNFα in the pathogenesis of sarcoidosis remains uncertain [43]. It is known that TNFα stimulates angiogenesis in many models [44-45]. TNFα is involved in promoting angiogenesis in vivo while inhibiting endothelial cell proliferation in vitro [15]. We demonstrated a strong correlation between serum TNFα level and the number of new vessels created after injection of MNC preincubated with the sera from ILD patients. TNFα has not only proinflammatory but also proangiogenic properties. The results of this study suggest that angiogenesis plays
a role in the pathogenesis of ILD. This raises a question whether hypoxia may be responsible for vascular cell proliferation and angiogenesis in interstitial lung diseases [46]. From all the studied cytokines in ILD patients, TNFα seems to exert the most important proangiogenic effect.

Conclusions

Our findings indicate that sera from ILD patients and from healthy people constitute a source of mediators modulating angiogenesis but the pattern of reaction varies in different diseases. Sera from SCL and HIS patients exert an inhibitory effect on angiogenesis while sera from AFL, SAR and IPF patients stimulate neovascularisation. TNFα as an important proinflammatory factor may stimulate angiogenesis in ILD.

References

1. Risau W (1997): Mechanisms of angiogenesis. Nature 386: 671-674.
2. Breier G (2000): Angiogenesis in embryonic development – a review. Placenta 21 (Suppl A): S11-S15.
3. Dunk C, Shams M, Nijar S, et al. (2000): Angiopoietin-1 and Angiopoietin-2 activate trophoblast Tie-2 to promote and migration during placenta development. Am J Pathol 156: 2185-2199.
4. Smith SK (2000): Angiogenesis and implantation. Hum Reprod 15 (Suppl 6): 59-66.
5. Hazzard TM, Stouffer RL (2000): Angiogenesis in ovarian follicular and luteal development. Baillieres Best Pract Res Clin Obstet Gynaecol 14: 883-900.
6. Witte MB, Barbul A (1997): General principles of wound healing. Surg Clin North Am 77: 509-528.
7. Hudlicka O, Brown M, Egginton S (1992): Angiogenesis in skeletal and cardiac muscle. Physiol Rev 72: 369-417.
8. Kerbel RS (2000): Tumor angiogenesis: past, present and the future. Carcinogenesis 21: 505-515.
9. Jackson JR, Seed MP, Kircher CH, et al. (1997): The codependence of angiogenesis and chronic inflammation. FASEB J 11: 457-465.
10. Spirin KS, Saghizadeh M, Lewin SL, et al. (1999): Basement membrane and growth factor gene expression in normal and diabetic human retinas. Curr Eye Res 18: 490-499.
11. Bouloumie A, Drexler HC, Lafontan M, Busse R (1998): Leptin, the product of OB. Gene, promotes angiogenesis. Circ Res 83: 1059-1066.
12. Takemura T, Hiraga Y, Oomichi M, et al. (1995): Ultrastructural features of alveolitis in sarcoidosis. Am J Respir Crit Care Med 152: 360-366.
13. Pepper MS, Mandriota SJ, Vassalli JD, et al.: Angiogenesis-
-regulating cytokines: activities and interactions. In: Current Topics in Microbiology and Immunology. Vol. 213/II: Attempts to Understand Metastasis Formation II. Ed. U Günthert,
W Birchmeier. Spring-Verlag. Berlin Heidelberg. 1996, 31-67.
14. Yoshida S, Ono M, Shono T, et al. (1997): Involvement of interleukin-8, vascular endothelial growth factor, and basic fibroblast growth factor in tumor necrosis alfa-dependent angiogenesis. Mol Cell Biol 17: 4015-4023.
15. Folkman J, Brem H: Angiogenesis and infammation. In: Inflammation: Basic Principles and Clinical Correlates. Second Edition. Ed. JI Gallin, IM Goldstein, R Snyderman. Raven Press Ltd. New York. 1992, 821-839.
16. Di Pietro LA (1997): Thrombospondin as a regulator of angiogenesis. EXS 79: 295-314.
17. Agostini C, Semenzato G (1998): Cytokines in sarcoidosis. Semin Respir Infect 13: 184-196.
18. Keane MP, Strieter RM (2002): The importance of balanced pro-inflammatory and anti-inflammatory mechanisms in diffuse lung disease. Respir Res 3: 5. Epub 2001 Oct 15.
19. Boyum A (1968): Isolation of mononuclear cells and granulocytes from human blood. Isolation of monuclear cells by one centrifugation, and of granulocytes by combining centrifugation and sedimentation at 1 g. Scand J Clin Lab Invest Suppl 97: 77-79.
20. Sidky YA, Auerbach R (1975): Lymphocyte-induced angiogenesis: a quantitative and sensitive assay of the
graft-vs.-host reaction. J Exp Med 141: 1084-1100.
21. Zielonka TM, Demkow U, Kowalski J, et al. (1997): Ocena aktywności angiogennej surowic chorych na śródmiąższowe choroby płuc. Pneumonol Alergol Pol 65: 754-760.
22. Ebina M, Shimizukawa M, Shibata N, et al. (2004): Heterogeneous increase in CD34-positive alveolar capillaries in idiopathic pulmonary fibrosis. Am J Respir Crit Care Med 169: 1203-1208.
23. Keane MP, Arenberg DA, Lynch JP 3rd, et al. (1997): The CXC chemokines, IL-8 and IP-10, regulate angiogenic activity in idiopathic pulmonary fibrosis. J Immunol 159: 1437-1443.
24. Weber J, Meyer KC, Banda P, et al. (1989): Studies of bronchoalveolar lavage cells and fluids in pulmonary sarcoidosis. I Enhanced capacity of bronchoalveolar lavage fluids from patients with pulmonary sarcoidosis to induced cell movement in vitro. Am Rev Respir Dis 140: 1450-1454.
25. Majewski S, Skopińska-Różewska E, Jabłońska S, et al. (1985): Modulatory effect of sera from scleroderma patients on lymphocyte-induced angiogenesis. Arthritis Rheum 28: 1133-1139.
26. Papanicolaou D, Wilder RL, Manolagas SC, Chrousos GP (1998): The pathophysiologic roles of interleukin-6 in human disease. Ann Intern Med 128: 127-137.
27. Brull DJ, Sanders J, Rumley A, et al. (2002): Impact of angiotensin converting enzyme inhibition on post-coronary artery bypass interleukin 6 release. Heart 87: 252-255.
28. Motro B, Itin A, Sachs L, Keshet E (1990): Pattern of interleukin 6 gene expression in vivo suggests a role for this cytokine in angiogenesis. Proc Natl Acad Sci U S A 87: 3092-3096.
29. Sahashi K, Ina Y, Takada K, et al. (1994): Significance of interleukin 6 in patients with sarcoidosis. Chest 106: 156-160.
30. Shimizu E, Funatsu H, Yamashita H, et al. (2002): Plasma level of interleukin-6 is an indicator for predicting diabetic macular edema. Jpn J Ophthalmol 46: 78-83.
31. Theoharides TC, Boucher W, Spear K (2002): Serum interleukin-6 reflects disease severity and osteoporosis in mastocytosis patients. Int Arch Allergy Immunol 128: 344-350.
32. Standiford TJ, Kunkel SL, Basha MA, et al. (1990): Interleukin-8 gene expression by pulmonary epithelial cell line. J Clin Invest 86: 1945-1953.
33. Koch AE, Polverini PJ, Kunkel SL, et al. (1992): Interleukine-8 (IL-8) as a macrophage-derived mediator of angiogenesis. Science 258: 1798-1801.
34. Hammond ME, Lapointe GR, Feucht PH, et al. (1995): IL-8 induced neutrophil chemotaxis predominantly via type I IL-8 receptors. J Immunol 155: 1428-1433.
35. Nickoloff BJ, Mitra RS, Varani J, et al. (1994): Aberant production of interleukin-8 and thrombospondin-1 by psoriatic keratinocytes mediates angiogenesis. Am J Pathol 144: 820-828.
36. Glynn PC, Henney EM, Hall IP (2001): Peripheral blood neutrophils are hyperresponsive to IL-8 and Gro-a in cryptogenic fibrosing alveolitis. Eur Respir J 18: 522-529.
37. Sinigaglia F (2000): IL-12 in lung diseases. Sarcoidosis Vasc Diffuse Lung Dis 17: 122-124.
38. Taha RA, Minshall EM, Olivenstein R, et al. (1999): Increased expression of IL-12 receptor mRNA in active pulmonary tuberculosis and sarcoidosis. Am J Respir Crit Care Med 160: 1119-1123.
39. Cavallo F, Di Carlo E, Butera M, et al. (1999): Immune events associated with the cure of established tumors and spontaneous metastases by local and systemic interleukin 12. Cancer Res 59: 414-421.
40. Voest EE, Kenyon BM, O’Reilly MS, et al. (1995): Inhibition of angiogenesis in vivo by interleukin 12. J Natl Cancer Inst 87: 581-586.
41. Burchett SK, Weaver MW, Westall JA, et al. (1988): Regulation of tumor necrosis factor/cachectin and IL-1 secretion in human mononuclear phagocytes. J Immunol 140: 3473-3481.
42. Pantelidis P, Fanning GC, Wells AU, et al. (2001): Analysis of tumor necrosis factor-alpha, lymphotoxin- alpha, tumor necrosis factor receptor II, and interleukin-6 polymorphisms in patients with idiopathic pulmonary fibrosis. Am J Respir Crit Care Med 163: 1432-1436.
43. Armstrong L, Foley NM, Millar AB (1999): Inter-relationship between tumour necrosis factor-alpha (TNF-αlpha) and TNF soluble receptors in pulmonary sarcoidosis. Thorax 54: 524-530.
44. Norby K (1996): TNF-αlpha and de novo mammalian angiogenesis. Microvasc Res 52: 79-83.
45. Liebovich SJ, Polverini PJ, Shepard HM, et al. (1987): Macrophage-induced angiogenesis is mediated by tumor necrosis factor. Nature 329: 630-632.
46. Humar R, Kiefer FN, Berns H, et al. (2002): Hypoxia enhances vascular cell proliferation and angiogenesis in vitro via rapamycin (mTOR)-dependent signaling. FASEB J 16: 771-780.