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International Journal of Experimental Pathology logoLink to International Journal of Experimental Pathology
. 2013 Nov 1;95(2):131–137. doi: 10.1111/iep.12061

Neovascularization is prominent in the chronic inflammatory lesions of Sjögren's syndrome

Margherita Sisto *,1, Sabrina Lisi *,1, Giuseppe Ingravallo †, Dario Domenico Lofrumento ‡, Massimo D'Amore §, Domenico Ribatti *
PMCID: PMC3960040  PMID: 24772480

Abstract

Angiogenesis is a common finding in chronic inflammatory diseases; however, its role in Sjögren's syndrome (SS) remains to be elucidated. Previous SS studies have demonstrated an increase in VEGF-A/VEGFR-2 system expression in minor salivary gland (MSG) biopsies from patients with SS, but differences in the new blood vessel formation between the different grades of disease severity have not been reported. Therefore, experiments were performed to demonstrate angiogenesis during different phases of primary SS (pSS) and to define the relationship between the microvessel density (MVD), macrophage infiltration and histiocyte distribution in SS MSG inflammatory lesions. In this series of experiments, immunohistochemistry was used to examine angiogenesis in serial sections of pSS MSG. Patients with pSS were classified accordingly with the grade of inflammatory lesions as I = low-grade (low focus score of 1 or 2), II = intermediate-grade (focus score of 3–6) and III = extensive inflammation in the MSG (high focus score of 12). Histological examination demonstrated that the MVD increased with the severity of the inflammatory lesions, and in addition, we found an increased infiltration of inflammatory and pro-angiogenic cells.These findings reveal that angiogenesis is intimately involved in the progression of pSS, may be central to the propagation of the chronic immune response observed in pSS and could represent a novel potential biomarker of pSS disease activity.

Keywords: Sjögren's syndrome, angiogenesis, CD31, CD68, Tryptase

Sjögren's syndrome (SS) is a chronic autoimmune disease of the exocrine glands with infiltration of lymphocytes and with a female predominance (Gallo et al. 2012). Dryness of the mouth and eyes results from destruction of the salivary and lachrymal glands. The exocrinopathy can be encountered alone (primary SS, pSS) or in the presence of other autoimmune disorders such as rheumatoid arthritis, systemic lupus erythematosus or progressive systemic sclerosis (secondary SS). Histologically, SS is characterized by extensive lymphocytic infiltration of the salivary and lachrymal glands (Tzioufas & Voulgarelis 2007). SS pathogenesis involves the interplay of multiple biological components, among which non-immune cells play a crucial role (Lisi et al. 2007, 2010, 2011, 2013a; Manoussakis & Kapsogeorgou 2007; Tzioufas & Voulgarelis 2007). In particular, endothelial cells play a key role in multiple aspects of chronic inflammation linked to SS (St Clair et al. 1992), including expression of cell adhesion molecules, chemokine secretion and recruitment of activated or memory T cells (Ichikawa et al. 1992). Angiogenesis is a complex process mediated by multiple cell types and is fundamental to many biological processes, including growth, development and repair (Ribatti & Crivellato 2012). Besides its well-known role in cancer, it has become clear that angiogenesis is also an integral part of a diverse range of non-neoplastic chronic inflammatory and autoimmune diseases, including atherosclerosis, rheumatoid arthritis, diabetic retinopathy, psoriasis, airway inflammation, peptic ulcers, Alzheimer's disease and SS (Carmeliet 2005; Sisto et al. 2012a,b2012b; Lisi et al. 2013a,b2013b). Indeed, angiogenesis is intrinsic to chronic inflammation and is associated with structural changes, including activation and proliferation of endothelial cells, and capillary and venule remodelling, all of which result in expansion of the tissue microvascular bed (Majno 1998). The anatomical expansion of the microvascular bed combined with its increased functional activation can foster further recruitment of inflammatory cells, and angiogenesis and inflammation have become chronically co-dependent processes (Bagli et al. 2004; Sisto et al. 2012a; Lisi et al. 2013a).

The angiogenic role played by the pathways involving the vascular endothelial growth factors and their receptors is well characterized. The vascular endothelial growth factor-A (VEGF-A) and its main receptor, vascular endothelial growth factor receptor-2 (VEGFR-2), are fundamental mediators of pathological angiogenesis, such as in neoplasia and chronic inflammation (Bagli et al. 2004; Carmeliet 2005; Sisto et al. 2012a; Lisi et al. 2013a), so that targeted blockade of VEGF-A/VEGFR-2 system is currently being used as a therapeutic approach to block angiogenesis in malignant tumours and to dampen inflammation (Folkman 1995). Studies from our laboratory reported evidence that, in SS, an increased production of pro-angiogenic factors from human salivary gland epithelial cells (SGEC) occurs via the VEGF-A/VEGFR-2 system activation and that targeting angiogenesis by VEGFR-2 blockade is an effective and entirely novel approach to block experimental SS (Sisto et al. 2012a,b2012b; Lisi et al. 2013a,b2013b).

However, the mechanisms responsible for angiogenesis in SS are not yet well defined. The current experiments were performed to study the relationship between the immunohistochemical expression of microvessel density (MVD) assessed by CD31/CD34 staining and macrophage (CD68) infiltration and histiocyte (tryptase) distribution in salivary gland biopsies from patients with pSS.

Materials and methods

Patient Population

A large number of patients with SS were examined for the pSS diagnosis. Among these, the Department of Pathology identified bioptic sections of minor salivary gland (MSG) belonging to different groups with several grades of inflammation. The department selected 34 biopsies and from these the biopsies belonging to the same number of patients were chosen, excluding dubious cases. Healthy control subjects (n = 9) were analysed for an abnormal salivary function and suspected Sjogren's syndrome, but which resulted in a normal biopsy. Other diagnostic tests were also included in the study. The patients gave their written consent, the study was approved by the local ethical review committee, and the experiments were conducted according to the tenets of the Declaration of Helsinki. After obtaining informed consent, MSG biopsy samples were taken from the patients with SS. Labial MSGs were harvested from the lower lip under local anaesthesia through normal mucosa, according to the explant outgrowth technique (Sens et al. 1985). Of the 34 patients with SS initially selected, only twenty-four were confirmed as coming from patients with primary SS and were included in the study. The patients with pSS were classified by the grade of inflammatory lesion, SS-I, low (focus score < 1, n = 8); SS-II, intermediate (focus score < 1, n = 8); and SS-III, advanced MSG lesions (high focus score > 1, n = 8). The patients all had definite disease according to the revised 2002 American-European criteria (Vitali et al. 2002). All patients had the clinical symptoms of dry eyes and mouth, a positive Schirmer's test (less than 5 mm wetting of a strip of filter paper per five minutes) and Rose Bengal staining (increased uptake of Rose Bengal dye in devitalized areas in the conjunctiva and cornea) along with the presence of at least one of the following autoantibodies: anti-Ro/SSA, anti-La/SSB, anti-nuclear antibodies and rheumatoid factor. None of the patients studied had received any glucocorticoid and/or immunosuppressive drug treatment until biopsy performance. At the time of biopsy, the median age of subjects in the control group was 49.8 years (range 21–66), the median age of patients in the group with low focus scores was 57.7 years (range 24.6–66.9), the median age of patients in the group with intermediate focus scores was 52.4 years (range 24.8–66.7), and the median age of patients in the group with high focus scores was 52.9 years (range 38.9–70.1).

Immunostaining for CD31, CD34, CD68 and tryptase in MSG from patients with SS

Immunostaining was performed as previously described (Sisto et al. 2012a). Briefly, paraffin-embedded salivary gland sections of patients with histological pSS were analysed for the intensity of stromal inflammatory reaction and scored semi-quantitatively by the density (nuclear counting) of lymphocytes and monocytes/macrophages revealed in the sections. The inflammatory reaction was categorized as: I, low; II, intermediate; and III, advanced MSG inflammatory lesions; and correlated with the histological focus score (Chisholm & Mason 1968). Serial 3-lm sections were cut other, deparaffinized, hydrated and blocked for endogenous peroxidase using 3% H2O2/H2O. After incubation with the primary antibodies against CD31 (Novocastra, Leica, Wetzlar, Germany), CD34 (Beckman Coulter, Brea, USA) CD68 and tryptase (both from Dako, Hamburg, Germany), the slides were incubated with the HRP-conjugated secondary antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Afterwards, the slides were incubated with diamino benzidine tetrahydrochloride (DAB) (Sigma, St.Louis, MO, USA) as substrate and counterstained with haematoxylin (Merck Eurolab, Dietikon, Switzerland). Negative controls without primary antibody were included in each experiment to verify antibody specificity. The images were captured under a light microscope. Immunoreactivity for CD31, CD34, CD68 and tryptase was assessed by both staining intensity and percentage of positive cells. The percentage of positive cells was subjectively divided into three grades by an observer who was blinded to the type of tissue.

Determination of MVD

Intraglandular microvessels were highlighted by immunostaining of the endothelial cells for CD31 and CD34 antigens. Microvessel density was assessed without knowledge of any patient's medical information. Slides were scanned in the light microscope at × 40 magnification, and three areas of maximal MVD, so-called hot spots, were identified. In each hot spot, microvessels (capillaries and small venules) were counted at × 400 magnification (each field representing an area of 0.375 mm2). The same observer evaluated every sample to minimize interexaminer variability. For each slide, the mean number of microvessels from these three areas, per 0.375 mm2, was calculated. Any brown staining in a single endothelial cell or a cluster of endothelial cells was considered as a countable microvessel. A visible vascular lumen and red blood cell were not the criteria to define a microvessel.

Data analysis

All data are expressed as mean ± SD. Differences between parameters were evaluated using Student's t-test. We subjected the results of measurements from all sets to an analysis of variance (anova). Differences were considered statistically significant at P < 0.05.

Results

The increased neovascularization is associated with the grade of the SS MSG inflammatory lesions

The number and the distribution of blood vessels in the different pSS MSG inflammatory lesions was quantified and then classified as I, low; II, intermediate; and III, advanced respectively. Eight biopsy specimens that belong to each of the three resultant pSS subgroups, as classified by the grade of inflammatory lesion (SS-I, low; SS-II, intermediate; and SS-III, advanced MSG lesions), were analysed. Immunohistochemistry using antibodies to CD31 was used to label blood vessels (Figure 1). The number of blood vessels inside the ‘hot spot’ area is shown in Figure 1. A relationship was found between the grade of salivary gland inflammation and the number of CD31-positive cells. Microscopic analysis of serial sections revealed that CD31 staining demonstrated a significant increase in the CD31-positive endothelial cells and in the MVD, which in turn resulted in association with the inflammatory grade (Figure 1, panels c–e). As shown in the tissue area marked by the square of the panel e, in the SS-III advanced MSG lesions, endothelial cell hypertrophy was indeed observed. The numbers of CD31-positive cells per mm2 were 4.3 ± 2.5 in healthy subjects (panel b), 9.7 ± 3.2 in group SS-I, 18.7 ± 7.5 in group SS-II and 27.0 ± 9.4 in group SS-III (respectively, showed in panels c–e). Statistical analysis showed that the number of CD31-positive cells were significantly different between the groups (P < 0.01, anova). In particular, in both intermediate and advanced lesions, CD31-positive cells were significantly augmented with respect to low infiltrate group (SS-II vs SS-I and SS-III vs SS-1 P < 0.01, Student's t-test).

Figure 1.

Figure 1

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Representative examples of MSG tissues that belong to the three SS subgroups, as classified by the grade of inflammatory lesion (SS-I, low; SS-II, intermediate; and SS-III, advanced MSG lesions). (c–e) Tissues stained brown by anti-CD31 antibody, which reveals the blood vessels (shown by arrowheads). The tissue area marked by the square incorporated in the panel e is representative of a blood vessel with hypertrophic endothelial cells. (h–j) Difference in the distribution of CD34+ cells in SS patients with different MSG inflammatory lesion severity. The sections were counterstained with haematoxylin (blue). Panels (a,f) negative controls for immunohistochemistry in which the primary antibody is not added. The secondary antibody is incubated with the sample in the same way reported in ‘Materials and Methods’ section. Panels (b–g): healthy control subjects. (Original magnifications: ×10 (a–j); ×20 (insert in e). Bar = 20 μm.

The number of CD31-positive blood vessels was 4.3 ± 2.5 in healthy subjects (panel b), 9.7 ± 3.2 in group SS-I, 18.7 ± 7.5 in group SS-II and 27.0 ± 9.4 in group SS-III. In addition, we measured angiogenesis by expression of CD34. However, it is noteworthy that CD34, a cell surface sialomucin, is expressed by haematopoietic precursors, eosinophils, mast cells and vascular endothelial cells and is suggested to play an integral role in mucosal inflammatory responses. In accordance with the CD34 distribution reported in the literature, we found that CD34 staining was present in a more diffuse pattern on several cell types in salivary gland tissues from patients with SS. In fact, as shown, CD34 positive stromal cells and myofibroblasts were observed; lymphocytes were CD31/CD34 negative (Figure 1, panels h–j). Our data demonstrate that an increase in CD34 positive staining occurs in accordance with the grade of inflammation observed in pSS MSG biopsies. Notably, the intensity of the CD34 stain in advanced and intermediate inflammatory lesions (II and III grades, panels i,j) was strongly comparable, whereas the low (I grade, panel h) displayed a very weak CD34 intensity, indicating that increased distribution of CD34 positive cells occurs in the inflamed tissues from patients with SS.

Rates of infiltration by macrophages and histiocytes in the chronic inflammatory lesions of SS

As an increase in macrophage and histiocyte infiltration associated with angiogenesis has been reported in a number of inflammatory processes (Sunderkotter et al. 1994; Ribatti et al. 2012), immunostainings, respectively, for CD68 and tryptase were performed in all study subject biopsies. The degree of infiltration by CD68-and tryptase-positive cells was counted manually in 5 different high-power fields with a high density of positive cells (at 400× magnification), with results expressed as the median number of positive cells per microscopic field. As shown in the Figure 2, panels c–e, all tested pSS MSG biopsy specimens displayed CD68-positive cells that had the typical morphological appearance of macrophages. In the SS-II (panel d) and SS-III (panel e) MSG specimens, ample numbers of macrophages were detected within the mononuclear cell infiltrates, and these were scattered or aggregated around ducts in lymphoepithelial lesions as well as spread throughout the remaining glandular parenchyma. In contrast, in SS-I (panel c), these cells were few and were scattered in the glandular parenchyma. Macrophages were significantly more frequent in the specimens from patients with an advanced grade of inflammation compared with those from low grade of inflammation; the infiltration of CD68-positive macrophages increased with the inflammatory grade from SS-I to SS-III. The number of CD68-positive macrophages was 3.7 ± 1.4 in healthy subjects (panel b), 8.4 ± 2.1 in group SS-I (panel c), 17.7 ± 6.5 in group SS-II (panel d) and 25.5 ± 5.3 in group SS-III (panel e). This increase in macrophages was statistically significant. (P < 0.05, anova) and showed a progressive increment in significnace levels from the low (I) to the advanced (III) inflmmatory stages. (SS-II vs SS-I P < 0.05; SS-III vs SS-1 P < 0.01, Student's t-test). As shown in Figure 2 (panels h–j), an increased number of tryptase-reactive histiocytes was demonstrated in the biopsy specimens with advanced pSS inflammatory lesions. The histiocyte count was significantly lower in the low grade of inflammation (panel h), in comparison with the intermediate grade (panel i) and resulted much higher in the advanced grade of inflammation (panel j). The number of tryptase+ histiocytes was 1.6 ± 1.3 in healthy subjects (panel g), 3.4 ± 1.8 in group SS-I (panel h), 6.8 ± 2.3 in group SS-II (panel i) and 12.5 ± 5.5 in group SS-III (panel j). Statistical analysis carried out by Student's t-test showed that the histiocyte count was significantly lower in the low grade of inflammation, in comparison with the intermediate grade (SS-I vs SS-II P < 0.05) and resulted much higher in the high grade of inflammation (SS-III vs SS-I P < 0.01).

Figure 2.

Figure 2

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Representative pictures of immunohistochemical stainings of paraffin-embedded MSG biopsies that were obtained from patients with SS classified by the grade of inflammatory lesion (I-III). Serial sections were treated according to the standard procedures for immunohistochemistry and stained brown for macrophage marker CD68 (c–e) and stained red for histiocyte marker tryptase (h–j). Panels a,f: negative controls for immunohistochemistry in which the primary antibody is not added. Panels b–g: healthy control subjects. Magnification a–j = 10×. The sections were counterstained with haematoxylin (blue). Bar = 20 μm.

Data analysis

The association between CD31, CD68 and tryptase expression in the I-III MSG inflammatory lesions was studied (Figure 3). After comparison of the mean values exhibiting the intensity for CD31, CD68 and tryptase expression between the four grades, a relationship was demonstrated between the macrophage and histiocyte infiltration, assessed by CD68 and tryptase expression, angiogenesis and the inflammatory grade of SS lesions and also measured by CD31 staining of new blood vessels. As shown, the specimens showing low CD31 (grade SS-I) also exhibited low CD68 and tryptase staining. On the contrary, the specimens showing moderate and strong CD31 expression (grades SS-II and SS-III) exhibited a CD68 and tryptase expression that varied from moderate to intense respectively.

Figure 3.

Figure 3

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Graph showing the expression of CD31 (blood vessels), CD68 (macrophages) and tryptase (histiocytes) in the SS MSG inflammatory lesions classified as I, low; II, intermediate; and III, advanced. Columns show the mean of the MVD, macrophage infiltration and histiocyte distribution per unit area, with standard deviation values. The statistical analysis was performed using the Student's t-test (P < 0.05*; P < 0.01**).

Discussion

In most of the studies published in the past few years, the authors have agreed that there are different molecular mechanisms implicated in the pathogenesis of SS. The salivary gland lesions of patients with SS are characterized by increased production of pro-inflammatory cytokines derived from infiltrating T cells. These cytokines probably take part in the amplification and perpetuation of inflammatory responses and tissue destruction through the modulation of activation, growth, differentiation and apoptosis of epithelial tissues (Abu-Helu et al. 2001). One of the earliest physiological responses to tissue injury or infection is an increase in vascular permeability and blood flow to the affected area, initiated by regional vasodilation and followed by enhanced angiogenesis to facilitate the wound healing process (Hoeben et al. 2004). It has now been clearly established that the microvascular changes associated with angiogenesis are key contributors to the tissue injury and remodelling processes that inevitably accompany chronic inflammation (Bagli et al. 2004; Sisto et al. 2012a; Lisi et al. 2013a). However, the important role played by angiogenesis in several chronic inflammatory diseases is still being elucidated. Few data are available on the role of angiogenesis in SS even if, recently, evidence has emerged suggesting an important role of the neovascularization in the pathogenesis and evolution of the SS disease. Published data demonstrated that a functional impairment of the arterial wall may sustain early phases of atherosclerotic damage in SS, and a combined effect of disease-related chronic inflammatory and immunological factors appears to support the dysfunction of endothelium and vascular smooth muscle cells respectively (Gerli et al. 2010). Observations from our laboratory support the existence of an important link between inflammation and angiogenesis in the pathogenesis of SS. We have detected a strongly positive staining for VEGF-A and VEGFR-2 proteins in the biopsy specimens from patients with SS (Sisto et al. 2012a), and a large number of pro-angiogenic factors resulted upregulated in SS salivary glands (Sisto et al. 2012a; Lisi et al. 2013b). Furthermore, most recently, we have successfully demonstrated a role of neuropilins, first described as mediators of neuronal guidance, in angiogenic processes linked to SS (Sisto et al. 2012b). Although current knowledge suggests that angiogenesis is increased in SS (Sisto et al. 2012a; Lisi et al. 2013b), direct evidence for the new blood vessel formation in SS is still lacking, and it is unclear whether this process is in relationship with the level of inflammation. In the present work we have confirmed conclusively that in SS there is sprouting of new vessels from the pre-existing ones and splitting of the resident mature vessels. This increased local angiogenesis might favour the subsequent active infiltration of inflammatory cells. The new blood vessel formation is associated with an increased macrophage and histiocyte infiltration that could itself have a critical role in the development of neovasculature. Increased neovascularization also seemed to be consistent with the severity of the inflammatory lesions from which the SS biopsies were derived, and correlated with the level of macrophage and histiocyte infiltration. These findings were in agreement with recent literature which has shown that inflammation could be implicated in neo-angiogenesis, but at the same time, the newly formed vessels support the chronic inflammatory state by transport of nutrients and oxygen (Lisi et al. 2013a). Conversely an increase in macrophage and histiocyte infiltration has been demonstrated in association with angiogenesis in a number of inflammatory processes (Sunderkotter et al. 1994; Ozdemir et al. 2002; Ranieri et al. 2009). Macrophages, given their ability to migrate within virtually all tissues of the body, are also ideal cells to regulate angiogenesis during tissue injury and repair. There is substantial evidence that macrophages and histiocytes play a significant role in both physiological and pathological angiogenesis (Nucera et al. 2011), releasing angiogenic cytokines (Lewis et al. 1995; Ranieri et al. 2009). Furthermore, inflammatory signals recruit lymphocytes and macrophages into areas of neovascularization, which further act as a source of angiogenic and arteriogenic factors (Lewis et al. 1995; Ranieri et al. 2009).

The increased angiogenesis demonstrated in SS and the link with the MSG inflammatory lesion grades are in accordance with studies in other autoimmune disorders, which support that neovascularization is higher at the site of inflammation (Carvalho et al. 2007; Maruotti et al. 2007). More recent studies have addressed the role of the vasculature in the invasive and destructive processes occurring in the joints in rheumatoid arthritis, where abundant blood vessels were found in the synovium, and the number of blood vessels was correlated with hyperplasia of synovial cells and with cartilage and bone degeneration (Taylor 2005). Studies have indicated that VEGF-A plays a role in the pathogenesis of multiple sclerosis (Koch & Distler 2007), while in psoriasis, high levels of VEGF-A have been found in lesional tissues (Flisiak et al. 2013). There is, furthermore, accumulating evidence that VEGF produced in the thyroid follicular epithelial cells and its receptors is also important in Graves' disease and in thyroiditis, characterized by a marked increase in blood flow and vasodilation (Figueroa-Vega et al. 2009).

Overall, the results of this study lead to the conclusion that, in pSS, an increment in the new blood vessel formation associated with the increased number of macrophages and histiocytes infiltrating in the stroma of the inflammatory lesions of MSG occurs, and this correlated with lesion severity. These are to our knowledge the first data supporting the relationship. The understanding of the role of angiogenesis in the pathogenesis of SS requires further studies, and we suggest that additional studies of in angiogenesis will provide invaluable information which will facilitate the development of novel molecular targets with potential for use in both diagnosis and therapy of Sjögren's syndrome.

Acknowledgments

We are grateful to M.V.C. Pragnell, B.A., for language revision.

Funding source

This work was supported by a grant (N°: 20216000056) from the Italian Ministry for Universities and Research.

Conflict of interests

None.

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