Increased spontaneous production of VEGF by CD4+ T cells in type 1 diabetes

Clinical Immunology (2010) 137, 261–270

available at www.sciencedirect.com

Clinical Immunology www.elsevier.com/locate/yclim

Increased spontaneous production of VEGF by CD4 + T cells in type 1 diabetes Natalia Marek a,⁎, Małgorzata Myśliwiec b , Krystyna Raczyńska c , Katarzyna Zorena d , Jolanta Myśliwska d , Piotr Trzonkowski a a

Department of Clinical Immunology and Transplantology, Medical University of Gdańsk, Poland Clinic of Pediatrics, Hematology, Oncology and Endocrinology, Medical University of Gdańsk, Poland c Department and Clinic of Ophthalmology, Medical University of Gdańsk, Poland d Department of Immunology, Medical University of Gdańsk, Poland b

Received 17 June 2010; accepted with revision 22 July 2010 Available online 11 August 2010 KEYWORDS +

CD4 T cells; VEGF; Type 1 diabetes; Diabetic retinopathy

Abstract In the present study we report that CD4+ T cells from patients with type 1 diabetes produce significantly higher amounts of VEGF than respective cells from the healthy individuals. Among CD4+ T cells memory subsets were the main source of VEGF. In addition, memory CD4+ T subsets were the most numerous in patients with diabetic retinopathy (DR). DR was also characterized by significant increase of VEGF concentration in serum and culture supernatants. Hence, these data indicate that there is a sustained spontaneous production of VEGF by CD4+ T cells in type 1 diabetes, which is additionally exacerbated in DR. In our opinion alterations in the proportions of CD4+ T cell subsets and their VEGF production may be a useful tool for early assessment of the risk of DR onset and progression. © 2010 Elsevier Inc. All rights reserved.

1. Introduction Destruction of pancreatic β cells by self-reactive T cells results in insulin deficiency and clinical symptoms of type 1 diabetes [1–4]. However, lack of insulin is not the only consequence of the flawed activity of the immune system in diabetes. A vast majority of diabetic patients develop vascular complications, among which diabetic retinopathy (DR) is a leading cause of blindness in young adults in developed countries [5,6]. Despite high ⁎ Corresponding author. Medical University of Gdańsk, Department of Clinical Immunology and Transplantology, ul. Dębinki 7, 80-211 Gdańsk, Poland. Fax: +48 058 349 14 36. E-mail address: [email protected] (N. Marek).

prevalence of DR, its pathogenesis is not fully understood. We and others have recently reported that inflammation [6–10] and changes in general and local concentration of vascular endothelial growth factor (VEGF) are thought to be the main risk factors of this angiopathy [7–13]. In addition, it has been revealed recently that DR onset might also be associated with altered leukocyte activation [6,13–15]. There are reports that retinal vessels in both humans and animals with diabetes are characterized by increased numbers of leukocytes in comparison with healthy individuals. Moreover, it has been revealed that these cells may lead to obliteration of retinal capillaries and endothelial damage. This phenomenon is probably associated with massive extravasation, as diabetes is characterized by the presence of numerous activated leukocytes in the retinal tissue [6,15–18]. Indeed, in

1521-6616/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.clim.2010.07.007

262

N. Marek et al.

the diabetic retina the regions of endothelial injury and neovascularization overlap with the areas of leukocyte infiltration [16,18]. Moreover, there are reports that the inhibition of leukocyte adhesion can prevent or at least decrease the leakage of blood–retina barrier and injury of retinal endothelium [6,13,18,19], which are crucial for DR development. CD4+ T cells are one of the major players in inflammation. This population comprises several subsets with different potential of proliferation and effector functions [20–22]. In addition, CD4+ T cells are efficient source of various mediators, which regulate functions of other leukocytes [23], and were found to synthesize VEGF in some pathological conditions [24,25]. Moreover, T cells homing to inflammatory sites are in direct contact with endothelial cells [26]. In such circumstances secretion of small amounts of VEGF is sufficient to exert a strong effect on the target cell and influence the local angiogenesis. In view of the above, we hypothesized that CD4+ T cells in type 1 diabetes might be a source of VEGF and therefore they can be associated with the development of DR. If true, assessment of VEGF expression in CD4+ T cells might serve as a marker of retinopathy in diabetic patients. Therefore, in the current study we analyzed the production of VEGF by in vitro cultured naive and memory CD4+ T subsets from diabetic adolescents with and without nonproliferative DR and measured VEGF concentration in serum and culture supernatants.

2. Materials and methods

of the Clinic of Pediatrics, Hematology, Oncology and Endocrinology of Medical University of Gdańsk. Control group consisted of age and gender-matched 30 healthy individuals. Health status was established on the basis of medical history, physical examination and laboratory data obtained during annual control screening examination of adolescents. The exclusion criteria were: chronic disease (other than diabetes), infection within 1 month preceding the study, intake of anti-inflammatory drugs or other drugs known to affect the immune system. All diabetic patients were on insulin therapy (0.87 ± 0.24 U/kg). DR was determined by visual acuity, intraocular pressure measurement, anterior segment estimation (slit lamp; TOPCON SL-82, Japan) and fluorescein angiography (digital camera-Topcon IMAGEnet2000, Japan). The eye fundus examination was performed with the +90D lens (Ocular Instruments Inc., Bellevue, WA, USA). All investigated patients with DR had non-proliferative DR (NPDR). NPDR was classified according to Early Treatment Diabetic Retinopathy Study (ETDRS) into three levels: mild NPDR, moderate NPDR and severe NPDR [27–30]. Hence, patients were divided into two subgroups: 1). mild NPDR (n = 15) and 2). moderate and severe NPDR (n = 11) in order to estimate the association between investigated parameters and DR progression. Standard laboratory parameters were determined in all investigated groups (Table 1). The research was approved by The Ethics Committee of The Medical University of Gdańsk and was conducted according to the principles expressed in the Declaration of Helsinki. Written informed consent was obtained from all participants or their parents.

2.1. Patients 2.2. Reagents The examined groups comprised 60 adolescents with type 1 diabetes mellitus without diabetic complications and 26 individuals with non-proliferative DR selected from patients

The following fluorochrome-conjugated monoclonal antibodies (mAbs) and appropriate isotype controls from BD

Table 1 Clinical and laboratory characteristics of the investigated groups. As indicated by the distribution of the variables data are presented as medians with minimum and maximum values within parentheses and means ± SD (‡). Healthy = control group, DM1 = individuals with type 1 diabetes without complications, DM1 + DR = individuals with type 1 diabetes and retinopathy, n = number of subjects, SBP = systolic blood pressure, DBP = diastolic blood pressure, BMI = body mass index, HbA1c = glycated hemoglobin. Parameter n (female/male) Age (years) Diabetes duration (years) BMI (kg/m2) Glucose (mg/dl) HbA1c (%) Triglycerides (mg/dl) Total cholesterol (mg/dl) LDL cholesterol (mg/dl) HDL cholesterol (mg/dl) CRP (mg/L) Albumin excretion rate (mg/24 h) Serum creatinine (μ mol/l) SBP (mm/Hg) DBP (mm/Hg)

Healthy 30 (15/15) 17.18(±3.98)‡ 20 (18–23) 86.00 (± 16.67)‡ 5.00 (4.50–5.80) 72 (50–90) 171 (150–190) 70 (50–95) 58.60 (± 11.05)‡ 0.3 (0.1–1.0) 1 (0.2–5.0) 0.6 (0.5–0.75) 100 (90–120) 70 (60–80)

DM1

DM1 + DR

60 (30/30) 16.31(± 2.92)‡ 4.89 (± 1.70)‡ 19.50 (16.5–26) 122.62(± 28.67)‡ 8.00 (6.60–12.00) 73.23 (39–163) 170 (109–257) 81 (58–199) 58.33 (± 11.76)‡ 1.00 (0.21–3.8) 9.56 (1.67–46) 0.68 (0.45–0.91) 110 (80–140) 70 (55–100)

26 (14/12) 17.68 (± 2.17)‡ 9.64 (± 1.32)‡ 20.40 (17.24–25.8) 144.00(± 31.15)‡ 9.25 (7.56–12.20) 90.00 (47–160) 187.5 (120–284) 98.5 (68–197) 49.83 (± 10.14)‡ 1.48 (0.12–3.78) 27.82 (6.24–51.48) 0.81 (0.59–1.01) 120 (90–128) 70 (60–95)

CD4+VEGF+ T cells in diabetes type 1

263

Figure 1 Percentage of CD4+ Tn, Tcm and Tem cells. CD4+ lymphocytes were identified according to their low values of SSC parameter and high expression of CD4 receptor (A). Expression of CD27 and CD45RO molecules was used to identify Tn (CD27+CD45−), Tcm (CD27+CD45+) and Tem (CD27−CD45+) subsets among gated CD4+ T cell population (B). Statistical comparison of percentage of CD4+ Tn (C), Tcm (D) and Tem (E) subsets in healthy (Healthy; n = 30) and diabetic adolescents without complications (DM1; n = 60) and with DR (DM1 + DR; n = 26) is depicted. Data were analyzed with Kruskal–Wallis ANOVA and are presented as medians (symbols inside the boxes), 10–90% percentiles (boundaries of the boxes) and minimum–maximum (error bars outside the boxes). Differences between investigated groups that were statistically significant (p b 0.05) are marked with “*”.

Biosciences, USA (unless otherwise stated) were used (short names of fluorochromes, clones and isotypes of mAbs in parentheses): anti-CD4-peridinin-chlorophyll-protein (PerCP; SK3, mouse IgG1,κ), anti-CD45RO-allophycocyanin (APC; UCHL1, mouse IgG2a,κ), anti-CD27 (APC-Cy7; 0323, mouse IgG1,κ; BioLegend, USA) and anti-VEGF-phycoeritrin (PE; 23410, mouse IgG2a; R&D, France). LPS from Escherichia coli (055:B5) (Sigma Aldrich, Germany) was used for in vitro experiments as a known stimulator of VEGF expression in immune cells [31,32]. LPS was solubilized in phosphate buffered saline (PBS), aliquoted and stored at −80 °C. Final concentration of the compound was 100 ng/ml. Fixation and Permeabilization Solution Kit with GolgiStop (protein transport inhibitor; BD Biosciences) was used for intracellular staining.

Unlabeled anti-VEGF (23410, mouse IgG2a; R&D, France) was used for competition assays (1.5 μg/106 cells).

2.3. Specimen preparation Peripheral blood mononuclear cells (PBMC) were obtained by Ficoll/Uropoline gradient centrifugation from blood collected into sterile heparinized tubes. Cells (106) from each sample were subjected to immediate surface staining to determine the percentage of Tn, Tcm and Tem cells within peripheral blood CD4+ lymphocyte population. Serum was obtained from blood samples by centrifugation at 500 ×g for 15 min and stored at −80 °C until analysis. Supernatants were collected after 12 h of the culture (before addition of GolgiStop into the culture medium) and stored at −80 °C until analysis.

264

N. Marek et al.

Figure 2 VEGF expression in CD4+ Tn, Tcm and Tem cells. Percentage of VEGF+ cells among CD4+ Tn, Tcm and Tem subsets from healthy (Healthy; n = 30) and diabetic adolescents without complications (DM1; n = 60) and with DR (DM1 + DR; n = 26) was measured in non-stimulated (Non-stimulated; A) and LPS-treated cultures of PBMC (LPS; B). Intensity of VEGF expression in non-stimulated (Nonstimulated; C) and LPS-treated cultures of PBMC (LPS; D) was calculated as ratio of median fluorescence intensity of the sample vs. negative signal of the isotype control. Data were analyzed with Kruskal–Wallis ANOVA and are presented as medians (symbols inside the boxes), 10–90% percentiles (boundaries of the boxes) and minimum–maximum (error bars outside the boxes). Differences between investigated groups that were statistically significant (p b 0.05) are marked with “*”.

2.4. Intracellular expression of VEGF in CD4+ Tn, Tcm and Tem cells PBMC were cultured for 16 h in RPMI1640 (Sigma Aldrich) with 5% FCS and antibiotics (Penicillin and Streptomycin, 100 U/ml and 100 μg/ml respectively; Sigma Aldrich, Germany) in concentration 106 cells/ml/well (37 °C, 5% CO2 and 95% humidity) in absence and presence of LPS. After 12 h culture 100 μl of supernatants was collected and 2 μl of GolgiStop was added for further 4 h. Subsequently, cells were collected and stained (30 min at room temperature) with mAbs (10 μl each mAb/106 cells) to determine CD4+ Tn, Tcm and Tem subsets on the basis of consecutive immunophenotypes: CD4 + CD27 + CD45RO − , CD4+CD27+CD45RO+ and CD4+CD27−CD45RO+, respectively [22,33–35]. After incubation, cells were washed, fixed, permeabilized and stained for detection of VEGF. Each time unstained cells and cells labeled for surface antigens in presence of isotype control were analyzed to subtract autofluorescence and exclude nonspecific binding of anti-VEGF mAbs. For competition assays unlabeled anti-VEGF mAb (1.5 μg/106 cells) was added to the cells before incubation with anti-VEGF-PE mAbs. Measurements were performed on the LSRII flow cytometer (BD Biosciences) equipped with the solid state Coherent Sapphire blue laser (20 mW, 488 nm),

the solid state UV laser-Lightwave Xcyte laser (20 mW, 355 nm) and the Helium–Neon gas laser (17 mW, 633 nm), which allowed multicolor analysis. In order to minimize differences between the individual samples, the intensity of intracellular VEGF expression was calculated as ratio of median fluorescence intensity (MFI) of the sample labeled with anti-VEGF PE-conjugated mAb to MFI of appropriate isotype control (positive signal/negative signal).

2.5. VEGF concentration in serum and supernatants Levels of VEGF in serum and supernatants were measured with Cytometric Bead Array flex sets (BD Biosciences, Pharmingen) according to the manufacturer instructions. The limit of detection determined by the manufacturer was 4.5 pg/ml. The intra-assay (1.6) and inter-assay (6.4) reproducibility was determined for the tests by evaluating 20 replicates from the quality control data of the laboratory.

2.6. Statistics Data were analyzed with the software Statistica 8.0 (Statsoft, Poland). As indicated by distribution of the variables, data were calculated with Kruskal–Wallis

CD4+VEGF+ T cells in diabetes type 1

265 individuals and diabetic adolescents without complications (Fig. 1).

3.2. Diabetes was associated with high spontaneous synthesis of VEGF by CD4+ T cells Analysis of the expression of VEGF in CD4+ T cells revealed that diabetes was associated with increased spontaneous production of VEGF by Tn, Tcm and Tem subsets. The percentage of CD4+VEGF+ T cells in non-stimulated cultures of PBMC from healthy adolescents was significantly lower than that in the cultures of PBMC from the patients. In addition, non-stimulated CD4+VEGF+ T cells from the control group exhibited lower intensity of VEGF synthesis, than corresponding cells from diabetic participants (Fig. 2). While VEGF expression in non-stimulated lymphocytes from diabetic patients was remarkably high, it was almost undetectable in cells from healthy adolescents. Stimulation with LPS resulted in the significant increase in the percentage of CD4+ VEGF+ Tcm and Tem cells in the cultures of PBMC from healthy individuals. In addition, LPS enhanced the intensity of VEGF expression in all CD4+ VEGF+ T subsets of the control participants. The cells from the diabetic patients did not respond to the stimulation, with the exception of CD4+ Tn cells of the patients without complications (Fig. 2; Suppl. Table 1). Nevertheless, both the percentage of VEGF + T cells and the intensity of their VEGF synthesis were still higher in cultures of PBMC from diabetic groups than from healthy participants (Fig. 2; Suppl. Table 1).

Figure 3 VEGF concentration in supernatant and serum samples. VEGF levels were measured in supernatants from non-stimulated (Non-stimulated; A) and LPS-treated (LPS; B) cultures of PBMC and in serum (C) from healthy (Healthy; n = 30) and diabetic adolescents without complications (DM1; n = 60) and with DR (DM1 + DR; n = 26). Data were calculated with Kruskal–Wallis ANOVA and are presented as medians (symbols inside the boxes), 10–90% percentiles (boundaries of the boxes) and minimum–maximum (error bars outside the boxes). Differences between investigated groups that were statistically significant (p b 0.05) are marked with “*”.

3.3. Memory subsets were the main source of VEGF among CD4+ T cells There were significant differences in the production of VEGF by the subsets of CD4+ T cells. In each investigated group, the percentage of VEGF+ cells within CD4+ Tn subset was significantly lower than that noted in memory subpopulations. In addition, CD4+VEGF+ Tn cells exhibited lower intensity of VEGF synthesis, than CD4+VEGF+ Tcm and Tem subsets (Fig. 2).

3.4. Retinopathy was associated with the highest levels of VEGF in culture supernatants and serum ANOVA, Mann–Whitney U test and Spearman's rank correlation for nonparametric data. Simple logistic regression was used to determine the crude associations between investigated variables and DR and to select variables for multilevel model. Values of p b 0.05 were considered statistically significant.

3. Results

Non-stimulated and LPS treated PBMC from adolescents with DR secreted significantly higher amounts of VEGF than the cells from patients without complications and healthy adolescents (Fig. 3A–B). Similar differences were found for the serum samples. Adolescents with DR were characterized by significantly higher serum concentration of VEGF than diabetic patients without complications and healthy individuals (Fig. 3C).

3.1. Retinopathy was associated with increased percentage of memory subsets within CD4+ T cell population

3.5. Serum levels of VEGF correlated with the intensity of VEGF expression in CD4+ T cells

DR patients were characterized by significantly higher numbers of Tcm and Tem cells in PBMC than healthy

There was a strong correlation between serum concentration of VEGF and VEGF expression in CD4+ T cell subsets from

266

N. Marek et al.

Figure 4 Association between serum and intracellular concentration of VEGF and serum CRP levels in patients without diabetic complications. Left panel depicts correlations between serum VEGF levels and intensity of VEGF expression in CD4+ Tn (A), Tcm (B) and Tem (C) cells (R = 0.66, R = 0.74, R = 0.77, respectively; p b 1 × 10− 6 for all comparisons). Data were calculated with Spearman's rank correlation. R = Spearman's rank correlation coefficient. Right panel shows CRP levels in the patient subgroups characterized by low (I) and high (II) serum and intracellular levels of VEGF. Comparisons between the subgroups were done with Mann–Whitney U test and are presented as medians (symbols inside the boxes), 10–90% percentiles (boundaries of the boxes) and minimum–maximum (error bars outside the boxes). Differences that were statistically significant (p b 0.05) are marked with “*”.

diabetic patients without complications (Fig. 4), as well as in those with DR (Fig. 5).

3.6. High levels of VEGF in serum and CD4+ T cells were associated with increased serum CRP concentration In addition, the levels of VEGF were significantly associated with CRP. There was a strong association between serum VEGF concentration, VEGF expression in CD4+ T cells and serum CRP. According to the serum and intracellular levels of VEGF both diabetic groups could be divided into two subgroups (I and II). The subgroups (II) with high serum VEGF concentration and high intracellular expression of VEGF were characterized by increased serum CRP (Figs. 4 and 5).

3.7. Increased synthesis of VEGF by PBMC was associated with higher risk of DR onset and progression High spontaneous secretion of VEGF by PBMC (N 14 pg/ml VEGF in culture supernatants) was found to significantly increase the risk of DR onset (Table 2). The association was even stronger after adjustment for standard DR risk factors, such as CRP and HbA1c. However, the strongest association with DR and the highest sensitivity and specificity of the assay (94% and 100%, respectively) were reached after adjustment for the percentage of CD4 + Tem subset (Table 2). HbA1c alone was found to be significantly weaker predictor of DR in the investigated patients than the level of VEGF in culture supernatants and did not reach statistical significance. In addition, simple logistic regressions revealed

CD4+VEGF+ T cells in diabetes type 1

267

Figure 5 Association between serum and intracellular concentration of VEGF and serum CRP levels in patients with DR. Left panel depicts correlations between serum VEGF levels and intensity of VEGF expression in CD4+ Tn (A), Tcm (B) and Tem (C) cells (R = 0.86, R = 0.87, R = 0.86, respectively; p b 1 × 10− 6 for all comparisons). Data were calculated with Spearman's rank correlation. R = Spearman's rank correlation coefficient. Right panel shows CRP levels in the patient subgroups characterized by low (I) and high (II) serum and intracellular levels of VEGF. Comparisons between the subgroups were done with Mann–Whitney U test and are presented as medians (symbols inside the boxes), 10–90% percentiles (boundaries of the boxes) and minimum–maximum (error bars outside the boxes). Differences that were statistically significant (p b 0.05) are marked with “*”.

that CRP and HbA1c had low specificities (53% HbA1c and CRP) and relatively low sensitivities (87% HbA1c and 72% CRP) as DR markers (Table 2). In addition, our study confirmed that increased serum VEGF levels (≥ 200 pg/ml) are strongly associated with risk of DR onset. Adjustment for the percentage of CD4+ Tem cells resulted with higher assay specificity and sensitivity (Table 2). When patients with DR were divided into two groups which differed in DR severity, strong association between intensity of VEGF expression in CD4+ Tn and Tcm cells (MFI ratio of VEGF) and DR progression was observed (Table 3). These parameters were significantly lower in patients with mild NPDR than in those with moderate and severe NPDR. Single and multiple logistic regression models confirmed predictive value of VEGF expression in CD4+ Tn and Tcm cells for DR progression (Table 3).

4. Discussion In the present study we found that CD4+ T cells from diabetic adolescents were characterized by increased spontaneous production of VEGF. VEGF+CD4+ T cells were more numerous in diabetic patients and exhibited higher intensity of VEGF synthesis than the cells from healthy individuals. Moreover, patients with DR were characterized by the highest levels of the cytokine, both in the culture supernatants and sera. As interplay between inflammation and angiogenesis is well established [25] and proinflammatory LPS is known to stimulate VEGF production by immune cells [31,32], LPS was used as a stimulant in the in vitro studies. Interestingly, only T cells from healthy volunteers responded to LPS with the significant increase in the intensity of VEGF production. Despite these changes, when compared with diabetic

268 Table 2

N. Marek et al. Assessment of the risk of retinopathy onset in adolescents with type 1 diabetes (n = 86).

Variable

OR

95% CI

p value

Sensitivity (%)

Specificity (%)

HbA1c (%) CRP (mg/l) Age (years) Serum VEGF (pg/ml) a Adjusted for CRP (mg/l) Adjusted for CD4+ Tem (%) CD4+ Tem (%) Adjusted for serum VEGF (pg/ml) a Adjusted for VEGF in supernatants b CD4+ Tn (%) Ratio CD4+ Tem/CD4+ Tn (%) VEGF in supernatants (pg/ml) b Adjusted for HbA1c (%) Adjusted for HbA1c (%) and age (years) Adjusted for CRP (mg/l) Adjusted for CD4+ Tem (%)

1.43 1.99 1.33 9.29 7.03 10.29 1.38 1.34 1.99 0.67 1.62 26.66 30.87 28.91 30.12 31.13

0.92–2.20 1.14–3.28 1.02–1.72 3.14–27.47 2.20–26.89 3.21–54.23 1.00–1.91 0.91–2.00 0.78–4.80 0.44–1.03 1.02–2.56 16.84–48.30 17.78–51.54 16.99–50.12 15.00–51.32 14.00–54.32

0.09 9 × 10− 3 ⁎ 0.01 ⁎ 1 × 10− 5 ⁎ 6 × 10− 5 ⁎

87 73 52 76 76 98 90 98 94 95 76 95 93 96 94 94

53 53 77 73 73 94 68 94 100 92 73 92 96 96 95 100

b 1 × 10− 6 ⁎ 1 × 10− 3 ⁎ b 1 × 106 ⁎ 5 × 10− 5 ⁎ b 1 × 10− 6 ⁎ 2 × 10− 4 ⁎ b 1 × 10− 6 ⁎ b 1 × 10− 6 ⁎ b 1 × 10− 6 ⁎ b 1 × 10− 6 ⁎ 5 × 10− 5 ⁎

Simple and multiple logistic regression model was used to analyze association between retinopathy and investigated parameters. OR = odds ratio; CI = confidence intervals; n = number of subjects. ⁎ Statistical significance (p b 0.05). a Serum VEGF ≥ 200 pg/ml. b Spontaneous secretion of VEGF by PBMC exceeding 14 pg/ml/.

patients, the percentage of CD4+VEGF+ T cells and the efficiency of VEGF synthesis after stimulation with LPS remained lower in healthy adolescents. These observations refer to the results of Bottomley et al., who found increased spontaneous secretion of VEGF in the cultures of PBMC from individuals with rheumatoid arthritis. In addition, the study

Table 3

revealed that LPS induced lower up-regulation of intracellular VEGF in rheumatoid arthritis PBMC, than in the cells from the healthy individuals [31]. Altogether, these observations suggest that CD4+ T cells in T cell-dependent autoimmune diseases, such as type 1 diabetes and rheumatoid arthritis [1– 4,36,37], are permanently activated. Therefore, the

Factors associated with the severity of retinopathy a in adolescents with type 1 diabetes (n = 26).

Variable

OR

95% CI

p value

Sensitivity (%)

Specificity (%)

HbA1c (%) CRP (mg/l) Serum VEGF (pg/ml) a VEGF in supernatants (pg/ml) b MFI ratio of CD4+VEGF+ Tn cells Adjusted for HbA1c (%) Adjusted for CRP (mg/l) Adjusted for serum VEGF (pg/ml) a MFI ratio of CD4+VEGF+ Tcm cells Adjusted for CRP Adjusted for HbA1c MFI ratio of CD4+ VEGF+ Tem cells CD4+ Tn (%) CD4+ Tcm (%) CD4+ Tem (%)

1.96 4.53 3.04 1.54 4.16 4.05 3.34 3.91 3.13 2.35 5.27 3.3 0.91 1.21 1.29

0.77–4.99 1.19–17.17 0.53–17.69 0.99–2.41 1.31–13.21 1.16–14.14 0.94–11.76 1.19–12.92 1.37–7.13 0.95–5.84 1.19–23.19 0.58–18.73 0.83–1.01 0.95–1.53 1.03–1.62

0.10 2 × 10− 3 ⁎ 0.17 5 × 10− 4 ⁎ 2 × 10− 3 ⁎ 8 × 10− 3 ⁎ 8 × 10− 3 ⁎ 9 × 10− 3 ⁎ 2 × 10− 4 ⁎ 9 × 10− 4 ⁎ 1 × 10− 4 ⁎

66 65 60 92 71 72 91 71 91 85 100 67 73 82 82

58 61 66 91 78 79 77 78 77 82 84 61 59 77 78

a

0.14 0.04 0.04 6 × 10− 4 ⁎

Simple and multiple logistic regression models were used to analyze association between two groups with different retinopathy severity and investigated parameters; gr.1 = mild non-proliferative diabetic retinopathy (n = 15); gr.2 = moderate and severe non-proliferative diabetic retinopathy (n = 11). OR = odds ratio; CI = confidence intervals; n = number of subjects. ⁎ Statistical significance (p b 0.05). a Serum VEGF ≥ 200 pg/ml. b Spontaneous secretion of VEGF by PBMC exceeding 14 pg/ml.

CD4+VEGF+ T cells in diabetes type 1 stimulation in vitro is unable to further up-regulate the production of VEGF in the cells from such patients. In view of the above, we may suppose that diabetes is associated with some kind of stimulation, which enhances the expression of VEGF in T cells. According to the recent studies, the production of VEGF increases in the inflammatory environment and can be regulated by cytokines. It has been shown that TNF-α and IFN-γ stimulated VEGF synthesis in human leukocytes, while IL-4 and IL-10 exhibited inhibitory effect [31]. Association between proinflammatory factors, VEGF and type 1 diabetes was also confirmed in our earlier studies [7–10,38]. We found the highest levels of VEGF in the sera of patients with the highest serum concentrations of IL-6 and TNF-α [7–9]. Presumably, in the current study, increased expression of VEGF in CD4+ T cells in diabetic adolescents resulted from in vivo activation of the lymphocytes by proinflammatory factors. Indeed, there was the correlation between intracellular and serum levels of VEGF and serum CRP values in diabetic patients. Increased production of VEGF in diabetes may result in severe consequences. In the current study it was proved that high levels of VEGF correlate with DR. This angiopathy was associated with the highest percentage of memory cells, which in turn were found to be the main source of VEGF. The interrelationship between memory cells and production of VEGF was also confirmed by the levels of VEGF in the sera and culture supernatants. Patients with DR were characterized by the highest VEGF levels in both sera and culture supernatants. Furthermore, there was a strong correlation between serum VEGF concentration and its intracellular expression. It is well established that VEGF is synthesized by a variety of cells [31,32,39–41] but, lymphocyte-derived VEGF may be crucial in the pathogenesis of DR. Early stages of DR are associated with increased leukocyte adhesion and increased local VEGF activity [13,16–19]. Lymphocyte extravasation is preceded by direct cell-to-cell contacts with endothelium [26,42,43]. In such conditions, the secretion of VEGF by adherent CD4+ T cells, even in small amounts, but directly on the surface of endothelial cells, can be a potent signal triggering retinal vessel permeability and DR. Even if CD4+ T cells are not the only population responsible for the inflammatory process in the diabetic retina, spontaneous secretion of VEGF by memory lymphocytes should be recognized as stable and persistent source of VEGF in this environment. Concluding, our results add new details to the understanding of diabetes, DR and biology of CD4+ T cell subsets. We found that VEGF production by memory lymphocytes is increased in type 1 diabetes and additionally enhanced in DR. Therefore, alterations in numbers and activation of CD4+ T cell subsets as a source of VEGF may be a useful tool for early assessment of the risk for DR onset and progression.

Acknowledgments The study was supported by the Polish Ministry of Science and Higher Education (grant no. N401 085 31/1973 and NN 407 173034). During this project PT was supported by HOMING programme of the Foundation for Polish Science (grant from Iceland, Liechtenstein and Norway through the EEA Financial

269 Mechanism). The authors do not have relevant conflict(s) of interest to disclose.

Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.clim.2010.07.007.

References [1] G.S. Eisenbarth, Type I diabetes mellitus a chronic autoimmune disease, N Engl J. Med. 314 (1986) 1360–1368. [2] K. Haskins, M. Portas, B. Bradley, D. Wegmann, K. Lafferty, T-lymphocyte clone specific for pancreatic islet antigen, Diabetes 37 (1988) 1444–1448. [3] N. Martin-Orozco, Y.H. Wang, H. Yagita, C. Dong, Programmed death (PD) ligand-1/PD-1 interaction is required for CD8+ T cell tolerance to tissue antigens, J. Immunol. 177 (2006) 8291–8295. [4] K.L. Wood, H.L. Twigg III, A.I. Doseff, Dysregulation of CD8+ lymphocyte apoptosis, chronic disease, and immune regulation, Front. Biosci. 14 (2009) 3771–3781. [5] D.S. Fong, L.P. Aiello, F.L. Ferris III, R. Klein, Diabetic retinopathy, Diab. Care 27 (2004) 2540–2553. [6] T.S. Kern, Contributions of inflammatory processes to the development of the early stages of diabetic retinopathy, Exp. Diabet. Res. 2007 (2007) 1–14. [7] K. Zorena, J. Myśliwska, M. Myśliwiec, A. Balcerska, P. Lipowski, K. Raczyńska, Interleukin-12, vascular endothelial growth factor and tumor necrosis factor-alpha in the process of neoangiogenesis of diabetic retinopathy in children, Klin. Oczna 109 (2007) 155–159. [8] M. Myśliwiec, A. Balcerska, K. Zorena, J. Myśliwska, P. Lipowski, K. Raczyńska, The assessment of the correlation between vascular endothelial growth factor (VEGF), tumor necrosis factor (TNF-alpha), interleukin 6 (IL-6), glycaemic control (HbA1c) and the development of the diabetic retinopathy in children with diabetes mellitus type 1, Klin. Oczna 109 (2007) 150–154. [9] M. Myśliwiec, A. Balcerska, K. Zorena, J. Myśliwska, P. Lipowski, K. Raczyńska, The role of vascular endothelial growth factor, tumor necrosis factor alpha and interleukin-6 in pathogenesis of diabetic retinopathy, Diabetes Res. Clin. Pract. 79 (2008) 141–146. [10] M. Myśliwiec, K. Zorena, A. Balcerska, J. Myśliwska, P. Lipowski, K. Raczyńska, The activity of N-acetyl-beta-Dglucosaminidase and tumor necrosis factor-alpha at early stage of diabetic retinopathy development in type 1 diabetes mellitus children, Clin. Biochem. 39 (2006) 851–856. [11] L.P. Aiello, R.L. Avery, P.G. Arrigg, B.A. Keyt, H.D. Jampel, S.T. Shah, L.R. Pasquale, H. Thieme, M.A. Iwamoto, J.E. Park, H.V. Nguyen, L.M. Aiello, N. Ferrara, G.L. King, Vascular endothelial growth factor in ocular fluid of patients with diabetic retinopathy and other retinal disorders, N Engl J. Med. 331 (1994) 1480–1487. [12] M.G. Petrovic, P. Korosec, M. Kosnik, J. Osredkar, M. Hawlina, B. Peterlin, D. Petrovic, Local and genetic determinants of vascular endothelial growth factor expression in advanced proliferative diabetic retinopathy, Mol. Vis. 14 (2008) 1382–1387. [13] A.M. Joussen, V. Poulaki, W. Qin, B. Kirchhof, N. Mitsiades, S.J. Wiegand, J. Rudge, G.D. Yancopoulos, A.P. Adamis, Retinal vascular endothelial growth factor induces intercellular adhesion molecule-1 and endothelial nitric oxide synthase expression and initiates early diabetic retinal leukocyte adhesion in vivo, Am. J. Pathol. 160 (2002) 501–509.

270 [14] A.M. Joussen, V. Poulaki, N. Mitsiades, W.Y. Cai, I. Suzuma, J. Pak, S.T. Ju, S.L. Rook, P. Esser, C.S. Mitsiades, B. Kirchhof, A. P. Adamis, L.P. Aiello, Suppression of Fas-FasL-induced endothelial cell apoptosis prevents diabetic blood–retinal barrier breakdown in a model of streptozotocin-induced diabetes, FASEB J. 17 (2003) 76–78. [15] A.M. Joussen, T. Murata, A. Tsujikawa, B. Kirchhof, S.E. Bursell, A.P. Adami, Leukocyte-mediated endothelial cell injury and death in the diabetic retina, Am. J. Pathol. 158 (2001) 147–152. [16] S. Schröder, W. Palinski, G.W. Schmid-Schönbein, Activated monocytes and granulocytes, capillary nonperfusion, and neovascularization in diabetic retinopathy, Am. J. Pathol. 139 (199) 181–100. [17] D.S. McLeod, D.J. Lefer, C. Merges, G.A. Lutty, Enhanced expression of intracellular adhesion molecule-1 and P-selectin in the diabetic human retina and choroid, Am. J. Pathol. 147 (1995) 642–653. [18] K. Miyamoto, S. Khosrof, S.E. Bursell, R. Rohan, T. Murata, A.C. Clermont, L.P. Aiello, Y. Ogura, A.P. Adamis, Prevention of leukostasis and vascular leakage in streptozotocin-induced diabetic retinopathy via intercellular adhesion molecule-1 inhibition, Proc. Natl Acad. Sci. USA 96 (1999) 10836–10841. [19] A.M. Joussen, V. Poulaki, N. Mitsiades, W.Y. Cai, I. Suzuma, J. Pak, S.T. Ju, S.L. Rook, P. Esser, C.S. Mitsiades, B. Kirchhof, A.P. Adamis, L.P. Aiello, Suppression of Fas-FasLinduced endothelial cell apoptosis prevents diabetic blood– retinal barrier breakdown in a model of streptozotocininduced diabetes, FASEB J. 17 (2003) 76–78. [20] M. Croft, L.M. Bradley, S.L. Swain, Naive versus memory CD4 T cell response to antigen. Memory cells are less dependent on accessory cell costimulation and can respond to many antigenpresenting cell types including resting B cells, J. Immunol. 152 (1994) 2675–2685. [21] F. Sallusto, D. Lenig, R. Forster, M. Lipp, A. Lanzavecchia, Two subsets of memory T lymphocytes with distinct homing potentials and effector functions, Nature 401 (1999) 708–712. [22] F. Sallusto, J. Geginat, A. Lanzavecchia, Central memory and effector memory T cell subsets: function, generation, and maintenance, Annu. Rev. Immunol. 22 (2004) 745–763. [23] S.G. Baidya, Q.T. Zeng, Helper T cells and atherosclerosis: the cytokine web, Postgrad. Med. J. 81 (2005) 746–752. [24] M.R. Freeman, F.X. Schneck, M.L. Gagnon, C. Corless, S. Soker, K. Niknejad, G.E. Peoples, M. Klagsbrun, Peripheral blood T lymphocytes and lymphocytes infiltrating human cancers express vascular endothelial growth factor: a potential role for T cells in angiogenesis, Cancer Res. 55 (1995) 4140–4145. [25] F. Mor, F.J. Quintana, I.R. Cohen, Angiogenesis-inflammation cross-talk: vascular endothelial growth factor is secreted by activated T cells and induces Th1 polarization, J. Immunol. 172 (2004) 4618–4623. [26] M.J. Yellin, J. Brett, D. Baum, A. Matsushima, M. Szabolcs, D. Stern, L. Chess, Functional interactions of T cells with endothelial cells: the role of CD40L-CD40-mediated signals, J. Exp. Med. 182 (1995) 1857–1864. [27] Grading diabetic retinopathy from stereoscopic color fundus photographs—an extension of the modified Airlie House classification: ETDRS report number 10. Early Treatment Diabetic Retinopathy Study Research Group, Ophthalmology 98 (1991) 786–806. [28] Classification of diabetic retinopathy from fluorescein angiograms: ETDRS report number 11. Early Treatment Diabetic Retinopathy Study Research Group, Ophthalmology 98 (1991) 807–822.

N. Marek et al. [29] Fundus photographic risk factors for progression of diabetic retinopathy: ETDRS report number 12. Early Treatment Diabetic Retinopathy Study Research Group, Ophthalmology 98 (1991) 823–833. [30] P. Parsons-Wingerter, K. Radhakrishnan, M.B. Vickerman, P.K. Kaiser, Oscillation of angiogenesis with vascular dropout in diabetic retinopathy by VESsel GENeration analysis (VESGEN), Invest. Ophthalmol. Vis. Sci. 51 (2010) 498–507. [31] M.J. Bottomley, N.J. Webb, C.J. Watson, P.J. Holt, A.J. Freemont, P.E. Brenchley, Peripheral blood mononuclear cells from patients with rheumatoid arthritis spontaneously secrete vascular endothelial growth factor (VEGF): specific up-regulation by tumour necrosis factor-alpha (TNF-alpha) in synovial fluid, Clin. Exp. Immunol. 117 (1999) 171–176. [32] S. Kiriakidis, E. Andreakos, C. Monaco, B. Foxwell, M. Feldmann, E. Paleolog, VEGF expression in human macrophages is NF-kappaB-dependent: studies using adenoviruses expressing the endogenous NF-kappaB inhibitor IkappaBalpha and a kinase-defective form of the IkappaB kinase 2, J. Cell Sci. 116 (2003) 665–674. [33] S. Damjanovich, R. Gáspár, G. Panyi, An alternative to conventional immunosuppression: small-molecule inhibitors of Kv 1.3 channels, Mol. Interven. 4 (2004) 250–254. [34] D.R. Palmer, U. Krzych, Cellular and molecular requirements for the recall of IL-4-producing memory CD4(+)CD45RO(+)CD27 (−) T cells during protection induced by attenuated Plasmodium falciparum sporozoites, Eur. J. Immunol. 32 (2002) 652–661. [35] K. Sugita, T. Hirose, D.M. Rothstein, C. Donahue, S.F. Schlossman, C. Morimoto, CD27, a member of the nerve growth factor receptor family, is preferentially expressed on CD45RA+ CD4 T cell clones and involved in distinct immunoregulatory functions, J. Immunol. 149 (1992) 3208–3216. [36] A. Dupuy d'Angeac, S. Monier, C. Jorgensen, Q. Gao, A. Travaglio-Encinoza, C. Bologna, B. Combe, J. Sany, T. Reme, Increased percentage of CD3+, CD57+ lymphocytes in patients with rheumatoid arthritis. Correlation with duration of disease, Arthritis Rheum. 36 (1993) 608–612. [37] A. Huber, F. Menconi, S. Corathers, E.M. Jacobson, Y. Tomer, Joint genetic susceptibility to type 1 diabetes and autoimmune thyroiditis: from epidemiology to mechanisms, Endocr. Rev. 29 (2008) 697–725. [38] J. Myśliwska, K. Zorena, M. Myśliwiec, E. Malinowska, K. Raczyńska, A. Balcerska, The −174GG interleukin-6 genotype is protective from retinopathy and nephropathy in juvenile onset type 1 diabetes mellitus, Pediatr. Res. 66 (2009) 341–345. [39] L. Azamfirei, S. Gurzu, R. Solomon, R. Copotoiu, S. Copotoiu, I. Jung, M. Tilinca, K. Branzaniuc, D. Corneci, J. Szederjesi, J. Kovacs, Vascular endothelial growth factor: a possible mediator of endothelial activation in acute respiratory distress syndrome, Minerva Anestesio (2010) [Epub ahead of print]. [40] R.S. Richardson, H. Wagner, S.R. Mudaliar, R. Henry, E.A. Noyszewski, P.D. Wagner, Human VEGF gene expression in skeletal muscle: effect of acute normoxic and hypoxic exercise, Am. J. Physiol. 277 (6 Pt 2) (1999) H2247–2252. [41] I. Friehs, A.M. Moran, C. Stamm, Y.H. Choi, D.B. Cowan, F.X. McGowan, P.J. del Nido, Promoting angiogenesis protects severely hypertrophied hearts from ischemic injury, Ann. Thorac. Surg. 77 (2004) 2004–2010. [42] E. Galkina, K. Ley, Leukocyte recruitment and vascular injury in diabetic nephropathy, J. Am. Soc. Nephrol. 17 (2006) 368–377. [43] L.M. Bradley, S.R. Watson, Lymphocyte migration into tissue: the paradigm derived from CD4 subsets, Curr. Opin. Immunol. 8 (1996) 312–320.