The role of effector T-cells in the pathogenesis op lupus nephritis Dolff, Sebastian Conrad Johannes

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CHAPTER 6

Urinary T-cells in active lupus nephritis show an effector memory phenotype

Sebastian Dolff1,2, Wayel H. Abdulahad1, Marcory C.R.F. van Dijk3, Pieter C. Limburg1, Cees G.M. Kallenberg1, Marc Bijl1

1

Department of Rheumatology and Clinical Immunology, University Medical Center Groningen, University of Groningen, The Netherlands

2

Department of Nephrology, University Hospital Essen, University Duisburg-Essen, Germany

3

Department of Pathology and Medical Biology, University Medical Center Groningen, University of Groningen, The Netherlands

Ann Rheum Dis. 2010 Nov; 69 (11): 2034-41.

Chapter 6

Abstract Background: Systemic lupus erythematosus (SLE) is accompanied by alterations in T-cell homeostasis including an increased effector response. +

-

Migrated effector memory T-cells (CD45RO CCR7 ;TEM) appear to be involved in tissue injury. The objective of this study was to investigate the distribution and phenotype of effector memory T-cells in the peripheral blood (PB), and their presence in renal biopsies and urine of SLE patients. We tested the hypothesis that these TEM-cells migrate to the kidney during active disease. Patients and Methods: Fourty-three SLE patients and twenty healthy controls +

+

(HC) were enrolled. CD4 TEM-cells and CD8 TEM-cells were analysed in PB and urine using flow cytometric analysis. In ten patients with active lupus nephritis a parallel analysis was performed on the presence of TEM-cells in kidney biopsies. +

Results: The percentage of circulating CD8 TEM cells in SLE patients was significantly decreased versus HC (33.9 ±18.3 % vs. 42.9 ±11.0 %, p=0.008). In patients with active renal involvement (n=12) this percentage was further decreased to 30.4±15.9 %, p=0.01. Analysis of the urinary sediment in active +

renal disease showed increased numbers of CD4 T-cells (134 ±71 cells/ml) +

and CD8 T-cells (287 ±220 cells/ml), respectively, while in HC and patients without active renal disease almost no T-cells were present. 73.6 ±8.3 % of +

+

urinary CD4 T-cells and 69.3 ±26.0 % of urinary CD8 T-cells expressed the TEM +

phenotype. CD8 cells were as well found in renal biopsies. Conclusion: The data presented are compatible with the hypothesis that CD8

+

effector memory cells migrate from the PB to the kidney and appear in the urine during active renal disease in SLE patients. These cells could serve as an additional marker of renal activity in patients with SLE.

98

Urinary effector memory T-cells in active lupus nephritis

Introduction Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by multiple organ manifestations. Inflammation of the kidney, in particular, is 1

associated with an unfavourable prognosis. Although the precise pathogenesis of lupus nephritis (LN) has not been fully elucidated, disturbances in T-cell homeostasis seem to contribute to the inflammatory pathology of LN.

2;3

+

During maturation of CD45RO memory T-cells expression of the lymph node homing chemokine receptor CCR7 distinguishes central memory T-cells +

+

+

-

(TCM; CD45RO CCR7 ) from effector memory T-cells (TEM; CD45RO CCR7 ). In several autoimmune diseases including SLE, disturbances have been described in the distribution of these T-cell subsets in the peripheral blood.

4-6

Remarkably,

+

CD8 TEM have been thought to play a crucial role in T-cell homeostasis due to their ability to produce cytokines and exert cytotoxic activity.

7

Further evidence for the important role of T-cells in SLE is given by the observation that mononuclear cells, predominantly T-lymphocytes, are a frequent histological finding in proliferative forms (ISN/RPS class III and IV) of +

+

LN. However, data regarding CD4 and CD8 cell counts and their ratio in renal biopsies from lupus patients are conflicting.

2;3

Especially, the presence of

+

periglomerular infiltrating CD8 T-cells has been shown to correlate with 2;3;8

histologic activity, clinical severity and bad prognosis of LN.

Additionally,

several studies demonstrated the presence of mononuclear cells in urine of patients with active IgA nephropathy, LN and Wegener’s granulomatosis.

9;10

Thus, analysis of urinary cells reflecting renal inflammation in SLE could be a useful tool in monitoring the course of renal disease. In line with this, we recently reported an increase of urinary TEM (CD4 +

+

-

CD45RO CCR7 ) cells in patients with ANCA-associated vascultis (AAV) with active renal disease.

11

Thus, analysis of urinary TEM-cells seems a promising

tool for detecting renal flares. Although T-cell infiltration in kidneys of SLE +

patients has been reported, analysis of the state of activation of urinary CD4 or +

CD8 T-cells has not been performed so far even though T-cells lacking CCR7 have a strong ability to migrate in vitro.

12

99

Chapter 6

We hypothesize that T-cells with effector function migrate from peripheral blood into the kidneys of SLE patients during active renal disease. This is reflected by the presence of these T-cells in the urinary sediment. To test this hypothesis of T-cell migration, we analyzed the peripheral blood and urine for the presence of effector memory T-cells lacking CCR7 by 4-color flow-cytometry and evaluated in parallel obtained renal biopsies of patients with active renal disease for the presence of T-cells.

100

Urinary effector memory T-cells in active lupus nephritis

Patients and methods Study population Fourty-three SLE patients with a mean age of 41 ±12 years fulfilling at least four of the American College of Rheumatology revised criteria for SLE and 20 sex matched healthy controls (age 36 ±10 years) were enrolled in this study.

13

Disease activity was assessed by SLEDAI (SLE Disease Activity Index). Twenty-four patients had inactive disease (SLEDAI score ≤ 4) and nineteen patients had active SLE (defined as SLEDAI score > 4). Mean disease activity for all patients was 6.3 ±5.6 (Table 1). 25 patients had a current or former renal biopsy consistent with LN while 18 had no history of renal involvement. Currently active LN (n=12) was defined by a proliferative (class III or IV) glomerulonephritis in a parallel obtained renal biopsy (n=11) or the presence of an active urinary sediment with glomerular erythrocyturia (n=1) (Table 2). Seven active LN patients presented with the first episode of LN. 12 patients did not receive any immuno-modulating medication at the time of analysis, 6 of them were newly diagnosed. 31 patients received immuno-modulating medication (Table 1).

Materials EDTA-blood and fresh urine samples were collected from patients and HC. Urine samples from patients which were nitrite positive on a dip stick test or with proof of bacterial contamination in the sediment were excluded. Percentages +

+

and absolute counts of CD4 and CD8 T-cells were assessed immediately after sampling by four-color flow cytometry in blood and urine samples. Paraffin-embedded sections of renal biopsy specimens obtained from eleven patients were included in the present study. Informed consent was obtained from the patients after approval by the Local Ethics Committee. The study was conducted according to the ethical guidelines of our institution and the Declaration of Helsinki.

101

Chapter 6

Table 1: Baseline characteristics and medication of all patients included (n=43). SLEDAI: systemic ¶

lupus erythematosus disease activity index, MMF: mycophenolate mofetil, renal activity defined by a current biopsy proving active renal disease, or the presence of an active urine sediment in patients with previously known renal involvement.

Antibodies The following antibodies were used in flow cytometry: phycoerythrin (PE)conjugated anti-CCR7 (clone 3D12), fluorescein (FITC)-conjugated antiCD45RO (clone UCHL-1), peridin-chlorophyll (PerCP)-conjugated anti-CD4 (clone SK3), allophycocyanin (APC)-conjugated anti-CD3 (clone UCHT1), MultiTEST

TM

four-color antibodies (CD3-FITC, CD8-PE, CD45-PerCP and CD4-

APC), and isotype matched control antibodies of irrelevant specificity. All were purchased from Becton-Dickinson ((BD), Amsterdam, The Netherlands).

102

Urinary effector memory T-cells in active lupus nephritis

Sample preparation and flow cytometry Immediately after voiding, 100 ml of urine was diluted 1:1 with cold phosphatebuffered saline (PBS) and processed as decribed before.

11

Briefly, isolated

mononuclear cells were resuspended in wash-buffer (1 % BSA in PBS) and mixed with appropriate concentrations of anti-CD45RO-FITC, anti-CCR7-PE, anti-CD4-PerCP, and anti-CD3-APC for 15 minutes at room temperature in the dark. In parallel, blood samples were labeled with the aforementioned monoclonal antibodies. Afterwards, cells were successively treated with 2 ml diluted FACS lysing solution (BD, Amsterdam, The Netherlands) for 10 minutes and samples were washed twice in wash-buffer and immediately analyzed by flow cytometry. Four-color staining was analyzed on FACS-Calibur (BD, 5

Amsterdam, The Netherlands) and data were collected for 10 events for each sample and plotted using Win-List software package (Verity Software House Inc., ME, USA). Positively and negatively stained populations were calculated by quadrant dot-plot analysis, as determined by the isotype controls. Representative examples are shown in Figure 1.

Quantification of effector memory T-cells T-cells were quantified in urine using TruCOUNT Netherlands). In brief, 20 µl of MultiTEST

TM

TM

tubes (BD, Amsterdam, The

four-color antibodies (CD3-FITC,

CD8-PE, CD45-PerCP, and CD4-APC) and 50 µl of sample (urine or blood) were added to bead-containing TruCOUNT

TM

tubes. The cell suspension was

processed and analysed as described elsewere.

11

Afterwards, the absolute

counts for TEM cells in 1 ml urine were calculated as decribed before.

11

Analysis and scoring of renal biopsies Biopsies taken at the time of analysis of blood and urine samples were processed. All biopsies were reviewed and classified by an experienced nephropathologist (MvD) according to the revised criteria for LN. The activity index (AI) and chronicity index (CI) were calculated for each specimen with maximum scores of 24 for the AI and 12 for the CI.

14

For this study 103

Chapter 6

methenaminesilver-stained slides (with HE-counterstaining), H&E and PAS stained slides, were used. The assessment was completed by determining the ISN/RPS2003 classification and activity and chronicity indices for LN. For these aspects of the assessment, the definitions of the classification systems and the activity and chronicity indices were used.

Table 2: Laboratory and histological data of 12 patients with active renal disease are shown. na: not assessed §

m = male, f = female

¶

histological ISN/RPS classification, nc: not classified

#

serum creatinine expressed as µmol/l

Immunohistochemistry staining All specimens were fixed in 10 % neutral buffered formalin and paraffin embedded. Five-micrometer-thick sections were deparaffinized in xylene and rehydrated in a series of different concentrations of ethanol. EDTA buffer, pH 8.2, for heat-induced epitope retrieval was applied for 1 h, followed by neutralization of endogenous peroxidase with 0.3 % H2O2. Incubation with a monoclonal mouse anti-human CD8 (DAKO, Glostrup, Denmark) was performed. Next, sections were washed and incubated with a HRP-conjugated TM

secondary antibody (Envison , DAKO, Glostrup, Denmark) for 30 min. at room

104

Urinary effector memory T-cells in active lupus nephritis

temperature. A DAB substrate was used for visualization. Washing with PBS was performed after each incubation step. CD4 (Monosan, Uden, Netherlands) was performed in the Benchmark Ultra (Ventana, Ventana Medical Systems S.A. CEDEX France) with citrate buffer heat inducted antigen retrieval and detected with the ultraView Universal Alkaline Phosphatase Red Detection Kit (Ventana). Finally, the slides were counterstained with haematoxylin and mounted with Kaiser’s glycerine gelatin (Merck, Darmstadt, Germany). Cells were separately counted for the interstitium and glomeruli. Cells with positive staining for CD8 and CD4 were counted per high powerfield (40 x magnification). The average value was calculated for each biopsy.

Statistical analysis Results are presented as mean ±SD and the nonparametric Mann-Whitney Utest was used for comparison of values between groups. Correlation with disease activity was assessed using Spearman’s rank correlation coefficient. Two-tailed P-values less than 0.05 were regarded as statistically significant.

105

Chapter 6

Results SLE

patients +

have

decreased

percentage

of

circulating

CCR7

-

+

CD45RO CD8 effector memory T-cells during active disease and even less during active renal disease +

-

We determined the percentages of naïve (CCR7 CD45RO ;Tnaive), central +

+

+

+

-

+

memory (CCR7 CD45RO ;TCM) and effector memory (CCR7 CD45RO ;TEM) subsets of CD4 and CD8 T-cells in peripheral blood of HC and SLE patients. +

No differences in the percentages of circulating Tnaive, TCM or TEM CD4 T-cells were found between HC and SLE patients. There was a significant difference in +

the percentages of TCM and TEM CD8 T-cells between HC and SLE patients (TCM: 5.8 ±3.8 % vs. 10.9± 10.3 % p=0.02; TEM: 42.9 ±11.0 % vs. 33.9± 18. %, p=0.008, Figure 2a and b). TCM were increased in SLE patients while TEM CD8

+

T-cells were decreased as compared to HC. There was no difference between +

the percentages of circulating naïve CD8 T-cells in peripheral blood of HC and SLE patients. Within the patient group there was no difference in the +

percentages of circulating naïve CD8 T-cells in peripheral blood between those with or without immunomodulating medication. In addition, subsets were compared between SLE patients with active and inactive disease. No differences were present between the percentages of +

circulating naïve (Tnaive), central memory (TCM) or effector memory (TEM) CD4 Tcells of HC as compared to active and inactive SLE patients, respectively +

(Figure 2c). Within the CD8 T-cell populations, a significant increase of circulating TCM cells was observed in SLE patients with active disease as compared to HC (TCM: 14.4 ±13.3 % vs. 5.8 ±3.8 % p=0.009). The TEM population was significantly decreased in active disease as compared to HC (TEM: 31.6 ±14.9 % vs. 42.9 ±11.0 % p=0.004, Figure 2d). +

+

Next, we assessed the percentages of CD4 and CD8 T-cell subsets in SLE patients in relation to the presence of active renal disease. The percentage of circulating TCM cells in SLE patients without active LN was significantly increased compared to HC (TCM: 10.1 ±9.0 % vs. 5.8 ±3.8 % p=0.02) whereas circulating peripheral TEM cells were decreased in patients with inactive renal 106

Urinary effector memory T-cells in active lupus nephritis

disease as compared to healthy controls (TEM: 35.2 ±19.2 % vs. 42.9 ±11.0 % p=0.03). They were even more decreased in patients with active renal disease (TEM: 30.4 ±15.9 % vs. 45.1 ±9.4 % p=0.01, Figure 2f) but not statistically significant compared to inactive disease. There was no difference between patients with a first episode of LN and those with relapsing renal disease regarding circulating memory T-cell subsets (data not shown).

Urinary T-cells with effector memory phenotype are associated with active renal disease in SLE In parallel to the analysis of peripheral blood we collected urine to quantify the +

+

absolute numbers of CD4 and CD8 cells (Figure 3a). The absolute count of +

CD4 T-cells was significantly increased in SLE patients with active lupus nephritis (LN) as compared to SLE patients without active LN and healthy controls, respectively (134 ±71 cells/ml vs. 15 ±30 cells/ml, p=0.0001 and vs. +

2 ±4 cells/ml, p=0.002). Furthermore, the absolute count of CD8 T-cells was significantly increased in SLE patients with active LN as compared to SLE patients without active LN and HC, respectively (287 ±220 cells/ml vs. 22 ±28 cells/ml, p