Elevated Levels of Fractalkine Expression and Accumulation of CD16+ Monocytes in Glomeruli of Active Lupus Nephritis

Elevated Levels of Fractalkine Expression and Accumulation of CD16ⴙ Monocytes in Glomeruli of Active Lupus Nephritis Shuhei Yoshimoto, MD,1 Kimihiko Nakatani, MD,1 Masayuki Iwano, MD,1 Osamu Asai, MD,1 Ken-ichi Samejima, MD,1 Hirokazu Sakan, MD,1 Miho Terada,2 Koji Harada, MD,1 Yasuhiro Akai, MD,1 Hideo Shiiki, MD,1 Masato Nose, MD,2 and Yoshihiko Saito, MD1 Background: Fractalkine (Fkn) is a chemokine that affects cells expressing its receptor, CX3CR1, including CD16-positive (CD16⫹) monocytes/macrophages (CD16⫹ Mos). The relationship of levels of glomerular Fkn expression and infiltration by CD16⫹ Mos with the severity and diversity of glomerular lesions in human lupus nephritis is not known. Study Design: Retrospective cross-sectional analysis of variables observed in biopsy specimens. Settings & Participants: 88 patients with systemic lupus erythematosus. Predictor: Histological class and severity of lupus nephritis according to the International Society of Nephrology/Renal Pathology Society and clinicopathologic factors. Outcomes: Outcome variables are assays related to the degree of glomerular Fkn expression and CD16⫹ Mo infiltration. Measurements: Immunohistological grading of Fkn staining, number of CD16⫹ Mos, and messenger RNA levels of Fkn and CD16 in glomeruli. Results: Patients with proliferative lupus nephritis (class III and IV glomeruli) showed significantly greater glomerular Fkn expression and CD16⫹ Mo counts than those with other classes. Infiltrating CD16⫹ Mos within glomeruli expressed CX3CR1. Moreover, glomerular Fkn expression significantly correlated with the histopathologic activity index and CD16⫹ Mo counts, and CD16⫹ Mo counts significantly correlated with serum levels of blood urea nitrogen, complement (CH50), and anti-DNA antibody; estimated glomerular filtration rate; and urinary protein excretion. Glucocorticoid therapy had a tendency to decrease both glomerular Fkn expression and CD16⫹ Mo counts. Limitations: Only frozen biopsy specimens (from 49 patients) were analyzed for the evaluation of glomerular Fkn expression. Conclusion: Disease activity and proliferative glomerular lupus nephritis lesions are associated with the glomerular Fkn expression and CD16⫹ Mo accumulation. Am J Kidney Dis 50:47-58. © 2007 by the National Kidney Foundation, Inc. INDEX WORDS: Fractalkine; lupus nephritis; CD16⫹ monocytes.

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ystemic lupus erythematosus (SLE) is the prototype human systemic autoimmune disease and is characterized by the production of pathogenic autoantibodies and tissue deposition of immune complexes, which leads to local release of inflammatory mediators and influx of inflammatory cells.1 Renal involvement is one of the most serious and highly variable complications of SLE,1 with diffuse proliferative lupus nephritis (DPLN; International Society of Nephrology/Renal Pathology Society [ISN/RPS] class IV) the most common and severe form of lupus nephritis.2 In the initiation and progression of lupus nephritis, monocytes/macrophages and/or T cells have a critical role. Immunohistochemical analyses showed that monocytes/macrophages and/or T cells infiltrate both glomeruli and interstitium in patients with lupus nephritis, and the extent of macrophage infiltration correlates with proteinuria and poor renal outcomes.3,4

Fractalkine (Fkn) is a unique member of the chemokine superfamily and is found in 2 distinct forms, 1 form anchored to the cell membrane and a soluble form generated by proteolytic cleavage from the membrane. 5 Fkn originally was detected in human umbilical vein endothelial cells and recently found to be expressed on tumor necrosis factor ␣– and interleukin 1␤–activated endothelial cells, denFrom the 1First Department of Internal Medicine, Nara Medical University, Kashihara, Nara; and 2Department of Pathology, Division of Pathogenomics, Ehime University Graduate School of Medicine, Toon, Ehime, Japan. Received November 2, 2006. Accepted in revised form April 19, 2007. Address correspondence to Kimihiko Nakatani, MD, First Department of Internal Medicine, Nara Medical University, 840, Shijo-cho, Kashihara-city, Nara, 634-8522, Japan. E-mail: [email protected] © 2007 by the National Kidney Foundation, Inc. 0272-6386/07/5001-0007$32.00/0 doi:10.1053/j.ajkd.2007.04.012

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dritic cells, neuronal cells, astrocytes, interstitial epithelial cells, mesangial cells, and smooth muscle cells.5-12 Fkn can function as both a potent chemoattractant (soluble form) or an adhesion molecule (membrane-anchored form) for cells expressing its receptor, CX3CR1.5,13 CX3CR1 expression and Fkn-dependent migration were observed in a wide variety of cells, including T cells, monocytes/macrophages, and natural killer cells.5-7,9,13,14 Moreover, Fkn expression and recruitment of CX3CR1-positive (CX3CR1⫹) cells reportedly had a key role in the accumulation of intrarenal inflammatory cells in patients with kidney disease.8,10-12,15 Recently, intermediate to high levels of CX3CR1 expression were detected in CD14highCD16ⴙ and CD14lowCD16ⴙ monocytes, corresponding to the previously defined CD16ⴙ monocytes (CD16ⴙ Mos).16,17 The CD16 antigen is the Fc␥ receptor III, and CD16ⴙ Mos are regarded as proinflammatory cells, the numbers of which increase in peripheral blood of patients with such disorders as sepsis and cancer.18 CD16ⴙ Mos also were identified in urine of patients with proliferative glomerulonephritis (GN), including acute GN, immunoglobulin A nephropathy, membranoproliferative GN, and lupus nephritis, suggesting that CD16ⴙ Mos may serve as effecter cells in proliferative GN.19 Notably, CD16ⴙ Mos appear to be preferentially recruited to the vessel wall through the chemoattractant property of locally expressed Fkn, suggesting that the CD16ⴙ Mo-Fkn pathway may contribute to vascular and tissue injury in cases of the aforementioned pathological conditions.16 Our aim in the present study is to determine levels of glomerular Fkn expression and CD16ⴙ Mo infiltration and assess the relationship of these levels with the severity and diversity of glomerular lesions in human lupus nephritis. We assessed glomerular expression of Fkn and CD16ⴙ Mo infiltration in patients with minimal mesangial proliferative glomeruli (ISN/ RPS class I), purely mesangial alterations (mesangiopathy, ISN/RPS class II), mesangial endocapillary proliferation involving less than 50% of glomeruli (ISN/RPS class III), diffuse mesangial endocapillary proliferation (DPLN; ISN/RPS class IV), and membranous GN (ISN/ RPS class V). In addition, to assess the poten-

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tial role of CD16ⴙ Mos in the progression of lupus nephritis, we correlated various clinical parameters and histopathologic active and/or sclerosing lesions with degree of glomerular CD16ⴙ Mo infiltration. Finally, to assess the importance of Fkn chemoattractance, we also examined the correlation between Fkn levels and infiltration of cells expressing CX3CR1 in glomeruli. METHODS Study Population Informed consent was obtained from all study participants. Eighty-eight patients with SLE diagnosed based on criteria set forth in 1982 and 1997 by the American Rheumatism Association were evaluated in this study.20,21 There were 15 men and 73 women with a mean age of 34.05 ⫾ 13.5 years (range, 15 to 70 years). Renal biopsy specimens were obtained from all patients and used to make the diagnosis of lupus nephritis. Serum levels of total protein, albumin, blood urea nitrogen, creatinine, complement (CH50), anti-DNA antibody and immune complex, and urinary protein excretion (proteinuria) were measured in all patients. Estimated glomerular filtration rate was calculated by means of the creatinine-based Modification of Diet in Renal Disease Study equation.22 Patient characteristics are listed in Table 1.

Histopathologic Studies Renal biopsy specimens were fixed in 10% neutral phosphate-buffered formalin, embedded in paraffin, and cut into 3-␮m thick sections, which were stained with hematoxylin and eosin, periodic acid–Schiff reagent, or periodic acid– silver methenamine. Sections were examined under a light microscope, and tissue histopathologic state was classified according to ISN/RPS criteria as follows: class I, minimal mesangial GN; class II, mesangial proliferative GN showing purely mesangial hypercellularity to any degree and/or mesangial matrix expansion; class III, proliferative GN involving less than 50% of glomeruli; class IV, diffuse segmental or global GN involving 50% or more of glomeruli; and class V, membranous GN. Activity and chronicity indexes of the histological appearance also were assessed based on National Institutes of Health scores.23 Two observers (H.S., M.N.) with no prior knowledge of the clinical course examined renal tissue to establish the diagnosis by using standard pathological methods.

Immunohistochemical Studies Frozen renal tissue sections suitable for analysis of Fkn expression were obtained from 49 of 88 patients. Fresh renal biopsy specimens were embedded in ornithine carbamoyltransferase and frozen in liquid nitrogen, after which 4-␮m thick sections were cut using a cryostat and washed with isotonic phosphate-buffered saline (pH 7.2). Fkn was immunohistochemically labeled using goat antihuman Fkn polyclonal antibodies (1:100 dilution; R&D, Minneapolis, MN),

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Table 1. Clinical Characteristics of Patients With Lupus Nephritis ISN/RPS Classification

No. of patients Age (y) Sex (F/M) Proteinuria (g/d) Serum total protein (g/dL) Serum albumin (g/dL) Blood urea nitrogen (mg/dL) Serum creatinine (mg/dL) Estimated GFR (mL/min) CH50 (U/mL) Immune complex titer Serum anti-DNA antibody titer Activity index score Chronicity index score

Class I

Class II

Class III

Class IV

Class V

7 39.0 ⫾ 11.1 6/1 0.13 ⫾ 0.19 7.10 ⫾ 0.75 3.79 ⫾ 0.53 12.00 ⫾ 3.65 0.56 ⫾ 0.10 98.3 ⫾ 23.1 37.29 ⫾ 5.77 2.84 ⫾ 3.00 8.04 ⫾ 6.05 0.71 ⫾ 0.76 0.29 ⫾ 0.49

29 35.9 ⫾ 15.1 25/4 0.25 ⫾ 0.58 6.55 ⫾ 0.82 3.78 ⫾ 0.40 11.55 ⫾ 4.16 0.61 ⫾ 0.17 95.3 ⫾ 33.3 30.62 ⫾ 10.16 3.27 ⫾ 2.13 10.51 ⫾ 14.20 0.69 ⫾ 0.66 0.21 ⫾ 0.41

10 36.7 ⫾ 14.9 9/1 1.96 ⫾ 1.10 5.27 ⫾ 0.53 3.20 ⫾ 0.34 13.90 ⫾ 3.51 0.65 ⫾ 0.20 91.0 ⫾ 40.3 30.30 ⫾ 9.92 5.16 ⫾ 8.18 20.18 ⫾ 43.29 4.50 ⫾ 1.27 1.10 ⫾ 0.57

30 29.1 ⫾ 11.0 23/7 4.42 ⫾ 4.85 5.44 ⫾ 1.20 3.00 ⫾ 0.75 25.70 ⫾ 22.84 1.08 ⫾ 0.87 71.4 ⫾ 33.7 14.56 ⫾ 8.74 13.68 ⫾ 16.89 426.28 ⫾ 906.96 8.83 ⫾ 2.67 2.03 ⫾ 0.61

12 37.0 ⫾ 13.9 10/2 3.25 ⫾ 5.69 6.49 ⫾ 1.11 3.37 ⫾ 0.66 13.08 ⫾ 4.78 0.56 ⫾ 0.14 102.4 ⫾ 25.0 29.00 ⫾ 15.27 1.61 ⫾ 0.31 7.52 ⫾ 8.46 1.33 ⫾ 0.78 0.83 ⫾ 0.72

Note: Values expressed as mean ⫾ SD. To convert serum creatinine in mg/dL to ␮mol/L, multiply by 88.4; blood urea nitrogen in mg/dL to mmol/L, multiply by 0.357. Estimated GFR was calculated by means of the creatinine-based Modification of Diet in Renal Disease Study equation. Abbreviations: ISN/RPS, International Society of Nephrology/Renal Pathology Society; GFR, glomerular filtration rate.

after which labeling was visualized by means of staining using a Dako LSAB⫹ Kit (Dako, Glostrup, Denmark) according to the manufacturer’s instructions. Two pathologists (M.T., K.N.) then decided immunohistological grading conjointly using a scale of 0 to 3, in which grade 0 indicates no staining; grade 1, 1% to 25% positively stained cells within glomeruli; grade 2, 25% to 50% positivity; and grade 3, greater than 50% positivity. ␣-Smooth muscle actin and CD31⫹ cells were immunohistochemically labeled using mouse antihuman smooth muscle actin monoclonal antibodies (1:100 dilution; Dako) or mouse antihuman CD31 monoclonal antibodies (1:100 dilution; Dako), after which labeling was visualized by means of staining using a Dako Envision Kit. CD16ⴙ Mos, CD3ⴙ T cells, and CD8ⴙ T cells also were detected immunohistochemically in 88 formalin-

Figure 1. Immunoperoxidase staining for fractalkine (Fkn) in renal biopsy specimens of human lupus nephritis. (A) Fkn staining of a normal glomerulus (class I) and (B) those showing mesangiopathy (class II), (C) proliferative glomerulonephritis (class III), (D) diffuse mesangial endocapillary proliferation (DPLN; class IV), (E) membranous glomerulonephritis (class V), and (F) DPLN treated with goat immunoglobulins instead of goat anti-Fkn antibody serving as a negative control.

fixed paraffin-embedded renal sections by using a mouse antihuman CD16 monoclonal antibody (1:800 dilution; Dako), rabbit antihuman CD3 polyclonal antibody (1:100 dilution; Dako), or mouse antihuman CD8 monoclonal antibody (1:100 dilution; Dako). A Dako CSA II Kit for CD16ⴙ Mos and Dako Envision Kit for CD3ⴙ T cells and CD8ⴙ T cells were used for signal amplification and visualization. We counted the number of CD16ⴙ Mos, CD3ⴙ T cells, and CD8ⴙ T cells within glomeruli (average glomeruli scored per patient, 14.1 ⫾ 7 [SD]), then calculated mean numbers of these cells per glomerulus. CX3CR1 expression on CD16ⴙ Mos also was detected in formalin-fixed paraffin-embedded renal serial sections by using a rabbit anti-CX3CR1 monoclonal antibody (1: 1,500 dilution; Chemicon, Temecula, CA). Again a Dako

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Figure 2. Immunoperoxidase staining of (A) fractalkine (Fkn), (B) ␣-smooth muscle actin, and (C) CD31 in serial renal sections affected by diffuse mesangial endocapillary proliferation. Fkn-expressing cells in glomeruli showed positive staining of ␣-smooth muscle actin or CD31.

Figure 3. Fractalkine (Fkn) expression in glomeruli affected by human lupus nephritis and correlation between glomerular fractalkine expression and histochemical parameters in lupus nephritis. (A) Fkn expression indexes in class I (n ⫽ 4), II (n ⫽ 16), III (n ⫽ 3), IV (n ⫽ 18), or V (n ⫽ 8) glomeruli. Kruskal-Wallis analysis of variance by ranks with Bonferroni adjustment was used for comparison between groups. *P ⬍ 0.001 (*1 ⫽ versus class I, *2 ⫽ versus class II, *5 ⫽ versus class V). #P ⬍ 0.001 (#1 ⫽ versus class I, #2 ⫽ versus class II, #5 ⫽ versus class V). (B) Correlation of Fkn glomerular expression with histopathologic activity index (AI) in lupus nephritis (classes I to V; r ⫽ 0.87; P ⬍ 0.001) and (C) proliferative lupus nephritis (class III ⫹ IV; r ⫽ 0.58; P ⫽ 0.0051), after which the relationship was evaluated using Spearman rank correlation.

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Figure 4. Immunoperoxidase staining for CD16⫹ monocytes/macrophages in renal biopsy specimens of human lupus nephritis. CD16 staining of (A) normal glomerulus (class I) and those showing (B) mesangiopathy (class II), (C) proliferative glomerulonephritis (class III), (D) diffuse mesangial endocapillary proliferation (DPLN; class IV), and (E) membranous glomerulonephritis (class V) and (F) glomerulus showing DPLN treated with mouse immunoglobulins instead of mouse anti-CD16 antibody as a negative control.

CSA II Kit was used for signal amplification and visualization.

Tissue Sampling by Laser Capture Microdissection We used laser capture microdissection (LCM) to sample target glomeruli per frozen renal specimen (collected glomeruli range per patient, 10 to 15) from 15 patients with SLE. Frozen tissue blocks were cut with a cryostat into 8-␮m serial sections, then thaw-mounted on uncoated glass slides. Sections were immediately fixed for 30 seconds in nuclease-free 75% ethanol, rehydrated for 30 seconds in nuclease-free distilled water, stained for 20 seconds using a HistoGene LCM Frozen Section Staining Kit (Arcturus, Mountain View, CA), and rinsed for 30 seconds in nucleasefree distilled water. Sections were dehydrated by means of sequential immersion in 75%, 95%, and 100% ethanol for 30 seconds each, then immersion in xylene for 5 minutes before tissue procurement by means of LCM. LCM of that targeted glomeruli was carried out using an Arcturus PixCell II LCM instrument according to manufacturer’s protocols.24,25

RNA Extraction and Reverse Transcription Samples collected by means of LCM were immediately processed using a PicoPure RNA isolation kit (Arcturus) according to the manufacturer’s instructions.24 Briefly, 10 ␮L of extraction buffer containing the sample was placed into an HS-CapSure/microcentrifuge assembly and incubated for 30 minutes at 42°C. After incubation, the assembly was briefly centrifuged and total cellular RNA was extracted into 10 ␮L of buffer. First-strand complementary DNA was made from total RNA by using a SuperScript Preamplification System (Invitrogen, Carlsbad, CA) with random hexamers. For real-time polymerase chain reaction (PCR), 1 ␮L of each first-strand reaction product was amplified with appropriate primers and the corresponding fluorescent probes specific for Fkn (assay ID: Hs00171086-ml) or ␤-actin (assay ID: Hs00242273-ml), designed by the Applied Biosystems “Assay-on-Demand” service (Foster City, CA). Fkn/␤-actin messenger RNA (mRNA) ratios were calculated for each sample.

Statistical Analysis Numerical results are expressed as mean ⫾ SD. Student paired t-test was used for normally distributed variables. Comparing groups, 1-way analysis of variance was used, followed by post hoc t-test with Fisher Protected Least Significant Differences adjustment. For variables with a skewed distribution, we used Kruskal-Wallis analysis of variance by ranks, with Bonferroni adjustment. Spearman rank correlation or Pearson correlation coefficient was used to assess relationships between glomerular CD16ⴙ Mo infiltration and Fkn expression or clinical and pathological parameters. P less than 0.05 is considered significant.

RESULTS

Fkn Expression in Glomerular Lesions in Lupus Nephritis

Immunohistochemical analysis of 49 frozen renal sections from 49 patients with SLE showed the presence of Fkn in the mesangial area and/or along the capillary wall within the glomerulus (Fig 1). In patients with SLE, no Fkn positivity was detected in classes I and V glomeruli (Fig 1). Conversely, Fkn was present in the mesangial area in class II glomeruli and both the mesangial area and along the capillary wall in class III and IV glomeruli (Fig 1). Immunohistochemical analysis of serial sections showed Fkn expression in ␣-smooth muscle actin⫹ and/or CD31⫹ cells (Fig 2), suggesting that mesangial and/or endothelial cells expressed Fkn in glomeruli of patients with lupus nephritis. Mean Fkn levels in glomeruli were significantly greater in classes III (n ⫽ 3) and IV (n ⫽ 18) glomeruli than in the other classes (ISN/RPS I/II/III/ IV/V, 0/0.19 ⫾ 0.40/1.33 ⫾ 0.58/1.78 ⫾ 0.55/0; P

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Semiquantification of Fkn and CD16 mRNA Expression in Lupus Nephritis

To confirm overproduction of Fkn and CD16 in proliferative lesions of patients with lupus nephritis, we next used LCM and real-time PCR for semiquantitative analysis of glomerular Fkn and CD16 mRNA expression. We found that relative glomerular Fkn and CD16 mRNA levels

Figure 5. Number of CD16⫹ cells in glomeruli affected by human lupus nephritis: average number of immunohistochemically identified CD16⫹ monocytes/macrophages (CD16⫹ Mos) in class I (n ⫽ 7), II (n ⫽ 29), III (n ⫽ 10), IV (n ⫽ 30), or V (n ⫽ 12) glomeruli. Kruskal-Wallis analysis of variance by ranks with Bonferroni adjustment was used for comparison between groups. *P ⬍ 0.02 (*1 ⫽ versus class I, *2 ⫽ versus class II, *5 ⫽ versus class V). #P ⬍ 0.0001 (#1 ⫽ versus class I, #2 ⫽ versus class II, #3 ⫽ versus class III, #5 ⫽ versus class V).

⬍ 0.001; Fig 3A), indicating high Fkn levels in proliferative glomerular lesions. In addition, there was a strong correlation between the glomerular Fkn expression index and histopathologic activity index (r ⫽ 0.87; P ⬍ 0.001; Fig 3B). Even in patients with proliferative lupus nephritis, glomerular Fkn expression index correlated with activity index (r ⫽ 0.58; P ⫽ 0.0051; Fig 3C). Infiltration of Glomeruli by CD16ⴙ Mos in Lupus Nephritis

When we investigated the extent to which glomeruli are infiltrated by CD16ⴙ Mos in patients with lupus nephritis, we detected CD16ⴙ Mos immunohistologically in glomeruli of all 88 patients (Fig 4). Average numbers of CD16ⴙ Mos per glomerulus in class IV glomeruli (n ⫽ 30) were significantly higher than in the other classes (ISR/RPS I/II/III/IV/V, 0.51 ⫾ 0.19/0.77 ⫾ 0.45/1.94 ⫾ 0.83/4.94 ⫾ 1.46/0.94 ⫾ 0.59; P ⬍ 0.02; Fig 5). In addition, average numbers of CD16ⴙ Mos in class III glomeruli (n ⫽ 10) were higher than those in class I (n ⫽ 7), II (n ⫽ 29), or V (n ⫽ 12; Fig 5). Numbers of infiltrating CD16ⴙ Mos were higher in proliferative glomerular lesions of patients with SLE.

Figure 6. Glomerular expression of fractalkine (Fkn) and CD16 messenger RNA (mRNA). Semiquantitative analysis of glomerular expression of (A) Fkn and (B) CD16 mRNA using laser capture microdissection and real-time polymerase chain reaction with class I (n ⫽ 3), II (n ⫽ 4), IV (n ⫽ 5), or V (n ⫽ 3) glomeruli. mRNA levels expressed in arbitrary units (AU). Kruskal-Wallis analysis of variance by ranks with Bonferroni adjustment was used for comparison between groups. *P ⬍ 0.05 (*1 ⫽ versus class I, *2 ⫽ versus class II, *5 ⫽ versus class V).

Fractalkine and CD16⫹ Monocytes in Lupus Nephritis

(normalized to ␤-actin mRNA) were significantly greater in class IV (n ⫽ 5) than class I (n ⫽ 3), II (n ⫽ 4), or V glomeruli (n ⫽ 3; P ⬍ 0.05), consistent with immunohistologic findings (Fig 6). Correlation Between Glomerular Fkn Expression and Numbers of Infiltrating CD16ⴙ Mos in Lupus Nephritis

We found a significant correlation between glomerular Fkn expression level and CD16ⴙ Mo

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counts (r ⫽ 0.79; P ⬍ 0.0001; Fig 7A). That finding was confirmed by means of LCM and real-time PCR analysis, which also showed a strong correlation between glomerular Fkn and CD16 mRNA levels (r ⫽ 0.88; P ⬍ 0.0001; Fig 7B). Even in patients with proliferative lupus nephritis, glomerular Fkn expression levels correlated with CD16ⴙ Mo counts (r ⫽ 0.53; P ⫽ 0.013; Fig 7C). In addition, immunohistochemical analysis showed that CD16ⴙ Mos within proliferative glomerular lesions of patients with

Figure 7. Correlation between glomerular fractalkine (Fkn) expression and CD16⫹ monocyte/macrophage (CD16⫹ Mo) infiltration in lupus nephritis. (A) Immunohistochemical analysis was carried out to determine the glomerular Fkn expression index and average number of CD16 ⫹ Mos within glomeruli (r ⫽ 0.79; P ⬍ 0.0001), after which the relationship was evaluated using Spearman rank correlation. (B) Correlation of glomerular Fkn expression with CD16 messenger RNA (mRNA) determined by means of laser capture microdissection and real-time polymerase chain reaction (r ⫽ 0.88; P ⬍ 0.0001). The relationship was evaluated using Pearson correlation coefficient. (C) Correlation of glomerular Fkn expression index and average number of CD16⫹ Mos within glomeruli of proliferative glomerulonephritis (class III ⫹ IV; r ⫽ 0.53; P ⫽ 0.013), after which the relationship was evaluated using Spearman rank correlation. Abbreviation: AU, arbitrary units.

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Figure 8. Immunoperoxidase staining of (right) CD16⫹ monocytes/macrophages (CD16⫹ Mos) and (left) CX3CR1 in serial renal sections affected by diffuse mesangial endocapillary proliferation. CD16⫹ Mos within glomerular lesions expressed CX3CR1.

lupus nephritis expressed CX3CR1 (Fig 8), suggesting that the chemoattractant property of Fkn stimulates glomerular infiltration by CD16ⴙ Mos in patients with lupus nephritis. Correlation Between Clinicopathologic Parameters and Glomerular CD16ⴙ Mo Counts in Lupus Nephritis

Of clinical parameters tested, glomerular CD16ⴙ Mo counts correlated positively with serum blood urea nitrogen levels (r ⫽ 0.42; P ⬍ 0.0001), serum anti-DNA antibody titers (r ⫽ 0.43; P ⬍ 0.0001), and proteinuria (r ⫽ 0.42; P ⫽ 0.0002) and negatively with total serum protein (r ⫽ ⫺0.46; P ⬍ 0.0001), serum albumin (r

⫽ ⫺0.48; P ⬍ 0.0001), and serum complement levels (CH50; r ⫽ ⫺0.59; P ⬍ 0.0001) and estimated glomerular filtration rate (r ⫽ ⫺0.34; P ⫽ 0.0013; Fig 9A to F). Moreover, even in patients with proliferative lupus nephritis (classes III and IV, n ⫽ 40), CD16ⴙ Mo counts significantly correlated with serum complement levels (CH50; r ⫽ ⫺0.62; P ⬍ 0.0001) and anti-DNA antibody titers (r ⫽ 0.36; P ⫽ 0.0299; Fig 9H and I). In addition, of pathological parameters tested, there was a strong correlation between activity index and CD16ⴙ Mo count (r ⫽ 0.94; P ⬍ 0.0001; Fig 9G). Even in patients with proliferative lupus nephritis, activity index strongly correlated with CD16ⴙ Mo count (r ⫽ 0.82; P ⬍

Figure 9. Correlation between glomerular CD16⫹ monocyte/macrophage (CD16⫹ Mo) counts and the indicated clinical parameters. Clinical parameters were characterized in (A-G) all patients with systemic lupus erythematosus (SLE; n ⫽ 88) or (H-J) those with proliferative lupus nephritis (n ⫽ 40). Each point represents an individual patient with SLE. Horizontal axis of each graph indicates average number of CD16 ⫹ Mos in 1 glomerulus. The relationship was evaluated using Pearson correlation coefficient for (A) serum albumin (r ⫽ ⫺0.48; P ⬍ 0.0001), (B) blood urea nitrogen (BUN; r ⫽ 0.42; P ⬍ 0.0001), (C) CH50 (r ⫽ ⫺0.59; P ⬍ 0.0001), (D) serum anti-DNA antibody titer (r ⫽ 0.43; P ⬍ 0.0001), (E) proteinuria (r ⫽ 0.42; P ⫽ 0.0002), (F) estimated glomerular filtration rate (GFR; r ⫽ ⫺0.34; P ⫽ 0.0013), and (G) histopathologic activity index (r ⫽ 0.94; P ⬍ 0.0001) in all patients with lupus nephritis (classes I to V, n ⫽ 88) and (H) CH50 (r ⫽ ⫺0.62; P ⬍ 0.0001), (I) serum anti-DNA antibody titer (r ⫽ 0.36; P ⫽ 0.0299), and (J) histopathologic activity index (r ⫽ 0.82; P ⬍ 0.0001) in patients with proliferative lupus nephritis (class III ⫹ IV, n ⫽ 40).

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Table 2. Number of T Cells in Glomeruli of Lupus Nephritis ISN/RPS Classification

CD3ⴙ T cell CD8ⴙ T cell

Class I (n ⫽ 7)

Class II (n ⫽ 29)

Class III (n ⫽ 10)

Class IV (n ⫽ 30)

Class V (n ⫽ 12)

0.40 ⫾ 0.31 0.04 ⫾ 0.09

0.58 ⫾ 0.40 0.04 ⫾ 0.10

0.75 ⫾ 0.43 0.03 ⫾ 0.04

1.61 ⫾ 1.90* 0.16 ⫾ 0.27

0.64 ⫾ 0.37 0.08 ⫾ 0.12

Note: Values expressed as mean ⫾ SD. Abbreviation: ISN/RPS, International Society of Nephrology/Renal Pathology Society. *P ⬍ 0.0001 versus classes I, II, III, and V.

0.0001; Fig 9J). Conversely, chronicity index proved to be unrelated to CD16ⴙ Mo count (data not shown). Thus, accumulation of CD16ⴙ Mos within glomeruli appears to be associated with SLE disease activity. Correlation Between Glomerular Fkn Expression and Number of Infiltrating T Cells in Lupus Nephritis

We also detected CD3ⴙ T cells and CD8ⴙ T cells immunohistologically in glomeruli of all 88 patients. Average numbers of CD3ⴙ T cells per glomerulus in class IV glomeruli (n ⫽ 30) also were significantly higher than in the other classes (ISR/RPS I/II/III/IV/V, 0.40 ⫾ 0.31/0.58 ⫾ 0.40/ 0.75 ⫾ 0.43/1.61 ⫾ 1.9/0.64 ⫾ 0.37; P ⬍ 0.0001; Table 2). Glomerular CD8ⴙ T-cell counts showed a tendency to increase, but it was not significant (Table 2). Next, we studied the association between T-cell infiltration and glomerular Fkn expression levels. We found a weak correlation between glomerular Fkn expression level and CD3ⴙ T-cell count in patients with lupus nephritis (r ⫽ 0.36; P ⫽ 0.014; Table 3). Glomerular Fkn expression level was not associated Table 3. Correlation of Fractalkine Expression Levels With T-Cell Counts in Glomeruli of Lupus Nephritis

CD3⫹ T cell CD8⫹ T cell

Decreased Glomerular Fkn Expression and CD16ⴙ Mo Infiltration After Glucocorticoid and/or Methylprednisolone Pulse Therapy

With patients’ consent, we obtained second renal biopsy specimens from 10 patients with DPLN (class IV) after 6 months of glucocorticoid and/or methylprednisolone pulse therapy. Laboratory findings showed that clinical parameters of these patients significantly improved after therapy. The histopathologic activity index of lupus nephritis significantly decreased after glucocorticoid and/or methylprednisolone pulse therapy (pre/post, 8.3 ⫾ 2.9/3.0 ⫾ 1.8; P ⫽ 0.005; Fig 10A). Moreover, numbers of infiltrating CD16ⴙ Mos significantly decreased (pre/ post, 4.8 ⫾ 1.8/1.6 ⫾ 0.6; P ⫽ 0.005; Fig 10B). Glomerular Fkn expression index showed a tendency to decrease, but it was not significant (pre/post, 2.0/0.25 ⫾ 0.5; P ⫽ 0.059; Fig 10C). DISCUSSION

ISN/RPS Classification Class III ⫹ IV (n ⫽ 21)

Class I-V (n ⫽ 49)

with CD8ⴙ T-cell infiltration (Table 3). In patients with proliferative lupus nephritis, there was no significant correlation between glomerular Fkn expression level and T-cell count (Table 3). These results indicate that glomerular Fkn expression may correlate with T-cell infiltration in glomeruli, but not strongly, like CD16ⴙ Mos.

r

P

r

P

0.36 0.25

0.014 0.083

0.17 0.26

0.446 0.238

Note: Relationship was evaluated using Spearman rank correlation. Abbreviation: ISN/RPS, International Society of Nephrology/Renal Pathology Society.

The present study shows for the first time that glomerular Fkn expression and infiltration by CD16ⴙ Mos markedly increase in patients with proliferative lupus nephritis (ISN/RPS classes III and IV). We also found that Fkn expression level correlates significantly with degree of CD16ⴙ Mo infiltration. Fkn levels and CD16ⴙ Mo counts are associated significantly with histological activity index in patients with lupus nephritis and the clinical severity of SLE.

56

Yoshimoto et al

Figure 10. Serial changes in fractalkine (Fkn) expression and CD16⫹ monocyte/macrophage (CD16⫹ Mo) counts in glomeruli from patients with systemic lupus erythematosus administered glucocorticoid and/or methylprednisolone pulse therapy. Each line represents an individual patient. (A) Histopathologic activity index before and after treatment (n ⫽ 10). (B) Average numbers of CD16 ⫹ Mo within glomeruli before and after treatment (n ⫽ 10). (C) Fkn expression index within glomeruli before and after treatment (n ⫽ 4). *P ⫽ 0.005. Abbreviation: AI, activity index.

In human lupus nephritis, as well as in animal models of the disease, leukocytes leave the glomerular capillaries to enter the extracapillary space under the influence of cell adhesion molecules and chemokines.26-29 Fkn is a mononuclear cell–directed cell-surface–anchored chemokine that triggers adhesion of mononuclear cell subsets expressing CX3CR1 to endothelial cells under flow conditions. Fkn expression was identified in glomerular capillaries and mesangial cells,8,11,12 indicating that it could be involved in various types of renal disease. Moreover, the previous finding that an antagonist of Fkn reportedly delayed disease initiation and progression in a murine lupus nephritis model29 suggested that Fkn may be an independent progression factor for lupus nephritis. This study shows that Fkn expression is upregulated markedly in patients with proliferative lupus nephritis, but not those with mesangiopathy or membranous GN, confirming the importance of Fkn in the progression of lupus nephritis. This study also shows the accumulation of CD16ⴙ Mos in patients with proliferative lupus nephritis. CD16ⴙ Mos engage in transendothelial migration in response to Fkn, but not in response to monocyte chemoattractant protein 1 (CCL2), which is in contrast to CD16⫺ monocytes.16,17 CX3CR1 expression in glomerular CD16ⴙ Mos and the association between Fkn expression and glomerular CD16ⴙ Mo infiltration strongly suggest that glomerular infiltration

by CD16ⴙ Mos is Fkn-dependent in patients with lupus nephritis. CD16ⴙ Mos are 1 macrophage/monocyte subset and produce high levels of proinflammatory cytokines, including tumor necrosis factor ␣, interleukin 1, and neurotoxic factors,30-32 which suggests they may function as immunostimulatory and proinflammatory cells. CD16ⴙ Mos previously were reported to be effecter cells involved in the acute inflammation common to patients with all types of proliferative GN.19 The present study shows that CD16ⴙ Mos accumulate within glomeruli of patients with lupus nephritis, which suggests that CD16ⴙ Mos likely contribute to the development of active proliferative GN through the production of proinflammatory cytokines. Consistent with that idea, we confirmed a strong correlation between numbers of glomerular CD16ⴙ Mos and SLE clinical activity or histopathologic activity index of patients with lupus nephritis. Although actions of CD16ⴙ Mos within glomeruli are not known, our data suggest they contribute significantly to the pathogenesis of renal damage in patients with SLE. Glomerular Fkn expression levels and CD16ⴙ Mo counts decreased in patients with DPLN after glucocorticoid and/or methylprednisolone pulse therapy. Although the precise mechanism by which glucocorticoid decreases Fkn production and CD16ⴙ Mo infiltration is not known, it is known that by inhibiting nuclear factor-␬B

Fractalkine and CD16⫹ Monocytes in Lupus Nephritis

activity, glucocorticoid suppresses expression of such proinflammatory cytokines as interleukin 1 and tumor necrosis factor ␣, which induce Fkn expression.33,34 Thus, the capacity of glucocorticoid to suppress the histopathologic activity index of lupus nephritis may in part reflect suppression of glomerular Fkn production and CD16ⴙ Mo accumulation. Fkn expression may represent an important role of T-cell trafficking. In T cells, the CD8ⴙ subset of T cells is reported to express the Fkn receptor CX3CR1.35,36 Cockwell et al11 showed that Fkn was expressed predominantly in glomeruli and/or tubulointerstitium of patients with vasculitic GN or acute allograft rejection. In addition, they showed colocalization of Fkn with macrophages and T cells in the interstitium of patients with acute allograft rejection. In our study, Fkn was expressed in glomeruli of patients with lupus nephritis, compatible with previous reports. In glomeruli of patients with lupus nephritis, especially those with proliferative lupus nephritis, the majority of infiltrating cells were macrophages/monocytes, although a few T cells also infiltrated in glomeruli. In our study, CD3ⴙ T-cell counts significantly increased in glomeruli of patients with proliferative lupus nephritis. Therefore, we also studied the relation between glomerular Fkn expression levels and T-cell infiltration. A significant association was found between Fkn expression level and CD3ⴙ T-cell count; however, the association was much weaker than that of CD16ⴙ Mos and there was no significant association of Fkn expression with CD8ⴙ T-cell infiltration. In addition, in patients with proliferative lupus nephritis, no association was observed between T-cell count and Fkn expression level. These results suggest that glomerular Fkn is a candidate for directing T cells in patients with lupus nephritis; however, its effect on T cells was much weaker than that on CD16ⴙ Mos. In conclusion, our findings suggest that glomerular Fkn expression and CD16ⴙ Mo accumulation are associated with active proliferative glomerular lesions in patients with lupus nephritis and SLE activity. Fkn appears to be an important mediator of glomerular infiltration by CD16ⴙ Mos. Fkn and CD16ⴙ Mos thus may have an important role in the pathogenesis of lupus nephritis and may represent targets for its treatment.

57 ACKNOWLEDGEMENTS The authors thank Hiromi Ohura and Miyako Sakaida of Nara Medical University for excellent technical assistance. Support: The study was supported in part by a grant for “Progressive Renal Disease” from the Ministry of Health, Labour and Welfare, Japan. Financial Disclosure: None.

REFERENCES 1. Cameron JS: Lupus nephritis. J Am Soc Nephrol 10:413-424, 1999 2. Huong DL, Papo T, Beaufils H, et al: Renal involvement in systemic lupus erythematosus. A study of 180 patients from a single center. Medicine (Baltimore) 78:148166, 1999 3. Alexopoulos E, Seron D, Hartley RB, Cameron JS: Lupus nephritis: Correlation of interstitial cells with glomerular function. Kidney Int 37:100-109, 1990 4. D’Agati VD, Appel GB, Estes D, Knowles DM II, Pirani CL: Monoclonal antibody identification of infiltrating mononuclear leukocytes in lupus nephritis. Kidney Int 30: 573-581, 1986 5. Bazan JF, Bacon KB, Hardiman G, et al: A new class of membrane-bound chemokine with a CX3C motif. Nature 385:640-644, 1997 6. Muehlhoefer A, Saubermann LJ, Gu X, et al: Fractalkine is an epithelial and endothelial cell-derived chemoattractant for intraepithelial lymphocytes in the small intestinal mucosa. J Immunol 164:3368-3376, 2000 7. Maciejewski-Lenoir D, Chen S, Feng L, Maki R, Bacon KB: Characterization of fractalkine in rat brain cells: Migratory and activation signals for CX3CR-1-expressing microglia. J Immunol 163:1628-1635, 1999 8. Ito Y, Kawachi H, Morioka Y, et al: Fractalkine expression and the recruitment of CX3CR1⫹ cells in the prolonged mesangial proliferative glomerulonephritis. Kidney Int 61: 2044-2057, 2002 9. Yoneda O, Imai T, Goda S, et al: Fractalkine-mediated endothelial cell injury by NK cells. J Immunol 164:40554062, 2000 10. Chakravorty SJ, Cockwell P, Girdlestone J, Brooks CJ, Savage CO: Fractalkine expression on human renal tubular epithelial cells: Potential role in mononuclear cell adhesion. Clin Exp Immunol 129:150-159, 2002 11. Cockwell P, Chakravorty SJ, Girdlestone J, Savage CO: Fractalkine expression in human renal inflammation. J Pathol 196:85-90, 2002 12. Chen YM, Hu-Tsai MI, Lin SL, Tsai TJ, Hsieh BS: Expression of CX3CL1/fractalkine by mesangial cells in vitro and in acute anti-Thy1 glomerulonephritis in rats. Nephrol Dial Transplant 18:2505-2514, 2003 13. Imai T, Hieshima K, Haskell C, et al: Identification and molecular characterization of fractalkine receptor CX3CR1, which mediates both leukocyte migration and adhesion. Cell 91:521-530, 1997 14. Fong AM, Robinson LA, Steeber DA, et al: Fractalkine and CX3CR1 mediate a novel mechanism of leukocyte capture, firm adhesion, and activation under physiologic flow. J Exp Med 188:1413-1419, 1998

58 15. Segerer S, Hughes E, Hudkins KL, Mack M, Goodpaster T, Alpers CE: Expression of the fractalkine receptor (CX3CR1) in human kidney diseases. Kidney Int 62:488495, 2002 16. Ancuta P, Rao R, Moses A, et al: Fractalkine preferentially mediates arrest and migration of CD16⫹ monocytes. J Exp Med 197:1701-1707, 2003 17. Geissmann F, Jung S, Littman DR: Blood monocytes consist of two principal subsets with distinct migratory properties. Immunity 19:71-82, 2003 18. Ziegler-Heitbrock HW: Heterogeneity of human blood monocytes: The CD14⫹ CD16⫹ subpopulation. Immunol Today 17:424-428, 1996 19. Hotta O, Yusa N, Ooyama M, Unno K, Furuta T, Taguma Y: Detection of urinary macrophages expressing the CD16 (Fc gamma RIII) molecule: A novel marker of acute inflammatory glomerular injury. Kidney Int 55:1927-1934, 1999 20. Tan EM, Cohen AS, Fries JF, et al: The 1982 revised criteria for the classification of systemic lupus erythematosus. Arthritis Rheum 25:1271-1277, 1982 21. Hochberg MC: Updating the American College of Rheumatology revised criteria for the classification of systemic lupus erythematosus. Arthritis Rheum 40:1725, 1997 22. Levey AS, Coresh J, Greene T, et al: Using standardized serum creatinine values in the Modification of Diet in Renal Disease Study equation for estimating glomerular filtration rate. Ann Intern Med 145:247-254, 2006 23. Austin HA III, Muenz LR, Joyce KM, Antonovych TT, Balow JE: Diffuse proliferative lupus nephritis: Identification of specific pathologic features affecting renal outcome. Kidney Int 25:689-695, 1984 24. Mikulowska-Mennis A, Taylor TB, Vishnu P, et al: High-quality RNA from cells isolated by laser capture microdissection. Biotechniques 33:176-179, 2002 25. Tadros Y, Ruiz-Deya G, Crawford BE, Thomas R, Abdel-Mageed AB: In vivo proteomic analysis of cytokine expression in laser capture-microdissected urothelial cells of obstructed ureteropelvic junction procured by laparoscopic dismembered pyeloplasty. J Endourol 17:333-336, 2003 26. Wuthrich RP: Vascular cell adhesion molecule-1 (VCAM-1) expression in murine lupus nephritis. Kidney Int 42:903-914, 1992

Yoshimoto et al 27. Nakatani K, Fujii H, Hasegawa H, et al: Endothelial adhesion molecules in glomerular lesions: Association with their severity and diversity in lupus models. Kidney Int 65:1290-1300, 2004 28. Perez de Lema G, Maier H, Nieto E, et al: Chemokine expression precedes inflammatory cell infiltration and chemokine receptor and cytokine expression during the initiation of murine lupus nephritis. J Am Soc Nephrol 12:13691382, 2001 29. Hasegawa H, Kohno M, Sasaki M, et al: Antagonist of monocyte chemoattractant protein 1 ameliorates the initiation and progression of lupus nephritis and renal vasculitis in MRL/lpr mice. Arthritis Rheum 48:2555-2566, 2003 30. Thieblemont N, Weiss L, Sadeghi HM, Estcourt C, Haeffner-Cavaillon N: CD14lowCD16high: A cytokineproducing monocyte subset which expands during human immunodeficiency virus infection. Eur J Immunol 25:34183424, 1995 31. Belge KU, Dayyani F, Horelt A, et al: The proinflammatory CD14⫹CD16⫹DR⫹⫹ monocytes are a major source of TNF. J Immunol 168:3536-3542, 2002 32. Randolph GJ, Sanchez-Schmitz G, Liebman RM, Schakel K: The CD16(⫹) (FcgammaRIII(⫹)) subset of human monocytes preferentially becomes migratory dendritic cells in a model tissue setting. J Exp Med 196:517527, 2002 33. Adcock IM, Brown CR, Gelder CM, Shirasaki H, Peters MJ, Barnes PJ: Effects of glucocorticoids on transcription factor activation in human peripheral blood mononuclear cells. Am J Physiol 268:C331-C338, 1995 34. Widmer U, Manogue KR, Cerami A, Sherry B: Genomic cloning and promoter analysis of macrophage inflammatory protein (MIP)-2, MIP-1 alpha, and MIP-1 beta, members of the chemokine superfamily of proinflammatory cytokines. J Immunol 150:4996-5012, 1993 35. Fong AM, Robinson LA, Steeber DA, et al: Fractalkine and CX3CR1 mediate a novel mechanism of leukocyte capture, firm adhesion, and activation under physiologic flow. J Exp Med 188:1413-1419, 1998 36. Chen S, Bacon KB, Li L, et al: In vivo inhibition of CC and CX3C chemokine-induced leukocyte infiltration and attenuation of glomerulonephritis in Wistar-Kyoto (WKY) rats by vMIP-II. J Exp Med 188:193-198, 1998