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Abstract

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES

Objective

Defective circulating dendritic cells (DCs) have been described in systemic lupus erythematosus (SLE) and correlated with high levels of interferon-α (IFNα). DCs are differentiated as being either myeloid or plasmacytoid, according to chemokine expression and the tendency to migrate toward inflamed tissue. We investigated the potential role of interleukin-18 (IL-18) in driving the glomerular migration of DCs in lupus nephritis (LN) and in affecting the ability of DCs to induce an imbalance in the Th1:Th2 ratio.

Methods

DC subsets were characterized by flow cytometry and defined as either myeloid or plasmacytoid according to the expression of CD11c/blood dendritic cell antigen 1 (BDCA-1) and CD123/BDCA-2, respectively. The serum Th1:Th2 profile was studied by enzyme-linked immunosorbent assay. IL-18 receptor (IL-18R) and other chemokine receptors were analyzed by flow cytometry. Glomerular levels of IL-18/IL-18R and the presence of plasmacytoid DCs and myeloid DCs were investigated by immunohistochemical analysis.

Results

The number of peripheral plasmacytoid DCs was decreased in patients with SLE compared with control subjects, and this defect in the number of DCs was correlated with LN. Patients with LN showed a prevalent Th1 response, with high production of IL-18, IL-12 and IFNγ. Only plasmacytoid DCs expressed IL-18R. Patients with severe LN showed a high accumulation of IL-18 within glomeruli in association with the presence of plasmacytoid DCs, whereas myeloid DCs were almost absent.

Conclusion

A deficient number of peripheral plasmacytoid DCs correlated with high levels of Th1 cytokines and was associated with LN. Both serum and glomerular IL-18 were increased in LN. It is suggested that the high level of expression of IL-18R by peripheral plasmacytoid DCs allows the DCs to relocate within glomeruli under IL-18 stimulation and triggers the resident T cells, thus promoting renal damage.

Lupus nephritis (LN) is a major complication of systemic lupus erythematosus (SLE) and is mediated by glomerular deposition of immune complexes that trigger a number of inflammatory events leading to tissue damage. Although autoantibody production, cytokines, and chemokines are required to promote glomerular inflammation (1–3), the separate pathogenic contributions of these factors to the development of LN have not been fully elucidated.

Impaired T cell function resulting either from an intrinsic defect or as a consequence of an underlying alteration of dendritic cells (DCs) has been demonstrated in experimental and human SLE (4). DCs are considered powerful antigen-presenting cells that activate naive T cells, regulate cytokine production, and interact with T cells, thus promoting autoimmunity.

A defect in the number of circulating DCs in conjunction with high production of serum interferon-α (IFNα) have been described in SLE (5). Two DC subsets, plasmacytoid DCs and myeloid DCs, have been identified on the basis of different antigen expression and levels of maturation, as well as the tendency to migrate toward peripheral tissue (6). Both subsets of DCs express a similar repertoire of adhesion molecules and chemokine receptors; however, most chemokine receptors of plasmacytoid DCs are not functional in circulating cells (7). In addition, myeloid DCs respond to several homeostatic and inflammatory chemokines such as CCR2, CCR5, and CXCR4, whereas responsiveness to CXCL12 by plasmacytoid DCs induces their activation and affects their migration. Although plasmacytoid DCs express high levels of both CCR7 and CXCR3, they fail to migrate in response to any of the specific receptors (8). Thus, additional chemoattractants are possibly involved in plasmacytoid DC trafficking within inflamed tissue.

Tumor necrosis factor α (TNFα) and interleukin-1 (IL-1) have been shown to be capable of stimulating DC activation and trafficking within the damaged skin of patients with SLE (9), whereas serum and glomerular accumulation of IL-18 occurs only in active LN (10). Also, plasmacytoid DCs exert a migratory function owing to the high expression of chemerin or the local accumulation of adenosine, which drives their trafficking toward inflamed tissue (11, 12). In addition, IL-18 receptor (IL-18R) is selectively expressed by peripheral plasmacytoid DCs, which are prone to responding to IL-18 (13). Because IL-18 is detected in the glomeruli of patients with LN, we reasoned that glomerular IL-18 may locally amplify the immune response by attracting the majority of IL-18R–positive cells, including both DCs and T cells. This implies that a defined role should be assigned to plasmacytoid DCs both in promoting a Th1 immune response and in increasing renal damage.

The aim of this study was to investigate circulating peripheral DC subsets in patients with active LN, as well as the levels of Th1 cytokines such as IL-18, IFNγ, and IL-12, to assess their functional roles.

PATIENTS AND METHODS

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES

Study population.

Thirty-five white patients with SLE (i.e., meeting ≥4 of the American College of Rheumatology criteria for the classification of SLE) (14, 15) and 41 matched healthy subjects were enrolled at the Department of Internal Medicine of the University of Bari. The local ethics committee approved the study, and informed consent was obtained from all patients and control subjects. Patients with other chronic renal diseases were excluded. Detailed demographic data were recorded, including a medication history and key laboratory parameters. Patients with SLE were grouped according to the presence (group A; n = 18) or absence (group B; n = 17) of renal involvement. Forty-one healthy control subjects comprised group C. Both serum and peripheral blood samples were collected at the time of renal biopsies. Sera were stored at −80°C and thawed for the enzyme-linked immunosorbent assay (ELISA). Biopsy specimens were available for all patients in group A and were obtained prior to treatment.

Thus, we included these patients in subsets numbered in relation to their class of nephritis according to the World Health Organization classification (16). Therefore, the patients were distributed as follows: subset II (class II; n = 1), subset III (class III; n = 5), subset IV (class IV; n = 8), and subset V (class V; n = 4). Because a single patient was classified as having class I LN, we arbitrarily included this patient in group B because of the absence of renal damage. The female:male ratio was 8.5, and the mean ± SD age of patients with SLE was 39.1 ± 13.1 years. Demographic characteristics of the patients with LN are summarized in Table 1.

Table 1. Characteristics of the patients with lupus nephritis*
Patient/sex/ageLaboratory parameterAutoantibodiesTreatment(s)
Urinary protein, mg/24 hoursUrinary castsUrinary RBCsANARNPdsDNA
  • *

    RBCs = red blood cells; ANA = antinuclear antibodies; dsDNA = double-stranded DNA; mPred. = methylated prednisone; IV = intravenous; IVIG = intravenous immunoglobulin; MTX = methotrexate; AZA = azathioprine; MMF = mycophenolate mofetil; CYC = cyclophosphamide; CSA = cyclosporin A.

1/F/292,200+++++++++mPred. IV, pred. 1 mg/kg/day, IVIG, MTX
2/F/351,750+++++−++mPred. IV, pred. 1 mg/kg/day
3/F/31300+−−++−++Pred. 1 mg/kg/day, AZA
4/M/37600+−−+−−++Pred. 1 mg/kg/day, AZA
5/F/191,000++−++−+++Pred. 0.5 mg/kg/day, MTX
6/F/271,200+−−++++++MMF, CYC IV, pred. 1 mg/kg/day, AZA
7/F/33300−−−+−−++CSA, pred. 1 mg/kg/day
8/F/28300++−+−−+Pred. 0.5 mg/kg/day, AZA
9/F/25500+−−++−++MMF, CYC
10/F/32300+−−+++++CSA, pred. 0.5 mg/kg/day
11/F/32400++−+−−+++CYC IV, AZA
12/F/362,500++++++mPred. IV, pred. 1 mg/kg/day
13/F/311,800+++++−+++CYC, MTX
14/F/301,500+−−++−+Pred. 1 mg/kg/day, AZA
15/F/29800+−−++−+Pred. 1 mg/kg/day
16/F/29400+−−+−−+++Pred. 0.5 mg/kg/day
17/F/33600+−−++++++Pred. 1 mg/kg/day
18/F/53800+−−+++++mPred. IV, pred. 1 mg/kg/day, AZA

Phenotypic characterization of DC subsets.

Peripheral blood mononuclear cells were purified by Ficoll-Hypaque sedimentation (Amersham Biosciences, Piscataway, NJ) for 45 minutes at 2,200 revolutions per minute and incubated for 30 minutes at 4°C with the following conjugated monoclonal antibodies (mAb): lineage (Lin) cocktail (anti-CD3, anti-CD14, anti-CD19, anti-CD16, anti-CD20, and anti-CD56; PharMingen, San Diego, CA), anti–HLA–DR (PharMingen), anti-CD123 (Miltenyi Biotec, Bologna, Italy), anti-CD11c (PharMingen), anti–blood dendritic cell antigen 1 (anti–BDCA-1), anti–BDCA-2, and anti–BDCA-4 (all from Miltenyi Biotec). In addition, to define the potential migratory behavior of DCs, the following chemokine receptors were analyzed: CCR2, CCR5, CCR7, and CXCR4 (all from PharMingen), whereas IL-18Rα (R&D Systems, Minneapolis, MN) was used to verify additional migratory molecules.

Two subsets of DCs were defined on the basis of their differing antigen expression. Briefly, myeloid DCs were defined as Lin−,CD11c+,BDCA-1+,CD123−,BDCA-2−,BDCA-4−, whereas plasmacytoid DCs were defined as Lin−,CD11c−,CD123+high,BDCA-2+,BDCA-4+. The plasmacytoid BDCA-2+,CD123+ and myeloid BDCA-1+,CD11c+ DCs were analyzed by 6-color cytometry (FACSCanto; Becton Dickinson, Oxford, UK), and a total of 50,000 events were analyzed with the FACSDiva software (Becton Dickinson). DCs were gated on the HLA–DR+,Lin− population, and because myeloid DCs express the CD123 antigen at a lower level than do plasmacytoid DCs, we gated the BDCA-2+,CD11c− cells and then the CD123+high,BDCA-2+ cells to define the dot plot representative of the plasmacytoid subset. Similarly, we gated the CD11c+,BDCA-2− cell population and defined the dot plot of CD11c+,BDCA-1+ cells as a myeloid subset of DCs. Mouse IgG1 and IgG2a mAb were used as isotype controls.

Cytokine ELISAs.

Th1 (IL-18, IL-12 p70, and IFNγ) and Th2 (IL-4) cytokines were measured by a sandwich ELISA in sera from patients and control subjects. Samples (1:10 dilution) were tested for the 18-kd bioactive isoform of IL-18 (Medical and Biological Laboratories, Nagoya, Japan), using a dedicated kit according to the manufacturer's instruction. Other cytokine measurements were assessed in our laboratory. Briefly, 96-well Nunc plates were coated overnight at 4°C with each mouse anti-human cytokine mAb (PharMingen). Following incubation with sera, a biotinylated anti-human antibody–specific cytokine (PharMingen) was used as secondary antibody, and streptavidin–alkaline phosphatase diluted 1:1,000 (Southern Biotechnology, Birmingham, AL) was added. The reaction was developed with an o-phenyldiamine chromogen solution (Sigma, St. Louis, MO), and optical density at 405 nm was measured with a Microplate Reader (Bio-Rad, Milan, Italy). Data were analyzed with dedicated software. The results were related to the standard curve obtained using the recombinant human cytokines in the relative tests (PharMingen).

Immunohistochemical analysis.

Both frozen and paraffin-embedded specimens from renal biopsy specimens obtained from patients and from a normal subject who underwent biopsy for persistent proteinuria were studied by immunohistochemistry to define the expression of IL-18 (Medical and Biological Laboratories), as well as the presence of DCs with an immature or mature phenotype. Anti-CD11c mAb (PharMingen) and anti–BDCA-2 (Miltenyi Biotec) were used to define myeloid DCs and plasmacytoid DCs, respectively, whereas anti-CD83 (R&D Systems) was used to investigate the presence of mature glomerular DCs. In addition, anti-CD68 (Vector, Burlingame, CA), anti-CD4 (Vector), and anti-CD19 (PharMingen) with the relative mAb were used to identify any additional cells infiltrating the glomeruli. Finally, both IL-18R (R&D Systems) and CCL21 (PharMingen) mAb were used to characterize glomerular plasmacytoid DCs.

Briefly, paraffin-embedded sections were treated in xylene and rehydrated by gradient of ethanol. After blocking with 2% horse serum, sections were incubated overnight with mouse mAb at appropriate concentrations. Binding of the secondary biotinylated horse anti-mouse IgG was detected with the Vectastain ABC system (Vector). Endogenous peroxidase activity was blocked with 3% H2O2 for 30 minutes. Next, 3,3′-diaminobenzidine was added for 10 minutes, and nuclei were counterstained with hematoxylin (DakoCytomation, Milan, Italy). Mouse IgG1 and IgG2a were used as isotype controls. To define the origin of both tubular and interstitial staining that was observed in several specimens, we conducted immunohistochemistry experiments with either a primary anti-rabbit IL-18 mAb or without the primary antibody, thus confirming that the positivity of tubular cells was prevalently attributable to the nonspecific cross-reactivity of the biotin-conjugated secondary antibody. However, these procedures never stained glomeruli. In each biopsy specimen, the number of positive cells within glomeruli was expressed as the mean value per glomerular cross-section (gcs). Staining was evaluated by light microscopy.

Statistical analysis.

The Mann-Whitney test was used to assess differences in serum cytokine levels and the number of circulating DCs. Correlation analyses were performed using Spearman's rank correlation test. P values less than 0.05 were considered significant.

RESULTS

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES

Reduction of circulating DCs in SLE.

Figures 1a and b show representative cytofluorimetric patterns of circulating DCs derived from patients in groups A and B and normal control subjects (group C). By analyzing the percentage of both plasmacytoid DCs and myeloid DCs, we determined that plasmacytoid DCs were reduced in patients with SLE (mean ± SD 0.61 ± 0.37%) compared with normal control subjects (1.12 ± 0.31%; P < 0.0001). In contrast, the percentage of circulating myeloid DCs was not statistically significantly different between patients and control subjects. In addition, the median ± SEM number of plasmacytoid DCs in group B patients (0.93 ± 0.24%) was similar to that in group C patients; thus, we investigated patients with organ involvement, focusing on LN, and showed that group A had a lower median ± SEM number of circulating plasmacytoid DCs compared with group B (0.33 ± 0.19% and 0.93 ± 0.24%, respectively; P = 0.0012) (Figures 1a and c).

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Figure 1. Phenotype pattern of circulating dendritic cell (DC) subsets. a and b, Representative dot plots for patients from groups A and B as well as normal control subjects (group C), showing low numbers of circulating CD123-positive/blood dendritic cell antigen 2 (BDCA-2)–positive plasmacytoid DCs (pDC) in group A compared with groups B and C (a), and similar numbers of peripheral CD11c-positive/BDCA-1–positive myeloid DCs (mDC) in groups A, B, and C (b). c and d, Levels of circulating plasmacytoid DCs (c) and myeloid DCs (d) in groups A and B. The number of plasmacytoid DCs was reduced in group A compared with group B (P = 0.0012) (c), whereas the occurrence of lupus nephritis (LN) did not influence the number of myeloid DCs (d). Data are shown as box plots. Each box represents the 25th to 75th percentiles. Lines outside the boxes represent the 10th and the 90th percentiles. Lines inside the boxes represent the median. e, Correlation between the mean and SD percent of either peripheral plasmacytoid DCs or myeloid DCs with different subsets of LN, showing a reduction in plasmacytoid DCs in subset IV (0.21 ± 0.08%) compared with subsets III (0.53 ± 0.22%) and V (0.41 ± 0.12%) (P < 0.05 for both).

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As shown in Figure 1e, by categorizing patients in group A according to the severity of LN, the lower number of plasmacytoid DCs was demonstrated in subset IV (mean ± SD 0.21 ± 0.08%) as compared with subset V (0.41 ± 0.12%; P < 0.05) and subset III (0.53 ± 0.22%; P < 0.05). In contrast, the percentage of myeloid DCs was independent of the severity of renal involvement (Figures 1b and d) and was further increased in subsets IV and V (Figure 1e) with respect to the other subsets of patients with LN. The mean ± SD percentages of both plasmacytoid DCs and myeloid DCs in patients (groups A and B) and control subjects (group C) are summarized in Table 2.

Table 2. Phenotypic characterization of circulating and glomerular dendritic cells (DCs)*
GroupPeripheral DCs, %Glomerular cells, cells/gcs
CD123+, BDCA-2+CD11c+, BDCA-1+BDCA-2+, IL-18R+CD11c+, IL-18R+BDCA-2+CD11c+IL-18+IL-18R+CD68+CD4+
  • *

    Values are the mean ± SD. gcs = glomerular cross-section; BDCA-2 = blood dendritic cell antigen 2; IL-18R = interleukin-18 receptor.

Group A (n = 18)0.33 ± 0.192.34 ± 1.2979.4 ± 135.1 ± 0.911.0 ± 61.2 ± 0.715.0 ± 7
 Subset II (n = 1)0.681.81205
 Subset III (n = 5)0.53 ± 0.221.69 ± 1.34.7 ± 1.706.1 ± 35.4 ± 210.4 ± 83.9 ± 0.6
 Subset IV (n = 8)0.21 ± 0.082.1 ± 1.616.5 ± 41.0 ± 0.320.7 ± 910.9 ± 314.7 ± 58.9 ± 4
 Subset V (n = 4)0.41 ± 0.122.3 ± 0.379.7 ± 21.8 ± 0.915.9 ± 710.6 ± 513.8 ± 67.3 ± 3
Group B (n = 17)0.93 ± 0.242.44 ± 1.081.8 ± 83.1 ± 0.40000
Group C (n = 41)1.12 ± 0.312.42 ± 0.4476.1 ± 93.4 ± 0.8

Overexpression of IL-18R and other chemokine receptors by plasmacytoid DCs.

The next set of experiments was devoted to investigating the expression of both IL-18R and other chemokines involved in DC migration. Cells were gated starting from the HLA–DR+,Lin− population, and both myeloid DCs (CD11c+,BDCA-1+) and plasmacytoid DCs (CD123+,BDCA-2+) were analyzed. As shown in Figure 2, a very low number of peripheral CD11c-positive myeloid DCs expressed IL-18R in a representative patient with SLE (mean ± SD 4.1 ± 0.6%) (Figure 2a), whereas peripheral plasmacytoid DCs expressed high levels of IL-18R (79.1 ± 13.0%). In group C, levels of peripheral myeloid DCs and plasmacytoid DCs were similar (3.6 ± 0.3% and 77 ± 16%, respectively) (results not shown). Thus, it is conceivable that only plasmacytoid DCs express IL-18R and may be chemoattracted by IL-18–positive cells. In addition, by measuring the mean fluorescence intensity (MFI) fold increase of various chemokine receptors compared with the relative isotype control (Figure 2c), we observed that plasmacytoid DCs overexpressed CXCR4 (MFI 4.7), CCR7 (MFI 5.3), and CCR5 (MFI 4.6) as compared with myeloid DCs (MFI 2.7, MFI 2.4, and MFI 1.7, respectively). The majority of these receptors, however, are expected not to be functional (17) nor directly implicated in the migration of plasmacytoid DCs, whereas the presence of IL-18R on plasmacytoid DCs (MFI 9.3) may represent a novel functional molecule involved in their migration.

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Figure 2. Representative pattern of interleukin-18 receptor (IL-18R) expression by circulating DCs, and the Th1/Th2 profile. a, Lack of expression of IL-18R by CD11c-positive myeloid DCs. b, Expression of IL-18R by CD123-positive plasmacytoid DCs. c, Expression of different chemokine receptors by myeloid DCs and plasmacytoid DCs. Compared with myeloid DCs, plasmacytoid DCs overexpressed CCR5, CCR7, CXCR4, and IL-18R. df, Serum levels of Th1 (IL-12, interferon-γ [IFNγ], and IL-18) and Th2 (IL-4) cytokines as measured by enzyme-linked immunosorbent assay. d, Patients with systemic lupus erythematosus (SLE) produced high amounts of both Th1 and Th2 cytokines as compared with control subjects. ∗ = P < 0.0001. e, Patients in group A produced more IL-12, IFNγ, and IL-18 compared with patients in group B, demonstrating that the difference in cytokine production was mostly attributable to the presence of nephritis. Conversely, serum levels of IL-4 were higher in group B compared with group A. ∗ = P < 0.05. f, Th1 levels in subsets of patients in group A based on the World Health Organization classification of lupus nephritis. Serum levels of IL-12 did not reflect the severity of renal damage, whereas serum levels of both IFNγ and IL-18 were increased in subsets IV and V compared with subset III. Values are the mean and SD. See Figure 1 for other definitions.

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Correlation of serum levels of Th1 cytokines and lupus nephritis.

Figure 2d shows IL-12, IFNγ, IL-18, and IL-4 serum levels in patients with SLE and normal control subjects. Levels of both Th1 and IL-4 were significantly increased in patients (for IL-12, mean ± SD 511.8 ± 90 pg/ml; for IFNγ, 628.1 ± 101 pg/ml; for IL-18, 596.5 ± 270 pg/ml; for IL-4, 987.2 ± 210 pg/ml) as compared with controls (P < 0.0001 for all). However, as shown in Figure 2e, the highest production of Th1 cytokines was attributable to renal damage, because patients in group A had higher titers of IL-12 (769 ± 112 pg/ml), IFNγ (715 ± 74 pg/ml), and IL-18 (814 ± 185 pg/ml) than those in group B (P < 0.05 for all). Conversely, patients in group B produced high amounts of serum IL-4 (1,090 ± 130 pg/ml; P < 0.05 versus group A).

A correlation between serum levels of Th1 cytokines and specific classes of LN was further investigated (Figure 2f). IL-12 levels were almost similar in the different subsets of LN, whereas levels of IFNγ (mean ± SD 893 ± 65 and 786 ± 79 pg/ml, respectively; P < 0.05) and IL-18 (871 ± 75 and 884 ± 39 pg/ml, respectively; P < 0.05) were elevated in subsets IV and V as compared with subset III (366 ± 62 pg/ml and 415 ± 35 pg/ml, respectively). In subset II, levels were reduced (430 pg/ml and 337 pg/ml, respectively) compared with the levels in subsets IV and V, although a statistical evaluation has not been done for the low number of patients included in subset II. In addition, elevations in the levels of both IFNγ and IL-18 were unrelated to other organ involvement, thus suggesting their direct correlation with the renal disease.

Glomerular IL-18 overexpression in LN.

To assess local production of IL-18 within the kidney, we studied its in situ expression in renal specimens from patients with LN, by immunohistochemistry. As shown in Figure 3, we demonstrated a prevalence of IL-18–positive cells in glomeruli from patients in subsets IV and V (Figures 3a and b), whereas specimens from patients in subset III (Figure 3c) showed a weak presence of the cytokine. Conversely, few IL-18–positive cells were detected in glomeruli from patients in subset II (results not shown), and specimens from control subjects (Figure 3d) were negative in all instances. IL-18–positive cells were located within the mesangial matrix, and the majority of infiltrating mononuclear cells expressed the cytokine.

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Figure 3. ad, Representative patterns of glomerular IL-18 and DC subset expression in LN. Expression of IL-18 was high in subset IV (a) and subset V (b) and weak in subset III (c). IL-18 was prevalently located within the mesangial matrix as well as in infiltrating mononuclear cells. In contrast, no evidence of IL-18 was observed in glomeruli of a control subject with minimal proteinuria and normal renal findings (d). eo, Representative patterns of CD11c-positive myeloid DCs (ei) and BDCA-2–positive plasmacytoid DCs (jo) in renal specimens from patients with different classes of LN. In a control subject with persistent proteinuria and normal renal findings (e and j) and in patient subset II (f and k), both myeloid DCs (e and f, respectively) and plasmacytoid DCs (j and k, respectively) were practically absent. In patient subset III, myeloid DCs were absent (g) and the presence of plasmacytoid DCs was minor (m). In patient subset IV (h and n) and patient subset V (i and o), remarkable amounts of BDCA-2+ cells were observed. See Figure 1 for definitions (original magnification × 50).

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Although a comparative analysis with regard to the glomerular content of IL-18 between different classes of LN was not performed because of the low number of patients with LN, a prevalent accumulation of IL-18–positive glomerular mononuclear cells was demonstrated in biopsy specimens from both subset IV and subset V (mean ± SD 20.7 ± 9 cells/gcs and 15.9 ± 7 cells/gcs, respectively) as compared with subset III (6.1 ± 3 cells/gcs; P < 0.05 for both). A low presence of IL-18 was also demonstrated in tubular epithelial cells. However, tubular expression paralleled that observed in normal kidney as well as that of control specimens processed either without the primary anti–IL-18 antibody or with an antibody from different species (mouse anti-rabbit IL-18). Detection of CD68 also revealed that in both subset IV and subset V, the majority of infiltrating mononuclear cells were represented by macrophages (14.7 ± 5 cells/gcs and 13.8 ± 6 cells/gcs, respectively) in conjunction with CD4+ T cells (8.9 ± 4 cells/gcs and 7.3 ± 3 cells/gcs, respectively) as compared with subset III (10.4 ± 8 cells/gcs and 3.9 ± 0.6 cells/gcs, respectively), whereas CD19+ B cells were virtually absent in all specimens, with the exception of a few positive spots in subset V specimens.

Accumulation of plasmacytoid DCs within glomeruli in LN.

To verify the ability of DCs to migrate from peripheral blood toward inflamed tissue under the stimuli induced by both chemokines and cytokines, we evaluated the expression of DCs within glomeruli of patients with different classes of LN, as well as in patients without renal damage and normal subjects. Figure 3 shows a representative immunohistochemical pattern of glomerular accumulation of both myeloid DCs (Figures 3e–i) and plasmacytoid DCs (Figures 3j–o). Both myeloid DCs and plasmacytoid DCs were absent in specimens derived from patients in subset II (Figures 3f and k) and in a subject from group C with persistent proteinuria in the absence of renal damage (Figures 3e and j). In addition, myeloid DCs were absent in patients with LN in subset III (Figure 3g), whereas a poor accumulation was demonstrated in those from both subset IV (mean ± SD 1 ± 0.3 cells/gcs) and subset V (1.8 ± 0.9 cells/gcs) (Figures 3h and i, respectively). In contrast, plasmacytoid DCs highly accumulated within glomeruli of patients in subset IV (16.5 ± 4 cells/gcs) followed by subset V (9.7 ± 2 cells/gcs) and subset III (4.7 ± 1.7 cells/gcs) (P < 0.05 by one-way analysis of variance) (Figures 3n, o, and m, respectively). All subsets showed a higher number of BDCA-2–positive plasmacytoid DCs as compared with CD11c-positive myeloid DCs (P < 0.05).

As shown in Figure 4a, an inverse correlation was demonstrated between the number of peripheral plasmacytoid DCs and local IL-18–producing cells (r = −0.5927, P < 0.05), thus suggesting the involvement of IL-18 in the chemoattraction of IL-18R–positive plasmacytoid DCs. In addition, a correlation between the number of glomerular BDCA-2–positive cells and IL-18R–positive cells (r = 0.697, P < 0.05) was also demonstrated in group A (Figure 4b). To further confirm the presence of IL-18R–positive cells within the kidney, we evaluated the expression of IL-18R in subsets III (Figure 4c), IV (Figure 4d), and V (Figure 4e), showing that the mean ± SD numbers of positive cells (5.4 ± 2 cells/gcs, 10.9 ± 3 cells/gcs, and 10.6 ± 5 cells/gcs, respectively) paralleled those of BDCA-2–positive cells (4.7 ± 1.7 cells/gcs, 16.5 ± 4 cells/gcs, and 9.7 ± 2 cells/gcs, respectively). However, it is conceivable that other cells such as T cells may express the receptor within the kidney.

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Figure 4. Correlation between plasmacytoid dendritic cells (pDC) and interleukin-18 (IL-18) production in lupus nephritis. a, Inverse correlation between the number of peripheral plasmacytoid DCs and IL-18–producing cells within the kidney. b, Relationship between IL-18 receptor (IL-18R)–positive and blood dendritic cell antigen 2 (BDCA-2)–positive cells. ce, Representative patterns derived from patient subsets III (c), IV (d), and V (e), depicting the accumulation of IL-18R–positive cells. f, Presence of CD83-positive cells in a representative patient from subset IV. gcs = glomerular cross-section. (Original magnification × 50.)

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Finally, the presence of CD83-positive cells sharing a typical plasmacytoid-like morphology (Figure 4f) was demonstrated exclusively within glomeruli of patients in subset IV (8.0 ± 3 cell/gcs), whereas CD83-positive cells were undetectable in the other subsets of LN (results not shown). Because myeloid DCs were almost absent in patients in subset V, in parallel with the low level of CD83 antigen, we speculated that the simultaneous presence of BDCA-2–positive and CD83-positive cells may reflect their plasmacytoid origin. Moreover, the results suggest that immature glomerular plasmacytoid DCs may undergo final local maturation under IL-18 stimulation, thus exerting their immunologic functions within the kidney.

DISCUSSION

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES

A defective number and impaired function of DCs have been described in SLE and correlated to high production of IFNα and an imbalance in Th1:Th2 cytokine homeostasis (18). Results from the present study indicate that the number of peripheral immature myeloid DCs and plasmacytoid DCs is decreased in SLE, and that the plasmacytoid DC defect correlates with LN activity. In addition, plasmacytoid DCs accumulate within the glomeruli of patients with LN, and the ability of these DCs to migrate is apparently related to IL-18R stimulation by high local production of IL-18.

The role of subsets of immature DCs in SLE is presently being debated. Previous studies demonstrated a reduced frequency of circulating CD11c-positive myeloid DCs. However, the skew from the typical Th2 immune response, the high IFNα serum levels (18), and the evidence of a role of Th1 cytokines in experimental and human SLE (10, 19, 20) emphasize the involvement of deregulated plasmacytoid DCs in the pathogenesis of SLE. Our results demonstrated a decreased number of circulating DCs in SLE, with a prevalent defect of plasmacytoid DCs in patients with LN. In addition, an inverse correlation between the number of peripheral plasmacytoid DCs and the severity of renal damage was detected. There are different possible explanations for the reduced proportion of peripheral DC subsets in SLE. The number of immature DC progenitors might be reduced, although a deficit of CD34-positive cells has not been demonstrated. DC clearance might be increased due to accelerated apoptosis, or DCs might be actively recruited toward inflamed tissue under the effect of both chemokine and cytokine stimulation, as shown in other autoimmune and inflammatory disorders (21).

Peripheral myeloid DCs and plasmacytoid DCs show differing chemokine repertoires and peculiar migratory behavior (7). Current evidence suggests that peripheral plasmacytoid DCs express receptors that permit their migration into lymphoid tissues, including L-selectins, CXCR4, CCR7, and α4 integrins (8). However, peripheral plasmacytoid DCs may also locate within inflamed peripheral tissue other than lymphoid sites, where they present antigens and stimulate T cell cytotoxicity (9). We observed that peripheral plasmacytoid DCs overexpress different chemokines as compared with myeloid DCs, including CXCR4, CCR5, and CCR7. However, the mechanisms by which peripheral plasmacytoid DCs migrate toward inflamed tissue are still undefined, because they are unresponsive to the above-mentioned molecules (22). In addition, CCL21 was not expressed within glomeruli of patients with LN, thus confirming its lack of involvement in the recruitment of CCR7-positive plasmacytoid DCs. Therefore, additional chemoattractants might be involved in the migratory machinery of plasmacytoid DCs.

We have studied IL-18R as a potential molecule involved in plasmacytoid DC migration. As reported, myeloid DCs produce IL-18, but only plasmacytoid DCs express the functional receptor and migrate in response to IL-18 stimulation (13). Our results are concordant with those of previous studies and confirm that CD123+,BDCA-2+ plasmacytoid DCs from both patients with SLE and control subjects constitutively express IL-18R, whereas myeloid DCs are negative in all instances. Therefore, circulating plasmacytoid DCs might be prone to stimulation induced by IL-18 production.

Human and experimental models have suggested that LN is a largely Th1-mediated disorder, and that high production of serum IL-18 and IFNγ is related to the severity of renal damage (10). IL-18 is mainly produced by macrophages, although myeloid DCs may contribute to elevated levels of IL-18. It is known that peripheral plasmacytoid DCs induce a prevalent Th2 immune response, but under the stimulation of IL-18 they switch T cells toward a Th1 phenotype through IFNγ and IL-12 overproduction (13). Our results also showed deficient production of serum IL-4 and high levels of IL-18, IFNγ, and IL-12 in patients with LN, although the latter abnormality is apparently unrelated to the severity of renal damage. Thus, it is conceivable that inflammatory cells including macrophages may release increased amounts of IL-18 and stimulate plasmacytoid DCs to promote a skewed Th1:Th2 ratio.

Up-regulation of IL-18 was associated with nephritic kidneys in lupus-prone MRL/lpr mice, with levels increasing in parallel with the severity of the disease (23). In addition, IL-18 exerts chemotactic functions, being also influenced by environmental factors that may amplify its inflammatory properties. Finally, IL-18 attracts IL-18R–positive cells such as plasmacytoid DCs and T cells. Thus, we hypothesize that high levels of glomerular IL-18 produced mainly by resident macrophages may recruit plasmacytoid DCs from peripheral blood, leading to subsequent glomerular accumulation.

Our findings emphasize that IL-18 is produced within nephritic glomeruli, and that its striking accumulation in the cytoplasm of resident mononuclear cells reflects its local production. As in other inflamed tissue, macrophages are the prevalent producers of the cytokine, and their number indeed paralleled the severity of renal damage. Therefore, persistent production of IL-18 by activated macrophages in LN may represent a persistent chemotactic trigger for the recruitment of inflammatory cells expressing the functional receptor (24).

T cells are the primary effectors of renal damage, while an accumulation of DCs at different stages of maturation has not been demonstrated. Previous studies (9) revealed the migration of plasmacytoid DCs toward peripheral inflamed tissues, including skin, in patients with SLE (25). This event has been correlated with their ability to transmigrate through high endothelial venules (26), independently of both chemokine and integrin expression. We have hypothesized that in LN, peripheral blood plasmacytoid DCs could be recruited within the kidney as an effect of the persistent inflammatory stimulation induced by IL-18, and that their prevalent glomerular accumulation correlates with the number of IL-18–positive cells. In addition, their ability to locate within glomeruli was also supported by the presence of IL-18R that was overexpressed by resident mononuclear cells, whereas functional chemokine expression was unrelated to the presence of the relative ligands within the kidney, as demonstrated for CCR7/CCL21. The expression of IL-18R by resident cells represents, at least in part, indirect support for the occurrence of plasmacytoid DCs in LN.

The primary role of IL-18 in plasmacytoid DC migration is also indicated by the absence within glomeruli of myeloid DCs that fail to express IL-18R. In vivo evidence suggests that IL-18 shapes the development of Th1 responses, and neutralization of IL-18 did not alter the Th1/Th2 phenotype of allogeneic T cells cocultured with myeloid DCs (27, 28). On the contrary, IL-18 induces chemotaxis of plasmacytoid DCs and enhances their allostimulatory capacity, promoting a prevalent Th1 response. Thus, we speculate that IL-18 may play a major role in the recruitment of IL-18R–positive plasmacytoid DCs within the kidney, and that glomerular plasmacytoid DCs may locally amplify the renal damage through long-term activation of T cells and Th1 cytokine production.

It has been reported that IL-18 induces maturative effects on the human KG-1 myelomonocytic cell line, although cultured monocytes are insensitive to IL-18 in vitro. The expression of CD83 by KG-1 cells confirmed its role as a marker for the identification of mature DCs (29), although its prevalent expression by plasmacytoid DCs and myeloid DCs or both is still controversial (30). CD83 defines myeloid DCs in different experimental and human models, although it was recently shown to be overexpressed by plasmacytoid DCs in other autoimmune diseases (21, 31). Thus, the presence of glomerular CD83-positive cells in severe LN in parallel with BDCA-2–positive plasmacytoid DCs in the absence of myeloid DCs suggested that CD83-positive DCs may be prevalently expressed by mature plasmacytoid DCs that undergo definitive maturation within glomeruli or by already mature DCs that migrate to the kidney in response to triggers of inflammation. IL-18R activation might not be the unique mechanism through which plasmacytoid DCs traffic inside the kidney. Indeed, environmental factors and the activation of adenosine in conjunction with chemerin overexpression may be involved in their activation and migration (12). Thus, the presence of renal plasmacytoid DCs may reflect their defect in the periphery in patients with SLE and supports the high glomerular levels of IL-18 as a primary chemotactic stimulus leading to glomerular accumulation and renal damage in active LN.

AUTHOR CONTRIBUTIONS

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES

Dr. Silvestris had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

Study design. Tucci, Silvestris.

Acquisition of data. Quatraro, Lombardi.

Analysis and interpretation of data. Tucci.

Manuscript preparation. Tucci, Dammacco, Silvestris.

Statistical analysis. Tucci, Pellegrino.

Acknowledgements

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES

We are grateful to Dr. G. Pannarale (Department of Nephrology, University of Bari) for providing the biopsy specimens.

REFERENCES

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. Acknowledgements
  8. REFERENCES