Helsinki University Central Hospital, Department of Medicine, Division of Nephrology Helsinki, Finland
Correspondence and offprint requests to: Carola Grönhagen-Riska, HUCH, PB 341, 00029 HUCH, Finland. Email: carola.gronhagen-riska{at}hus.fi
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Abstract |
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Methods. Amounts of IL-1ß and IL-1ra excreted in 24-h urine samples collected from 241 IgAN, 26 HSN patients and from 33 healthy controls were determined. Results were expressed as cytokine/creatinine (ng/mmol) ratios.
Results. Urinary IL-1ß excretion by the IgAN and HSN patients was no greater than urinary IL-1ß excretion by healthy controls. Urinary IL-1ra excretion by the IgAN patients was lower than urinary IL-1ra excretion by healthy controls (P < 0.05) and by the HSN patients (P < 0.01). In both patients and controls women had significantly higher IL-1ra, IL-1ß excretion levels and IL-1ra/IL-1ß ratios. The differences in urinary excretions of IL-1ra by the healthy controls and by the IgAN and HSN patients were significant in both sexes. Excretion of IL-1ß or IL-1ra did not correlate with excretion of urinary protein, duration of the disease or any histopathological variable. However, histopathological changes in renal biopsy specimens from patients with IL-1ra/IL-1ß ratios above normal were significantly milder than in renal biopsy specimens from patients with low or normal IL-1ra/IL-1ß ratios.
Conclusion. Urinary IL-1ra levels in IgAN patients were lower than urinary IL-1ra levels in healthy controls or HSN patients, a finding which may indicate that the two diseases have a different pathogenesis. Whether the male predominance in IgAN and HSN and the worse outcomes in males that have been reported previously in IgAN and HSN are connected with the lower excretion of IL-1ra and consequently lower IL-1ra/IL-1ß ratios in male patients than in female patients needs more thorough investigation.
Keywords: female/male; HenochSchönlein nephritis; IgA nephropathy; interleukin-1ß; interleukin-1 receptor antagonist
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Introduction |
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Interleukin-1 (IL-1) is a pro-inflammatory cytokine produced by activated monocytes/macrophages. It has been shown that IL-1 is involved in mesangial cell proliferation and in extracellular matrix production, crescent formation, endothelial cell activation, promoting intravascular coagulation and leucocyte adhesion [2]. Evidence suggests that IL-1 plays a role in the pathogenesis of IgAN and HSN. It has been shown that IL-1 is expressed in the glomeruli of IgAN patients [2,3]. Wu et al. [4] reported that urinary IL-1 activity was higher in IgAN and HSN patients than in others.
IL-1 receptor antagonist (IL-1ra) inhibits IL-1 by competing for receptor binding. Binding of IL-1ra to cell receptors, unlike the binding of IL-1 to cell receptors, does not result in any observable intracellular response [5]. In many inflammatory conditions, both in man and animals, it has been shown that high concentrations of endogenous IL-1ra or administrations of IL-1ra reduce recovery times or mortality [5].
There is evidence that the ratio of IL-1 to IL-1ra is important in the regulation of inflammatory responses and high urinary IL-1ra/IL-1ß ratios have been found to be associated with good outcomes in renal-transplant recipients [6].
We studied urinary concentrations of IL-1ß and IL-1ra in adults with IgAN and HSN, and in healthy control individuals. We also determined whether there were associations between urinary concentrations of IL-1ß and IL-1ra and degrees of histopathological damage and various clinical factors.
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Subjects and methods |
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There were no statistically significant differences between the groups of IgAN and HSN patients in relation to age, sex distribution, renal function, proteinuria or existence of hypertension. There were no statistically significant differences between the groups of patients with regard to histopathological findings (Table 1). Nine of 26 HSN patients had been exhibiting extrarenal symptoms when the 24-h urinary sample had been collected.
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Urine and serum samples, and histopathological evaluation
IL-1ß and IL-1ra assays. Twenty-four-hour urinary samples were collected and samples were stored at 20°C until analysis. Urinary IL-1ra levels were determined by an enzyme immunoassay, IL-1ß levels by means of radioimmunoassay, as described in detail elsewhere [6,7].
The limit of detection of the assay method for IL-1ra was 20 ng/l and for IL-1ß 5 ng/l. Concentrations below the limits of detection were recorded as equal to the limit of detection. IL-1ra/IL-1ß ratios were calculated. IL-1ra and IL-1ß excretion levels were related to concomitant urinary creatinine (Ucr) excretion levels.
Excretion levels were determined for the healthy control individuals. The 2.5th percentile of the levels for these control individuals was defined as the lower limit of normal and the 97.5th percentile was defined as the upper limit of normal [8].
Other laboratory analyses. Serum creatinine and albumin levels, microscopy of centrifuged urine specimen, and 24-h levels of excretion of protein, albumin and creatinine in the urine, were determined using routine methods. Creatinine clearance (Ccr) was calculated from serum creatinine levels using the CockcroftGault formula.
Histopathological evaluation. All biopsy samples were re-examined without any knowledge of patient histories. All 56 specimens contained at least five glomeruli. Mesangial cell proliferation and matrix increase, tubular atrophy, interstitial fibrosis, interstitial inflammatory cell infiltration and hyaline arteriolosclerosis were graded semi-quantitatively from 0 to 3. Percentages of glomeruli with necrosis and/or crescents were calculated. Sums of grades relating to mesangial cell proliferation and matrix increase were defined as glomerular score, sums of grades relating to tubular atrophy, interstitial fibrosis and interstitial cell infiltration as tubulointerstitial score and sums of glomerular and tubulointerstitial scores and grade of arteriolosclerosis as total histopathological score.
Statistical analyses
Values are expressed as medians (ranges). Means ± SEM are given when appropriate. The significances of differences between continuous variables relating to two groups were calculated using the MannWhitney U-test and the significances of differences in distributions of binary variables between groups were calculated using Pearsons 2 test. Correlations between variables were assessed using Spearmans correlation test (continuous variables) or Pearsons
2 test (categorical variables). Linear regression was used to assess relationships between categorical and continuous variables. Values of P < 0.05 were considered to indicate statistical significance in all cases [8].
All the statistical analyses were performed using SPSS 9.0 for Windows.
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Results |
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Neither urinary IL-1ra nor IL-1ß levels correlated significantly with degree of proteinuria (r = 0.038, P = NS and r = 0.023, P = NS, respectively), numbers of urinary erythrocytes (r = 0.039, P = NS and r = 0.056, P = NS), time since diagnostic renal biopsy (r = -0.032, P = NS and r = 0.090, P = NS) or age of the patient (r = 0.102, P = NS and r = 0.037, P = NS). No histopathological variable correlated with IL-1ra or IL-1ß excretion (data not shown).
Excretion of IL-1ra and IL-1ß, and IL-1ra/IL-1ß ratios were significantly higher in women than in men, in both groups of patients and in the controls. Levels of IL-1ra and IL-1ß excretion were, therefore, also investigated separately in women and men, in both groups of patients and in the controls. Results are shown in Figure 2.
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Discussion |
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IL-1ra and IL-1 are produced by monocytes/macrophages and polymorphonuclear cells in response to an identical stimulus. IL-1ra specifically inhibits IL-1 activity by competing for receptor binding, and is one of the most powerful endogenous anti-inflammatory agents. IL-1ra has been found to suppress IgAN in mice [15]. IL-1ra has also been used effectively in the treatment of animal models of antibody-mediated glomerulonephritis [16].
The balance between IL-1 and IL-1ra is believed to be important in the regulation of inflammatory responses. For example, the urinary IL-1ra/IL-1ß ratio has been found to be lower in recipients of renal transplants who showed signs of rejection than in those who did not [6].
Results of studies on IL-1ra gene polymorphism have also demonstrated the importance of IL-1ra in HSN and IgAN. An increased rate of IL1RN*2 carriage has recently been found in IgAN patients with macroscopic haematuria and in HSN patients. It was suggested that IL1RN*2 constitutes a genetic link between IgAN and HSN [17]. In another study the long-term survival rate among IgAN patients was shown to be lowest in those carrying IL1RN*2 [18]. The results of these studies did not demonstrate what role, if any, IL1RN*2 plays in relation to IL-1ra levels in serum or urine.
In the groups of IgAN and HSN patients and normal subjects we studied, we found no statistically significant differences in relation to any clinical or histopathological feature between IgAN and HSN patients, except in the history of extrarenal manifestations. The observed differences in the urinary excretion of IL-1ra may, therefore, be of importance in relation to the pathogenetic mechanisms of the two diseases.
In the study reported here, IL-1ß and IL-1ra were detectable in both healthy subjects and patients with IgAN and HSN. No statistically significant differences between the groups in relation to urinary excretions of IL-1ß were found. However, excretion of amounts of IL-1ß above the normal range occurred only in the IgAN patients.
The results of the study reported show that IgAN patients excreted less IL-1ra than HSN patients and healthy control individuals. HSN is a leukocytoclastic form of vasculitis with perivascular accumulation of inflammatory cells, mostly polymorphonuclear leukocytes and mononuclear cells. This may be why IL-1ra production was higher in HSN patients than in IgAN patients. It could also be that concentrations of other cytokines, such as IL-4, TGF-ß, IL-10, contributing to production of IL-1 and IL-1ra differed between the patients with HSN and IgAN. On the basis of results of studies on IL-1ra polymorphism our findings may have reflected greater carriage of IL1RN*2 by the HSN patients than by the IgAN patients, i.e. the differences between the diseases may be genetically determined.
Levels of IL-1ß and IL-1ra did not correlate with histopathological findings. This observation is in accordance with previous findings that IL-1ß did not correlate with the degree of mesangial proliferation in IgAN patients [13] and that cells expressing IL-1ß were observed in interstitium [2]. However, patients with IL-1ra/IL-1ß ratios above the normal range exhibited significantly milder interstitial changes, less severe increase of mesangial matrix and lower histopathological scores than other patients.
We found that levels of urinary excretion of IL-1ß and IL-1ra were significantly higher in women than in men. Lynch et al. [19] found no differences between the sexes in relation to excretion of IL-1ß by healthy individuals but their findings relating to IL-1ra were similar to ours.
As women are more susceptible to a number of autoimmune diseases, e.g. SLE and rheumatoid arthritis, attention has increasingly been paid to the effects of sex hormones on the functioning of immune cells and cytokine production. It is known that serum levels of cytokines are affected by sex hormones, but that response differs between organs [20]. No differences have been observed between men and women in relation to plasma IL-1ß immunoreactivity, but plasma IL-1ß binding capacity is higher in men than in women. Soluble IL-1 receptor II (IL-1RII) concentrations are highest in men, lowest in women in luteal phase [21]. Soluble IL-1RII can bind secreted IL-1 before it reaches target cells, preventing activating response and acting as a natural inhibitor of IL-1-induced inflammation [22]. Some 65% of patients with IgAN and HSN are male. Whether the male predominance in IgAN and HSN and the poorer outcomes in men than in women that have been reported previously are associated with the lower excretion of IL-1ra by men than by women, and consequential lower IL-1ra/IL-1ß ratios in men than women, requires more thorough investigation. It is also not yet known whether there are differences between the sexes in relation to other cytokines/growth factors in other inflammatory diseases.
In conclusion, we found that IL-1ra excretion in the urine of IgAN patients was lower than IL-1ra excretion in the urine of HSN patients and healthy individuals. Histopathological changes in renal biopsy specimens from patients with high IL-1ra/IL-1ß ratios were milder than histopathological changes in renal biopsy specimens from patients with low/normal IL-1ra/IL-1ß ratios. We also found that levels of excretion of IL-1ß and IL-1ra were significantly higher in women than in men.
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Acknowledgments |
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Conflict of interest statement. None declared.
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References |
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