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Background Several tumour necrosis factor (TNF) based therapeutics have already been approved for human use and several others are emerging. Therefore, we determined the mRNA expression levels of the TNF superfamily ligands (TNFSF) – e.g. TNF-α, lymphotoxin (LT)-α, LT-β, Fas-L (CD95-L), TNF-related apoptosis-inducing ligand (TRAIL), TNF-related weak inducer of apoptosis (TWEAK), 4-1BBL, OX40-L (CD252) and amyloid precursor protein (APP) in healthy human and mouse solid organs. Methods We used quantitative real time-PCR to analyse mRNA expression levels of TNFSF ligands. Murine models of acute ischemic renal injury, chronic oxalate nephropathy, and immune complex glomerulonephritis were used. Renal injury was assessed by PAS staining, and infiltrating immune cells were analysed by immunohistochemistry. Data was analysed using non-parametric ANOVA (non-parametric; Kruskal-Wallis test). Results We observed significant differences in the mRNA expression levels of TNFSF ligands in human and mouse solid organs. Furthermore, we determined their mRNA expressions during acute and chronic kidney injuries in mice. Our data demonstrate that the mRNA expression levels of TNFSF vary depending on the type of tissue injury – for example, acute ischemic renal injury, chronic crystalline nephropathy, and immune complex glomerulonephritis. In addition, we observed that mRNA expressions of TNFSF ligands are differentially regulated during the course of a transient ischemic renal injury (IRI) and chronic kidney modelling. We observed that TNF-α, LT-β, and 4-1BBL were significantly upregulated during the progression of IRI and crystal-induced chronic kidney disease (CKD), whereas only 4-1BBL and TNF-α were significantly upregulated and LT-β was significantly downregulated during the progression of immune complex glomerulonephritis. The mRNA expression of Fas-L was higher during IRI whereas it decreased in a time dependent manner during the progression of crystal-induced CKD. Conclusion We conclude that the injury- and species-specific differences of TNFSF ligands must be considered in order to avoid the misinterpretation and wrong conclusions during data extrapolation between species.

Tumor necrosis factor-α (TNF) is a cytokine mediating inflammatory kidney diseases such as immune complex glomerulonephritis. Its two receptors, TNFR1 and TNFR2, play distinct roles in this process, with TNFR2 strongly required for induction of disease. In contrast to soluble TNF (sTNF), transmembrane TNF robustly activates TNFR2. Thus, we examined the functional role of transmembrane TNF by inducing heterologous nephrotoxic serum nephritis in wild-type and transgenic TNFΔ1-9,K11E knock-in mice expressing transmembrane TNF but no sTNF (memTNF mice). Compared to wild-type, nephritis was exacerbated in memTNF mice on day 5, indicated by increased albuminuria, higher serum urea levels, and more pronounced glomerular deposits, together with higher numbers of dying and proliferating glomerular cells. This was associated with greater loss of glomerular endothelial cells, increased podocyte stress, and signs of augmented necroptosis in memTNF kidneys. Aggravation of nephritis was dependent on transmembrane TNF expression in parenchymal cells, but not leukocytes. Surprisingly, increased kidney injury was associated with reduced renal leukocyte infiltration in memTNF mice, which correlated with decreased renal mRNA expression of pro-inflammatory mediators. This effect was also present in isolated memTNF glomeruli stimulated with interleukin-1β in vitro. Thus, uncleaved transmembrane TNF is an important mediator of renal tissue damage characterized by increased renal cell death and loss of glomerular endothelial cells in murine glomerulonephritis. In contrast, sTNF predominantly mediates renal leukocyte recruitment and inflammation. These findings highlight the importance of transmembrane TNF in inflammatory kidney disease as a possible therapeutic target.

BACKGROUND: The outcomes of immunosuppressive therapy, when added to supportive care, in patients with IgA nephropathy are uncertain. METHODS: We conducted a multicenter, open-label, randomized, controlled trial with a two-group, parallel, group-sequential design. During a 6-month run-in phase, supportive care (in particular, blockade of the renin-angiotensin system) was adjusted on the basis of proteinuria. Patients who had persistent proteinuria with urinary protein excretion of at least 0.75 g per day were randomly assigned to receive supportive care alone (supportive-care group) or supportive care plus immunosuppressive therapy (immunosuppression group) for 3 years. The primary end points in hierarchical order were full clinical remission at the end of the trial (protein-to-creatinine ratio

Recent findings suggest that autoimmune disorders predispose to a diminished capacity to taste and smell. This has been shown for patients with systemic lupus erythematosus as well as for patients with rheumatoid arthritis (RA). Granulomatosis with polyangiitis (GPA), with its particular manifestations in the upper respiratory tract, may therefore have an even higher impact on these senses. The aims of this study were to evaluate the gustatory and olfactory function in patients with GPA, to compare them to sex- and age-matched healthy controls, and to correlate these findings with their GPA disease severity.Patients with established GPA were analysed by standardized assessments for gustatory and olfactory functions and examined for disease activity, stage of disease, and treatment.Forty-four GPA patients were tested for their chemosensory functions. Compared to age- and sex-matched healthy controls, GPA patients showed significantly decreased olfactory scores along with diminished scores for their gustatory functions. The diminished sense of smell in GPA patients correlated significantly with elevated C-reactive protein (CRP) values whereas the gustatory impairment correlated with the duration and extent of the disease.Our results indicate that olfactory and gustatory functions are significantly decreased in GPA. As the olfactory function of these patients was comparable to patients with RA, chemosensory impairment may not simply be a consequence of the involvement of the upper respiratory tract, but rather a common complication of systemic autoimmune diseases.

Renal dendritic cells (DCs) form an interstitial network contributing to inflammatory and adaptive immune responses in the kidney. The presence and functional role of DC-like glomerular CD11c(+) mononuclear phagocytes is a matter of debate. Using compartment-specific flow cytometry we found that healthy mouse kidneys contained 1.3 CD11c(+) cells per 100 glomeruli and these increased by 4.6-fold and 13-fold after TNF stimulation and immune complex deposition, respectively. Compartment-specific mRNA expression revealed a predominantly glomerular expression of TNF receptors, chemokines, and adhesion molecules; all upregulated after TNF exposure. Intraperitoneal TNF injection induced influx of neutrophils and mononuclear phagocytes including DC-like CD11c(+) cells into both the glomerular and tubulointerstitial compartments, but reduced in TNF receptor (Tnfr) 1-deficient mice. Additionally, Tnfr2 deficiency impaired glomerular infiltration of CD11c(+) cells, but not neutrophils. Interstitial CD11c(+) cells infiltrated in the presence of Tnfr1 or Tnfr2. TNF exposure also induced similar maturation of glomerular and interstitial CD11c(+) cells as demonstrated by increased surface expression of MHC II, CD54, and costimulatory molecules CD40, CD80, and CD86. Thus, by compartment-specific flow cytometry we could demonstrate the constitutive presence of DC-like CD11c(+) mononuclear phagocytes in normal mouse glomeruli and their TNF-induced accumulation and activation.

Macrophages convert proinflammatory or anti-inflammatory signals of tissue microenvironments into response mechanisms. These response mechanisms largely derive from evolutionary conserved defense programs of innate host defense, wound healing, and tissue homeostasis. Hence, in many settings these programs lead to renal inflammation and tissue remodeling (ie, glomerulonephritis and sclerosis or interstitial nephritis and fibrosis). There is abundant experimental evidence that blocking macrophage recruitment or macrophage activation can ameliorate renal inflammation and fibrosis. In this review we discuss experimental tools to target renal macrophage recruitment by using antagonists against selectins, chemokines, integrins, or other important cytokines that mediate renal injury via macrophage recruitment, some of these already having been used in clinical trials.

Tumor necrosis factor (TNF) alpha is a potent proinflammatory cytokine and important mediator of inflammatory tissue damage. In addition, it has important immune-regulatory functions. Many experimental studies and clinical observations support a role for TNF in the pathogenesis of acute and chronic renal disease. However, given its dual functions in inflammation and immune regulation, TNF may mediate both proinflammatory as well as immunosuppressive effects, particularly in chronic kidney diseases and systemic autoimmunity. Blockade of TNF in human rheumatoid arthritis or Crohn's disease led to the development of autoantibodies, lupus-like syndrome, and glomerulonephritis in some patients. These data raise concern about using TNF-blocking therapies in renal disease because the kidney may be especially vulnerable to the manifestation of autoimmune processes. Interestingly, recent experimental evidence suggests distinct roles for the 2 TNF receptors in mediating local inflammatory injury in the kidney and systemic immune-regulatory functions. In this review the biologic properties of TNF and its receptors, TNF receptors 1 and 2, relevant to kidney disease are summarized followed by a review of the available experimental and clinical data on the pathogenic role of the TNF system in nonimmune and immune renal diseases. Experimental evidence also is reviewed that supports a rationale for specifically blocking TNF receptor 2 versus anti-TNF therapies in some nephropathies, including immune complex-mediated glomerulonephritis.

Recruitment of leukocytes is a characteristic feature of tissue injury in systemic lupus erythematosus, including lupus nephritis. Locally secreted chemokines and their receptors are important mediators of leukocyte recruitment to the specific sites of immune complex injury, and contribute to renal inflammatory disease in the initiation and progression phase. Therefore, chemokines and chemokine receptors represent potential therapeutic targets in lupus nephritis. In this review we summarize available experimental and human data supporting their functional role in lupus nephritis. Moreover, interventional studies with chemokine and chemokine receptor antagonists that show the therapeutic potential of chemokine antagonists in experimental models of lupus nephritis and potentially in human renal disease are discussed.

Recently, divergent reports on the role of mast cells (MC) in different glomerular diseases have brought our attention to their role in an accelerated model of anti-glomerular basement membrane (GBM) glomerulonephritis (GN). Genetically MC-deficient Kit(W)/Kit(W-v) mice, MC-reconstituted Kit(W)/Kit(W-v) mice and Kit+/+ control mice were subjected to anti-GBM GN. Kit(+/+) mice developed moderate proteinuria and glomerular damage following the induction of anti-GBM nephritis. In contrast, proteinuria and glomerular damage were dramatically increased in MC-deficient Kit(W)/Kit(W-v) mice. MC-reconstituted Kit(W)/Kit(W-v) mice showed proteinuria and glomerular damage comparable to Kit+/+ mice. A significant increase in infiltrating T cells and macrophages was detected in MC-deficient Kit(W)/Kit(W-v) mice as compared to Kit+/+ control mice and MC-reconstituted Kit(W)/Kit(W-v) mice. Accordingly, we observed an increase of TGF-beta1 mRNA in kidneys from Kit(W)/Kit(W-v) mice. Interestingly, we did not detect MC in the kidney using either Giemsa staining or RT-real-time PCR, but MC were found in the regional lymph nodes. Finally, mortality of Kit(W)/Kit(W-v) mice was significantly increased after the induction of anti-GBM GN due to uremia. Our report provides the first direct evidence that MC are protective in anti-GBM GN, possibly by modulating the influx of effector T cells and macrophages to inflammatory sites in the kidney.

Many forms of glomerulonephritis lead to progressive renal disease and end-stage renal failure. Infiltrating leukocytes are key effectors that cause glomerular injury. Locally secreted chemokines mediate glomerular leukocyte recruitment, effector functions, and the subsequent glomerular damage during the initiation and progression phases of glomerulonephritis. In vivo studies demonstrated that chemokine blockade is an effective anti-inflammatory strategy in glomerulonephritis. However, chemokine antagonism might also alter innate and adaptive immune responses and exacerbate disease under certain conditions.

Chemokines, in concert with cytokines and adhesion molecules, play multiple roles in local and systemic immune responses. In the kidney, the temporal and spatial expression of chemokines correlates with local renal damage and accumulation of chemokine receptor-bearing leukocytes. Chemokines play important roles in leukocyte trafficking and blocking chemokines can effectively reduce renal leukocyte recruitment and subsequent renal damage. However, recent data indicate that blocking chemokine or chemokine receptor activity in renal disease may also exacerbate renal inflammation under certain conditions. An increasing amount of data indicates additional roles of chemokines in the regulation of innate and adaptive immune responses, which may adversively affect the outcome of interventional studies. This review summarizes available in vivo studies on the blockade of chemokines and chemokine receptors in kidney diseases, with a special focus on the therapeutic potential of anti-chemokine strategies, including potential side effects, in renal disease.

Chemokines and chemokine receptors are involved in the resolution or progression of renal disease. Locally secreted chemokines mediate leukocyte recruitment during the initiation and amplification phase of renal inflammation. In turn, the infiltrating leukocytes contribute to renal damage by releasing inflammatory and profibrotic factors. Rapid down modulation of the chemokine signal will support resolution of acute inflammation, whereas progression occurs if ongoing or repeated renal injury maintains continuous local chemokine secretion and leukocyte influx into the glomerulus or the interstitial space. In glomerular injury proteinuria itself as well as glomerular secreted cytokines stimulate downstream tubular epithelial cells to also secrete chemokines. During primary tubular injury, tubular epithelial cells directly become a major site of chemokine production. This in turn supports leukocyte infiltration and activation. Infiltrating leukocytes stimulate fibroblast proliferation and matrix synthesis, leading to widening of the interstitial space. The specific and intricate renal vascular architecture renders the organ susceptible to ischemic damage as interstitial volume increases. Ischemia in turn serves as a stimulus for chemokine and cytokine production and matrix synthesis. The mutual stimulation between fibroblasts and infiltrating leukocytes supports progressive tubular damage, renal fibrosis, and glomerulosclerosis. Potentially this vicious circle leading to progression of chronic nephropathies offers the opportunity for therapeutic intervention. Interfering with the chemokine network that mediates leukocyte recruitment may represent a promising therapeutic option for progressive renal disorders and renal fibrosis. This article summarizes the present data on the role of chemokines in acute and chronic renal disease with special emphasis on their potential role in mediating resolution or progression of renal disease as well as on therapeutic options.

In hepatitis C virus (HCV) infection, morbidity and mortality often result from extrahepatic disease manifestations. We provide evidence for a role of receptors of the innate immune system in virally induced inflammation of the endothelium in vitro and in vivo. Corresponding to the in vitro finding of an HCV-dependent induction of proinflammatory mediators in endothelial cells, mice treated with poly (I:C) exhibit a significant reduction in leukocyte rolling velocity, an increase in leukocyte adhesion to the vessel wall and an increased extravasation of leukocytes. HCV directly promotes activation, adhesion and infiltration of inflammatory cells into the vessel wall by activation of endothelial viral receptors. Poly (I:C) induces the expression of TLR3 in vivo and hereby allows for amplification of all of the aforementioned responses upon viral infection. Proinflammatory effects of viral RNA are specifically mediated by TLR3 and significantly enhanced by tumor necrosis factor alpha (TNFα). HCV-RNA induces the endothelial expression of TNFα and TNFα receptor subtype 2 and we provide evidence that leucocyte adhesion and transmigration in response to activation of viral RNA receptors seem to depend on expression of functional TNFR2. Our results demonstrate that endothelial cells actively participate in immune mediated vascular inflammation caused by viral infections.

The nephrotoxic serum nephritis (NTN) model is an integral part of experimental glomerulonephritis (GN) research. Here, we discuss how the murine NTN model can be induced and effectively used to mimic an immune complex-mediated GN. Further, we differentiate between heterologous and autologous models by comparing pathophysiology and phenotypic manifestations.

Cytokines of the TNF superfamily, particularly TNF itself, are important mediators of inflammatory leukocyte recruitment and activation in parenchymal organs. In inflammatory kidney diseases, leukocytes accumulate in glomeruli and the tubulointerstitium, leading to glomerulonephritis and tubulointerstitial nephritis, respectively. In particular, glomeruli can be the target of organ-threatening leukocyte-mediated inflammation. As microvasculatures of the glomerulus and the tubulointerstitium differ markedly in their structural and functional properties, recruitment and subsequent activation of leukocytes to these sites occur via distinct mechanisms. To understand the pathways and mediators of leukocyte-driven inflammation in the kidney it is therefore essential to analyze glomerular and tubulointerstitial leukocyte recruitment in a compartment-specific way. The protocol presented here describes an easy and rapid technique that allows compartment-specific quantitation and qualitative analysis of leukocytes present in glomeruli and tubulointerstitial tissue by flow cytometry after separation of these tissue compartments.

The chemokine receptors CCR2 and CCR5 play important roles in the recruitment of monocytes/macrophages and T cells. To better understand the role of both receptors in murine models of inflammatory diseases and to recognize potential problems when correlating these data to humans, we have generated mAbs against murine CCR2 and CCR5. In mice CCR2 is homogeneously expressed on monocytes and on 2–15% of T cells, closely resembling the expression pattern in humans. In contrast to humans, murine NK cells are highly CCR5 positive. In addition, CCR5 is expressed on 3–10% of CD4 and 10–40% of CD8-positive T cells and is weakly detectable on monocytes. Using a model of immune complex nephritis, we examined the effects of inflammation on chemokine receptor expression and found a 10-fold enrichment of CCR5+ and CCR2+ T cells in the inflamed kidneys. The activity of various chemokines and the antagonistic properties of the mAbs were measured by ligand-induced internalization of CCR2 and CCR5 on primary leukocytes. The Ab MC-21 (anti-CCR2) reduced the activity of murine monocyte chemotactic protein 1 by 95%, whereas the Ab MC-68 (anti-CCR5) blocked over 99% of the macrophage-inflammatory protein 1α and RANTES activity. MC-21 and MC-68 efficiently blocked the ligand binding to CCR2 and CCR5 with an IC50 of 0.09 and 0.6–1.0 μg/ml, respectively. In good correlation to these in vitro data, MC-21 almost completely prevented the influx of monocytes in thioglycollate-induced peritonitis. Therefore, both Abs appear as useful reagents to further study the role of CCR2 and CCR5 in murine disease models.

Interleukin (IL)-1β contributes to renal injury in immune complex glomerulonephritis. However, production of mature IL-1β depends on activation of the inflammasome that cleaves pro-IL-1β into its secretable form. A functional role of the NLRP3-containing inflammasome, which responds to various endogenous danger signals, was found in tubulointerstitial nephropathies, but its function in glomerular disease has not been established. To determine whether NLRP3 and its adapter molecule ASC contribute to glomerulonephritis, we induced T-cell-dependent autologous nephrotoxic serum nephritis in Nlrp3- and Asc-deficient mice. Renal expression of NLRP3/ASC inflammasome components and pro-IL-1β increased during nephrotoxic serum nephritis and was abundant in renal dendritic cells. This was associated with renal production of mature IL-1β, indicating inflammasome activation. Nlrp3 and Asc deficiency significantly attenuated glomerular injury, renal leukocyte infiltration, and T-cell activation. Production of mature IL-1β was abrogated in Asc-deficient mice, consistent with a loss of inflammasome-dependent IL-1β activation. Surprisingly, renal IL-1β secretion remained intact in Nlrp3-deficient mice, indicating noncanonical pro-inflammatory effects of NLRP3 in autologous nephrotoxic serum nephritis. This may include NLRP3-mediated glomerular release of pro-inflammatory high-mobility group box 1 protein as a noncanonical function of NLRP3/ASC in glomerulonephritis. Thus, therapeutic blockade of the NLRP3/ASC/IL-1β axis may be beneficial in glomerulonephritis.

TNF is an important mediator of glomerulonephritis. The two TNF-receptors TNFR1 and TNFR2 contribute differently to glomerular inflammation in vivo, but specific mechanisms of TNFR-mediated inflammatory responses in glomeruli are unknown. We investigated their expression and function in murine kidneys, isolated glomeruli ex vivo, and glomerular cells in vitro. In normal kidney TNFR1 and TNFR2 were preferentially expressed in glomeruli. Expression of both TNFRs and TNF-induced upregulation of TNFR2 mRNA was confirmed in murine glomerular endothelial and mesangial cell lines. In vivo, TNF exposure rapidly induced glomerular accumulation of leukocytes. To examine TNFR-specific inflammatory responses in intrinsic glomerular cells but not infiltrating leukocytes we performed microarray gene expression profiling on intact glomeruli isolated from wildtype and Tnfr-deficient mice following exposure to soluble TNF ex vivo. Most TNF-induced effects were exclusively mediated by TNFR1, including induced glomerular expression of adhesion molecules, chemokines, complement factors and pro-apoptotic molecules. However, TNFR2 contributed to TNFR1-dependent mRNA expression of inflammatory mediators in glomeruli when exposed to low TNF concentrations. Chemokine secretion was absent in TNF-stimulated Tnfr1-deficient glomeruli, but also significantly decreased in glomeruli lacking TNFR2. In vivo, TNF-induced glomerular leukocyte infiltration was abrogated in Tnfr1-deficient mice, whereas Tnfr2-deficiency decreased mononuclear phagocytes infiltrates, but not neutrophils. These data demonstrate that activation of intrinsic glomerular cells by soluble TNF requires TNFR1, whereas TNFR2 is not essential, but augments TNFR1-dependent effects. Previously described TNFR2-dependent glomerular inflammation may therefore require TNFR2 activation by membrane-bound, but not soluble TNF.

How bacterial or viral infections trigger flares of autoimmunity is poorly understood. As toll-like receptor (TLR)-9 activation by exogenous or endogenous CpG-DNA may contribute to disease activity of systemic lupus erythematosus, we examined the effects of CpG-oligodeoxynucleotides (ODN) or DNA derived from Escherichia coli (E. coli) on the course of nephritis in MRL(lpr/lpr) mice. In kidneys of these mice, TLR9 localized to glomerular, tubulointerstitial, and perivascular infiltrates. After intraperitoneal injection labeled CpG-ODN localized to glomerular and interstitial macrophages and dendritic cells in nephritic kidneys of MRL(lpr/lpr) mice but not in healthy MRL controls. Furthermore, murine J774 macrophages and splenocytes from MRL(lpr/lpr) mice, but not tubular epithelial cells, renal fibroblasts, or mesangial cells, expressed TLR9 and up-regulated CCL5/RANTES mRNA upon stimulation with CpG-ODN in vitro. In vivo both E. coli DNA and CpG-ODN increased serum DNA autoantibodies of the IgG2a isotype in MRL(lpr/lpr) mice. This was associated with progression of mild to crescentic glomerulonephritis, interstitial fibrosis, and heavy proteinuria. CpG-ODN increased renal CCL2/MCP-1 and CCL5/RANTES expression associated with increased glomerular and interstitial leukocyte recruitment. In contrast control GpC-ODN had no effect. We conclude that TLR9 activation triggers disease activity of systemic autoimmunity, for example, lupus nephritis, and that adaptive and innate immune mechanisms contribute to the CpG-DNA-induced progression of lupus nephritis.

Renal co-morbidity is common in patients with rheumatic disease based on regular assessment of serum and urine parameters of renal function. When patients present with both arthritis and renal abnormalities the following questions have to be addressed. Is kidney disease a complication of rheumatic disease or its management, or are they both manifestations of a single systemic autoimmune disease? Is rheumatic disease a complication of kidney disease and its management? How do rheumatic disease and kidney disease affect each other even when they are unrelated? The present review provides an overview of how to address these questions in daily practice.

Objectives To study the role of interleukin 22 (IL-22) in rheumatoid arthritis (RA). Methods IL-22 serum levels were measured in patients with early, treatment-naive RA (n=49) and in 45 age- and sex-matched healthy individuals as controls. Patients were assessed clinically and radiographically at baseline and followed up for 2 years. Correlations of IL-22 serum levels were sought with parameters of disease activity, serological markers, demographic factors and the incidence of erosions. IL-22 production by peripheral blood T cells was investigated by intracellular flow cytometry. Results 24 of 49 patients with RA demonstrated elevated IL-22 levels compared with the range of healthy controls. At baseline, a high percentage of these patients (8/24, 33%) demonstrated bone erosions, whereas only one patient (4%) from the group with normal IL-22 had erosions. During the 2 years of follow-up, six additional patients with increased IL-22 at baseline developed erosions. In contrast, none of the patients in whom IL-22 levels were normal developed erosions despite similar treatment regimens. Multivariate regression analysis accounting for other parameters predictive for erosions, such as the presence of rheumatoid factor or anti-cyclic citrullinated peptide antibodies and disease activity, showed that elevated IL-22 baseline levels were independently and significantly associated with erosive RA. Cellular analysis demonstrated enhanced expression of IL-22 from CD4 T cells in RA. Conclusion IL-22 is elevated in the serum of half of the patients with RA. Elevated serum IL-22 allows discrimination between patients with different radiographic progression and indicates a possible involvement of IL-22 in the pathophysiology of RA.

Chemokines remain attractive therapeutic targets for modulating inflammatory diseases in all areas of medicine including acute and chronic kidney disease. Industry has launched huge programs for the development of chemokine antagonists, and clinical trials with chemokine and chemokine receptor antagonists are ongoing. However, chemokine biology remains an area of unexpected discoveries. Here we discuss a number of questions which need to be addressed to further explore the potential of chemokine antagonism in renal inflammation: Why does renal expression of chemokines and chemokine receptors not always correlate with their functional significance? Why does chemokine antagonism only partially reduce renal leukocyte counts? Will antagonist combinations be more effective in reducing renal inflammation? What are the functional roles of homeostatic chemokines and atypical, nonsignaling chemokine receptors in renal inflammation? And finally, what classes of chemokine antagonists are available to address these questions experimentally?

The monocyte chemoattractant protein CCL2 is crucial for monocyte and T cell recruitment from the vascular to the extravascular compartment at sites of inflammation. CCL2 is expressed in human lupus nephritis and was shown to mediate experimental lupus; therefore, CCL2 antagonists may be beneficial for therapy. This study describes the l-enantiomeric RNA oligonucleotide mNOX-E36, a so-called Spiegelmer that binds murine CCL2 with high affinity and neutralizes its action in vitro and in vivo. The mirror image configuration of the Spiegelmer confers nuclease resistance and thus excellent biostability. mNOX-E36 does not induce type I IFN via Toll-like receptor-7 or cytosolic RNA receptors, as recently shown for certain synthetic D-RNA. Autoimmune-prone MRL(lpr/lpr) mice that were treated with a polyethylene glycol form of mNOX-E36 from weeks 14 to 24 of age showed prolonged survival associated with a robust improvement of lupus nephritis, peribronchial inflammation, and lupus-like inflammatory skin lesions. Thus, mNOX-E36-based inhibition of CCL2 represents a novel strategy for the treatment of autoimmune tissue injury, such as lupus nephritis.

Objectives. Leucocyte infiltration is the hallmark of vasculitis, chemokines being mainly responsible for leucocyte migration into inflamed tissues. The objective was to evaluate the local expression of chemokines and chemokine receptors in biopsies of patients with giant cell arteritis (GCA) compared with arteries from patients with polymyalgia rheumatica (PMR). We studied the expression of CCR5, CXCR3 and that of the Duffy antigen/receptor of chemokine (DARC), a chemokine internalizing receptor (interceptor), in parallel to the expression of the CCR5 ligand RANTES/CCL5. Methods. Paraffin-embedded tissue sections from six patients with GCA and five patients with PMR were available for immunohistological analysis of chemokine receptor expression. RANTES/CCL5 mRNA was detected in tissue sections by in situ hybridization. Results. In patients with biopsy-proven giant cell arteritis, CCR5 and CXCR3 were highly expressed by infiltrating leucocytes in involved tissue sections. Predominant clustering of CCR5 + and CXCR3 + leucocytes was found in the adventitia and was co-localized with the expression of CCL5/RANTES mRNA. Interestingly, we found marked expression of DARC on adventitial high endothelial venules in vasculitis lesions of patients with GCA, while in arteries from patients with PMR DARC was only expressed on a low number of vessels with flat lining endothelium. Conclusions. The co-localization of infiltrating CCR5 + and CXCR3 + leucocytes together with CCL5/RANTES and DARC in vasculitis lesions suggests a role for these chemokine receptors in leucocyte infiltration, possibly supported by DARC-mediated vascular presentation of chemokines.

Immune complex glomerulonephritis (GN) often deteriorates during infection with viruses and bacteria that, in contrast to mammals, have DNA that contains many unmethylated CpG motifs. Balb/c mice with horse apoferritin-induced GN (HAF-GN) were treated with either saline, CpG-oligodeoxynucleotides (ODN), or control GpC-ODN. Only CpG-ODN exacerbated HAF-GN with an increase of glomerular macrophages, which was associated with massive albuminuria and increased renal MCP-1/CCL2, RANTES/CCL5, CCR1, CCR2, and CCR5 mRNA expression. CpG-ODN induced a Th1 response as indicated by serum anti-HAF IgG(2a) titers, mesangial IgG(2a) deposits, and splenocyte IFN-gamma secretion. Messenger RNA for the CpG-DNA receptor Toll-like reeptor 9 (TLR9) was present in kidneys with HAF-GN but not in normal kidneys. The source of TLR9 mRNA in HAF-GN could be infiltrating macrophages or intrinsic renal cells, e.g., mesangial cells; but, in vitro, only murine J774 macrophages expressed TLR9. In J774 cells, CpG-ODN induced the chemokines MCP-1/CCL2 and RANTES/CCL5 and the chemokine receptors CCR1 and CCR5. It is concluded that CpG-DNA can aggravate preexisting GN via a shift toward a Th1 response but also by a novel pathway involving TLR9-mediated chemokine and chemokine receptor expression by macrophages, which may contribute to the enhanced glomerular macrophage recruitment and activation. This mechanism may be relevant during infection-triggered exacerbation of human immune-complex GN and other immune-mediated diseases in general.

Lupus nephritis is characterized by immune complex deposition and inflammatory cell infiltration. Therefore, the initiation and progression of lupus nephritis in MRL/MpJ Fas(lpr/lpr) (MRL/lpr) mice were investigated, with a focus on the expression of several chemokines and chemokine receptors. Mice were monitored for proteinuria from 6 to 20 wk of age, and kidneys were examined every 2 wk by light microscopy, electron microscopy, and immunohistologic analyses. Furthermore, the expression of chemokines, chemokine receptors, and proinflammatory cytokines was analyzed in ribonuclease protection assays. MRL/lpr mice demonstrated increased expression of monocyte chemoattractant protein-1, regulated upon activation, normal T cell-expressed and -secreted protein, inducible protein of 10 kD, and macrophage inflammatory protein-1beta at week 8. At that time point, levels of circulating and glomerular immune complexes were increased, and no proteinuria or histopathologic signs of renal damage could be observed. As assessed in immunohistochemical and in situ hybridization analyses, monocyte chemoattractant protein-1 and regulated upon activation, normal T cell-expressed and -secreted protein expression was preferentially located in the glomeruli and interstitium. Mononuclear cell infiltration of the kidney was observed by weeks 10 to 12. At week 12, the renal expression of chemokine receptor 1 (CCR1), CCR2, and CCR5 was increased, mice became proteinuric, and renal damage was histologically evident. Finally, the expression of proinflammatory cytokines was detected (weeks 12 to 14). In summary, (1) chemokines are upregulated before inflammatory cell infiltration, proteinuria, and kidney damage are observed; (2) chemokine generation is restricted to sites of subsequent inflammatory cell infiltration, i.e., glomeruli and interstitium; (3) chemokine receptor expression parallels mononuclear cell infiltration; and (4) proinflammatory cytokines are upregulated later, in parallel with inflammatory cell infiltration and the onset of proteinuria. These results support the hypothesis that chemokines initiate leukocyte infiltration and precede proteinuria and renal damage in MRL/lpr mice.

Introduction Elevated serum levels of the proinflammatory cytokine tumor necrosis factor alpha (TNFα) correlate with an increased risk for atherothrombotic events and TNFα is known to induce prothrombotic molecules in endothelial cells. Based on the preexisting evidence for the impact of TNFα in the pathogenesis of autoimmune disorders and their known association with an acquired hypercoagulability, we investigated the effects of TNFα and the role of the TNF receptor subtypes TNFR1 and TNFR2 for arteriolar thrombosis in vivo. Methods Arteriolar thrombosis and platelet-rolling in vivo were investigated in wildtype, TNFR1-/-, TNFR2-/- and TNFR1-/R2-/- C57BL/6 mice using intravital microscopy in the dorsal skinfold chamber microcirculation model. In vitro, expression of prothrombotic molecules was assessed in human endothelial cells by real-time PCR and flow cytometry. Results In wildtype mice, stimulation with TNFα significantly accelerated thrombotic vessel occlusion in vivo upon ferric chloride injury. Arteriolar thrombosis was much more pronounced in TNFR1-/- animals, where TNFα additionally led to increased platelet-endothelium-interaction. TNFα dependent prothrombotic effects were not observed in TNFR2-/- and TNFR1-/R2- mice. In vitro, stimulation of human platelet rich plasma with TNFα did not influence aggregation properties. In human endothelial cells, TNFα induced superoxide production, p-selectin, tissue factor and PAI-1, and suppressed thrombomodulin, resulting in an accelerated endothelial dependent blood clotting in vitro. Additionally, TNFα caused the release of soluble mediators by endothelial cells which induced prothrombotic and suppressed anticoagulant genes comparable to direct TNFα effects. Conclusions TNFα accelerates thrombus formation in an in vivo model of arteriolar thrombosis. Its prothrombotic effects in vivo require TNFR2 and are partly compensated by TNFR1. In vitro studies indicate endothelial mechanisms to be responsible for prothrombotic TNFα effects. Our results support a more selective therapeutic approach in anticytokine therapy favouring TNFR2 specific antagonists.

The chemokine CC chemokine ligand (CCL)5/RANTES as well as its respective receptor CCR5 mediate leukocyte infiltration during inflammation and are up-regulated early during the course of glomerulonephritis (GN). We tested the effects of the two CCL5/RANTES blocking analogs, Met-RANTES and amino-oxypentane-RANTES, on the course of horse apoferritin (HAF)-induced GN. HAF-injected control mice had proliferative GN with mesangial immune complex deposits of IgG and HAF. Daily i.p. injections of Met-RANTES or amino-oxypentane-RANTES markedly reduced glomerular cell proliferation and glomerular macrophage infiltration, which is usually associated with less glomerular injury and proteinuria in HAF-GN. Surprisingly, however, HAF-GN mice treated with both analogs showed worse disease with mesangiolysis, capillary obstruction, and nephrotic range albuminuria. These findings were associated with an enhancing effect of the CCL5/RANTES analogs on the macrophage activation state, characterized by a distinct morphology and increased inducible NO synthetase expression in vitro and in vivo, but a reduced uptake of apoptotic cells in vivo. The humoral response and the Th1/Th2 balance in HAF-GN and mesangial cell proliferation in vitro were not affected by the CCL5/RANTES analogs. We conclude that, despite blocking local leukocyte recruitment, chemokine analogs can aggravate some specific disease models, most likely due to interactions with systemic immune reactions, including the removal of apoptotic cells and inducible NO synthetase expression.