Background
Originally chemokines (
chemotactic cyto
kines) were described as key mediators for the selective migration of leukocytes into sites of tissue injury [
1]. Later on chemokines and chemokine receptors have also been described as important mediators in noninflammatory processes, including normal cellular trafficking, hematopoesis, angiogenesis, organ development, tissue remodelling, and tumor metastasis [
2‐
4]. To date more than 40 different human chemokines are characterized. The chemokine superfamily is separated into the C, CC, CXC, and CX3C subfamilies (Where X represents any intervening amino acid residue between the first two cysteines in the amino acid sequence) [
5,
6]. Chemokines mediate their biological activity by ligation and interaction with seven-transmembrane-spanning G protein-coupled receptors (i.e. C, CC, CXC, and CX3C receptors) [
7].
In the kidney expression of chemokines and chemokine receptors are important for the initiation and regulation of inflammatory glomerular diseases [
8]. Expression of the chemokines monocyte chemoattractant protein-1 (MCP-1/CCL2), regulated upon activation, normal T cell expressed and secreted (RANTES/CCL5), interleukin-8 (IL-8), and interferon-γ (IFN-γ)-inducible protein of 10 kD (IP-10/CXCL10) by human mesangial cells (MC) was shown by our group [
9] and others [
1,
10]. Inducible expression of the chemokine receptor CCR1 by human MC was previously described [
9]. The expression of the chemokine receptor CXCR3 on human MC was published by Romagnani and colleagues [
11]. A high level of expression of this receptor by MC was seen by immunohistochemistry in kidney biopsies from patients with IgA nephropathy, membranoproliferative glomerulonephritis or rapidly progressive glomerulonephritis. Recently our group showed that SLC/CCL21 is constitutively expressed by glomerular podocytes and CCR7 constitutively expressed by MC [
12].
In the kidney the well-regulated relationship among resident cell proliferation and apoptosis is important for the development of the sophisticated glomerular architecture during ontogenesis as well as maintaining normal function of adult human glomeruli. Dysfunction of the balance between glomerular cell proliferation and apoptosis after leukocyte infiltration has been discussed for many inflammatory kidney diseases [
13].
The finding of expression of chemokine receptors and their respective ligands by intrinsic renal cells not only under inflammatory conditions led to the hypothesis of an involvement of these receptors in glomerular homeostasis. Therefore the influence of chemokines on mesangial cell growth was investigated. We describe the different effects of the chemokines SLC/CCL21, IP-10/CXCL10, Mig/CXCL9, RANTES/CCL5 and the amino-terminally modified RANTES/CCL5 analogue Met-RANTES on mesangial cell proliferation and apoptosis, suggesting novel functions of chemokine/chemokine receptor pairs on local immunomodulation, glomerular regeneration and homeostasis.
Discussion
Proliferation and apoptosis play a pivotal role in a variety of biological processes, such as morphogenesis during the embryonic stage, cell selection during lymphoid development, tissue repair after injury, regression of inflammation, elimination of cells at risk of developing into a tumor and lymphocyte-mediated killing [
19]. A balance of proliferation and apoptosis is essential for the tissue homeostasis [
20,
21].
Apoptosis as mechanism of controlled cell death is a well-controlled process and progresses through a series of morphological and biochemical phases, including chromatin condensation and activation of proteolytic enzymes [
22,
23]. A number of mediators involved in the induction of apoptosis have been identified during recent years. Probably the most thoroughly characterized death receptor is the cell membrane receptor Fas (CD 95), a member of the TNF receptor family. Cross-linking of Fas, either via specific antibodies or via its specific ligand, activates a cascade reaction of caspases, which are responsible for induction of membrane alterations, breakdown of cellular constituents and DNA, and finally cell death [
19].
In the kidney beside the damage induced by infiltrating inflammatory cells the relationship among resident cell proliferation and apoptosis in glomeruli determines the outcome in various glomerulonephritides [
13]. Several groups reported that apoptosis plays an important role in the repair process in experimental and human glomerulonephritis [
24,
25]. Apoptosis has an additional role in the sclerosing process in the glomeruli [
26]. Cell number abnormalities are frequent in renal diseases, and range from the hypercellularity of postinfectious glomerulonephritis to the cell depletion of chronic renal atrophy. Death ligands and receptors, such as TNF and Fas-ligand, pro-apoptotic and anti-apoptotic Bcl-2 family members and caspases have all been shown to participate in apoptosis regulation in the course of renal injury [
27]. Some reports suggest that altered apoptotic signaling and regulatory mechanisms contribute to further progressive renal impairment, tubular atrophy, interstitial fibrosis, and glomerulosclerosis in a model of focal and segmental glomerulosclerosis in rats [
28].
In the glomerulus a balance between endothelial, mesangial and visceral epithelial cells and their extracellular matrix is essential for structural and functional integrity. During glomerular injury function and morphology of these cells are altered. Intrinsic cell proliferation in the glomerulus is regulated by a large number of mediators and growth factors like IL-10 [
29], insulin-like growth factors [
30] and platelet derived growth factor [
31]. Some of these factors also influence apoptosis in the glomerulus [
32].
The basis for the experiments performed in this work was the hypothesis that chemokines and chemokine receptors expressed by intrinsic renal cells may be involved both in the maintenance of glomerular homeostasis in normal adult human kidney and regulation of glomerular cell numbers during disease states. We previously showed constitutive expression of CCR7 protein and its ligand secondary lymphoid tissue chemokine (SLC/CCL21) in human renal tissue. In immunohistochemistry we found a clear staining pattern for SLC/CCL21 on podocytes and CCR7 on MC during nephrogenesis and in adult kidney. Also constitutive mRNA expression has been shown for CCR7 in cultured MC and for SLC/CCL21 in isolated human glomeruli. Furthermore it was demonstrated that mesangial CCR7 is functionally active since for example SCL/CCL21 induced a dose-dependent migration of MC [
12]. We therefore investigated the influence of chemokines on MC growth and found an significant increase in proliferation of MC after stimulation with SLC/CCL21 in a dose-dependent manner. To study the role of SLC/CCL21 in MC apoptosis cell death was induced by activating mesangial Fas/CD95 receptors. SLC/CCL21 was found to prevent MC apoptosis as shown by cell cycle analysis and Hoechst stain. Since caspase-3 assays revealed impaired activity it can be assumed that this molecule is involved in chemokine-influenced intracellular apoptosis pathways in MC. The finding of an anti-apoptotic function of SLC/CCL21 is novel. At present SLC/CCL21 is known to be constitutively produced by high endothelial venules and stromal cells within T cell zones of lymph nodes [
33]. Its corresponding receptor CCR7 is expressed on naive T cell subpopulations and up-regulated by maturing dendritic cells [
34]. Therefore this chemokine/chemokine receptor pair was described as an prototypic model for the homing of immune cells to lymphoid tissue [
35,
36]. Anti-apoptotic effects of chemokines seem not to be restricted to SLC/CCL21. The CX3CR1-binding chemokine fractalkine which is constitutively expressed on neuronal cells has been demonstrated as survival factor for brain microglia in Fas-induced cell death [
37].
A role of the chemokine receptor CXCR3 in inflammatory glomerular disease has been proposed before by the group of Romagnani et al. since a mesangial expression of CXCR3 in biopsies from patients with mesangioproliferative glomerulonephritis could be demonstrated [
11]. We therefore investigated the effects of the CXCR3 ligands IP-10/CXCL10 and Mig/CXCL9 on MC and also found a concentration dependent increase of proliferation of MC after stimulation with these chemokines. Interestingly, in contrast to the effect observed with SLC/CCL21 both IP-10/CXCL10 and Mig/CXCL9 had no effect on Fas induced apoptosis of MC.
The third chemokine receptor of interest was CCR1 since our group has demonstrated functionally active expression of CCR1 on human MC, inducible after stimulation with a combination of the proinflammatory cytokines TNF-α, IL-1β and IFN-γ [
9]. Futhermore upregulation of CCR1 expression is also known in an animal model for immune complex glomerulonephritis [
38]. In contrast to the effects observed with CCR7 and CXCR3 ligands, stimulation of MC with the CCR1 ligand RANTES/CCL5 had no effect on cell proliferation and apoptosis. In this context an article of Topham et al. is of special interest. This group demonstrated that CCR1 may have anti-inflammatory functions since mice negative for CCR1 showed enhanced Th1 immune responses and worsened histology in a model of nephrotoxic serum nephritis [
39]. Our group showed in a model of horse apoferritin (HAF)-induced glomerulonephritis that CC chemokine ligand 5/RANTES chemokine antagonists aggravate glomerulonephrtis despite reduction of glomerular leukocyte infiltration. These findings were associated with an enhancing effect of the CCL5/RANTES analogs on the macrophage activation state
in vitro and
in vivo. The humoral response and the Th1/Th2 balance in HAF-glomerulonephritis and mesangial cell proliferation
in vitro were not affected by the CCL5/RANTES analogs [
40]. Therefore also the effects of the CCR1 blocker Met-RANTES were studied but showed no influence on MC proliferation and apoptosis.
Authors' contributions
All authors were involved in experimental procedures and manuscript preparation.