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Keywords:

  • CCL2;
  • CCR2;
  • chemokine;
  • glomerulonephritis;
  • macrophage depletion

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

The CCL2/CCR2 chemokine/receptor axis directs the chemotaxis of infiltrating monocytes/macrophages and T cells and plays a pivotal role in tissue damage and fibrosis in kidney diseases. The eradication of the activated leucocytes should diminish the production of inflammatory mediators, limit tissue damage and ameliorate disease. A recombinant fusion protein (OPL-CCL2-LPM) comprised of the human CCL2 (monocyte chemoattractant protein-1) chemokine fused to a truncated form of the enzymatically active A1 domain of Shigella dysenteriae holotoxin (SA1) has been developed. The CCL2 portion binds specifically to CCR2-bearing leucocytes and the fusion protein enters the cells, where the SA1 moiety inhibits protein synthesis resulting in cell death. The compound was tested in a model of anti-thymocyte serum (ATS)-induced mesangioproliferative glomerulonephritis (ATS-GN). Male rats were injected with ATS on day 0 and treated intravenously with vehicle, 50 or 100 µg/kg of OPL-CCL2-LPM Q2D from days 2, 4, 6 and 8. Urine and blood were collected on days 0, 5 and 9. Animals were sacrificed on day 9. No treatment-related effects on body weight or signs of clinical toxicity were observed. Urine protein levels were decreased in treated animals. At the highest dose, histopathological analyses of kidney sections revealed maximum reductions of 36, 31, 30 and 24% for macrophage count, glomerular lesions, α-smooth muscle actin and fibronectin respectively. These results indicate a significant protective effect of OPL-CCL2-LPM in this model of nephritis.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Chemokine ligands and receptors are pivotal modulators of the activation, trafficking and proliferation of relatively disease-specific leucocyte subtypes (based on chemokine receptor expression) that contribute to tissue destruction and disease [1–5]. Tissue damage is induced by the release of proinflammatory and profibrotic mediators such as chemokines, cytokines, matrix metalloproteinases and reactive oxygen and nitrogen species from activated leucocytes and stimulated renal cells at the site of inflammation [6–8].

Macrophages/monocytes (MΦ) and certain T cell subpopulations modulated by the CCL2/CCR2 chemokine ligand-receptor axis have been found to be key players in the pathology of most human glomerular and tubulointerstitial diseases, including acute and chronic forms of glomerulonephritides [2–8]. Human biopsy studies have shown that intrinsic renal cells are a major source of CCL2 but the expression of the cognate receptor CCR2 is restricted to infiltrating MΦ and a small number of T cells [9–12]. Markers of renal damage, disease progression and stage of disease such as proteinuria, fibrosis, tubular atrophy and loss of renal function correlate with increased expression of CCL2 by intrinsic renal cells as well as increased numbers of CCR2 expressing MΦ and T cells [9–15]. Elevated numbers of glomerular and interstitial MΦ can be considered a marker of progressing renal damage, end-stage renal disease and transplant rejection [12,15–17]. In addition, urinary CCL2 levels from patients with a range of renal diseases have been found to correlate with disease activity index, histological index and the degree of proteinuria [9,14,15,18,19].

Several approaches targeting the CCL2/CCR2 axis with the goal of inhibiting the infiltration of pathological leucocytes have been found to be beneficial in animal models of a range of glomerulonephritides. These include the use of blocking antibodies to CCL2 and CCR2, chemokine receptor antagonists, gene therapy, anti-sense and immunosuppressive drugs (for reviews see [3–5,8,14,20]).

Another approach has been to target activated infiltrating leucocytes with cell-depleting agents [21–26]. Depletion of pathological leucocyte subtypes offers a comprehensive approach to therapeutic intervention as not only are inflammatory promoting cells eradicated, but in addition production of proinflammatory and tissue-damaging mediators from non-targeted cells is substantially decreased. Many studies have helped elucidate the roles of leucocytes, in particular MΦ, in several kidney disease models including ATS-GN, and have shown leucocyte depletion to be functionally and pathologically protective and to facilitate tissue repair [21–26].

Along with these observations, the fidelity of CCL2 for CCR2 alone and the differential and inducible expression of this receptor on pathological leucocyte subtypes in inflammatory conditions such as renal disease make CCR2 an attractive and selective therapeutic target. To exploit this chemokine-ligand/receptor axis we have developed OPL-CCL2-leucocyte population modulator (LPM), a fusion protein comprised of CCL2 linked to a truncated form of the enzymatic SA1 subunit which is designed to target activated CCR2+ leucocytes for eradication [21]. The chemokine ligand binds to its cognate receptor on targeted cells and the complex is internalized [21]. The SA1 enzyme is a ribosome inactivating protein and once internalized inhibits protein synthesis by hydrolyzing the N-glycosidic bond of a specific adenine residue in 28S rRNA, preventing elongation factors from binding to ribosomes and resulting in cell death [27]. In addition, the SA1 enzyme is an adenosine glycosidase that depurinates multiple forms of polynucleotides including DNA and is basis for its apoptotic effects [28]. The free enzyme has no inherent functional capacity to traverse the cell membrane [29].

The fusion and other chimeras comprised of different chemokine ligands are referred to as LPMs as they are designed to target discrete pathological leucocyte populations rather than complete major heterogeneous leucocyte groups, some of which do not express the targeted chemokine receptor. Additionally, we hypothesize that the eradication of inflammatory leucocytes leads to a decrease in the inflammatory mediator milieu, dampens inflammation, halts the further recruitment and activation of immune cells and ameliorates further tissue damage. Resolution of the inflammatory events begin with the participation of reparatory immune cell populations including MΦ and a homeostatic microenvironment can be restored [7,24,30]. Here we describe the safety and beneficial treatment effects of OPL-CCL2-LPM-mediated depletion of activated MΦ in the rat ATS-GN model.

Materials and methods

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Assessment of OPL-CCL2-LPM bioactivity

Peripheral blood mononuclear cell preparations and fluorescence-activated cell sorter.  Peripheral blood was obtained from healthy volunteers and peripheral blood mononuclear cells (PBMCs) prepared by Ficoll (Amersham Pharmacia Biotech, Piscataway, NJ, USA) gradient centrifugation. The cells were maintained in RPMI-1640 medium (Gibco/Invitrogen, Carlsbad, CA, USA). Fluorokine kits (cat. no. NFCP0; containing biotinylated hCCL2, avidin–fluorescein and biotinylated soyabean trypsin inhibitor protein control), anti-hCCR2-phycoerythrin (PE) (cat. no. FAB151P) and anti-hCCL2 neutralizing antibody (cat. no. MAB279) were from R&D Systems (Minneapolis, MN, USA). OPL-CCL2-LPM biotinylated using an EZ-Link NHS-LC biotinylation kit (cat. no. 21336), according to the manufacturer's instructions (Pierce Chemical Company, Rockford, IL, USA). Cells were washed with and resuspended in phosphate-buffered saline (PBS) at 4 × 106 cells/ml at 4°C left unstained or stained with anti-CCR2-PE, or incubated with biotinylated protein control, biotinylated CCL2 or biotinylated OPL-CCL2-LPM followed by secondary avidin–fluorescein staining following the R&D Systems Fluorokine kit protocols. PBMCs were analysed by flow cytometry gating for monocytes using forward- and side-shift light scattering.

Ribosomal inactivating protein assay.  The in vitro ribosomal inhibitory activity the SA1 moiety of OPL-CCL2-LPM was measured using a rabbit reticulocyte lysate cell-free protein synthesis (translation) assay, according to the manufacturer's instructions (Promega, Madison WI, USA). Briefly, the lysate contains the cellular components necessary for protein synthesis: tRNA, ribosomes, amino acids and initiation, elongation and termination factors and Luciferase control RNA. Translation reactions (quadruplet) are initiated with nuclease-treated lysate in the presence of increasing concentrations of test proteins and were incubated at 30°C for 90 min. A detection reagent (Bright-Glo™; Promega) was added and the relative luminescence for the reaction mixtures were read in a FLUOstar luminometer (BMG Lab Technologies, Durham, NC, USA) and the data analysed using GraphPad Prism 4 software. The ribosomal inactivating concentration (RIC50) of test proteins are the concentrations needed to reduce the synthesis of luciferase by 50%.

Cell viability assay.  THP-1 monocyte cells were grown according to the manufacturer's instructions (American Type Culture Collection, Manassas, VA, USA) in complete media containing RPMI-1640 media supplemented with 10% fetal bovine serum (Invitrogen, Carlsbad, CA, USA) and plated at a density of 3–4 × 104 cells/ml in 96-well plates. Vehicle or different concentrations of OPL-CCL2-LPM were added to plate wells in triplicate and incubated for 24 h at 37°C (5% CO2). The CellTiter-GloTM Luminescent Cell Viability Assay Kit (Promega) was used (as per the manufacturer's instructions) to assay cell viability as a measure of cell-based cytotoxicity. The proprietary CellTiter-Glo™ reagent is added to the wells and generates a luminescent signal upon cell lysis which is proportional to the amount of ATP present. The signal is directly proportional to the number of metabolically active cells remaining in the wells. Luminescence was measured using a FLUOstar luminometer and the data analysed using GraphPad Prism 4 software. Triplicate values were averaged and background luminescence subtracted. The ATP content in the presence of increasing concentrations of OPL-CCL2-LPM is represented as a percentage of vehicle control.

Animals and assessment of renal function

Animal experiments were authorized by the institution's Institutional Animal Care and Use Committee and performed in an Association for Assessment and Accreditation of Laboratory Animal Care-accredited facility and handled according to the guidelines provided in the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Twenty-four male Sprague–Dawley rats obtained from Harlan Laboratories (Houston, TX, USA) were allowed to acclimatize for 7 days with access to water ad libitum. Food was available throughout the study except for the periods where animals were housed in the metabolic cages, a precaution taken to avoid food-related protein from contaminating the urine samples. On day 0 rats were weighed (150–165 g) and set up in metabolic cages for 24 h for basal urine collection. Urine volumes were recorded and the urine processed and quantified for creatinine and protein. Rats were anaesthetized, and 0·5–1·0 ml blood was taken from a marginal tail vein at the initiation, mid-point (day 5) and termination points of the study. The blood was clotted and serum retained for measurement of blood, urea, nitrogen (BUN) and creatinine. Urine and blood collections were also taken on days 5 and 9 for analyses. Terminal time-point blood was obtained from the vena cava. Glomerular filtration rate (GFR) was calculated from urine/plasma creatinine concentration multiplied by urine collection volume and is expressed as ml/min. Body weight and health status of the rats was monitored daily.

The ATS induction of glomerulonephritis

On day 1 of the study 24 rats were injected intravenously (i.v.) with 20 mg/100 g body weight of anti-thymocyte (Thy1) IgG fraction (ATS; Probetex Inc., San Antonio, TX, USA) in PBS and returned to their cages. The rats were then divided randomly into three groups (groups 1–3; n = 8) and received i.v. doses of vehicle (50 mM sodium citrate buffer pH 6·2 containing 0·05 mM ethylenediamine tetraacetic acid), 50 µg/kg or 100 µg/kg of OPL-CCL2-LPM, respectively, on days 2, 4, 6 and 8. All rats were killed at day 9 and the kidneys were collected and cortex sliced for formalin fixation or flash-frozen in liquid nitrogen for subsequent histological analyses.

Immunohistochemical and histological examination

The kidney cortices were processed for staining and immunohistochemistry. Paraffin sections were stained with haematoxylin and eosin (H&E) in order to assess the extent of glomerular lesions and hypercellularity. Other sections were stained by immunoperoxidase histochemistry for ED-1 antibody (Chemicon Corporation, Temecula, CA, USA) to detect infiltrating glomerular and interstitial MΦ; anti-fibronectin (clone IST-9; Serotec, Harlan Bioproducts for Science, Indianapolis, IN, USA) as a marker for extracellular matrix (ECM) deposition and synthesis and anti-α-smooth muscle actin (α-SMA; clone 1A4 from Sigma, St Louis, MO, USA) as a marker of mesangial cell activation. After incubation with biotin-donkey anti-mouse IgG as second antibody (Chemicon) reaction product was developed using an avidin–biotin–peroxidase complex amplification technique according to the manufacturer's instructions (Vector Laboratories, Burlingame, CA, USA). A total of 25 glomeruli for each rat (eight per group) were scored in a blinded manner for each of the staining analyses by an experimental nephrologist.

Glomerular MΦ numbers were assessed by counting the number of ED-1+ cells/glomerulus in a total of 25 glomeruli from each section then averaging macrophages/glomerulus. Interstitial MΦ numbers were determined by counting the number of ED-1+ cells per five random fields using a 40× objective by bright-field microscopy. The counts were averaged and expressed as macrophages/field.

Glomerular lesions were evaluated by use of a modified semi-quantitative scoring system [31,32]. Briefly, the severity of the glomerular lesion was graded from 0+ to 4+ according to the percentage of glomerular involvement: 0 = normal glomerular architecture, 1 = small nodules occupying combined area ≤ one-third glomerular area, 2 = nodules occupying combined area of approximately one-half glomerular area, 3 = nodules occupying combined area of approximately two-thirds glomerular area and 4 = segmental nodules and global mesangial hypercellularity.

Glomerular expression of α-SMA and fibronectin were graded semi-quantitatively using a similar scoring system to that described above: 0 = normal fine, negligible staining in a typical mesangial pattern, 1 = slight increase in intensity and/or small nodular staining occupying combined area of one-third glomerular area, 2 = moderate increase in intensity and/or small nodular staining occupying combined area of one-half glomerular area, 3 = strong increase in intensity and/or small nodular staining occupying combined area of two-thirds glomerular area, 4 = strong increase in intensity and/or small nodular staining occupying the majority of the glomerular structure [31,32].

Statistical analysis

All values are expressed as the mean ± the standard error of the mean. The P-values for the glomerular H&E, SMA-α and fibronectin scores were are from the ordinal multinomial model test and the ED-1+ MΦ P-values are from a one-way analysis of variance test. Differences are considered significant if P < 0·05.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Human monocytic cells express CCR2 and bind CCL2 and OPL-CCL2-LPM

The expression of CCR2 and binding of biotinylated CCL2 and OPL-CCL2-LPM on human primary monocytes and the human THP-1 leukaemia cell line was analysed by fluorescence-actived cell sorter. Both cell types were found to express CCR2 (Fig. 1a,c) with the profiles indicating lower expression levels on THP-1 cells. Both cell types also bind CCL2 and the LPM (Fig. 1b,d). CCL2 bound with a higher affinity than the LPM on both cell types, and as expected showed a more heterogeneous binding profile on primary cells. The binding of both ligands was reduced dramatically upon the inclusion of CCL2 neutralizing antibody (data not shown). Similar patterns of hCCL2 and LPM binding were shown on CCR2 expressing monocytes in PBMC preparations from dogs, rats and cynomologus monkeys (data not shown).

image

Figure 1. Peripheral blood mononuclear cells (PBMCs) were stained and gated for monocytes during fluorescence-activated cell sorter (FACS) analysis, as described in the Materials and methods section. Monocytes and cultured monocytes (THP-1 cells) were stained with CCR2-phycoerythrin (PE) (a and c respectively). Unstained cells are shaded in light grey (c). Monocytes (b) and THP-1 cells (d) bound biotinylated ligands; control protein (first peak), OPL-CCL2-leucocyte population modulator (LPM) (second peak) and CCL2 (third peak).

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In vitro bioactivity of OPL-CCL2-LPM

The in vitro ribosome inactivating activity of the SA1 enzymic moiety of the fusion protein was verified using a cell-free protein synthesis system as outlined in the Materials and methods section. The RIC50 concentration range for OPL-CCL2-LPM is routinely between 15 and 30 pM (Fig. 2a). A cell viability assay verifies that CCL2 targets its cognate receptor (CCR2) present on THP-1 cells. The fusion protein is internalized and kills the cells. Calculated IC50 values for OPL-CCL2-LPM lots are in the range of 55–60 µg/ml (Fig. 2b). Inclusion of CCL2 neutralizing antibody in the assay mixtures abolished the LPM cytotoxic activity, verifying that the ligand is responsible for receptor binding and internalization (data not shown).

image

Figure 2. In vitro bioactivity of OPL-CCL2-leucocyte population modulator (LPM). (a) Ribosome inactivating activity of OPL-CCL2-LPM in a protein synthesis cell-free assay system. (b) The cytotoxic effect of the fusion protein on THP-1 monocytic cells. Values are plotted as the average ± standard error of the mean.

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General health status

All animals gained weight throughout the study, with no appreciable differences between control and test rats. BUN and serum creatinine levels were within the normal range for all animals throughout the study.

Toxicology and renal functional analyses

Independent Good Laboratory Practice toxicology studies using normal same-strain Sprague–Dawley rats revealed that the administration of OPL-CCL2-LPM in cumulative doses of up to 22·5 mg/kg over 29 days had no adverse effects on blood chemistry, renal function or organ pathology including the kidneys (data not shown).

In this study, H&E examination of liver, heart, spleen, lung and brain showed no signs of cytotoxicity in vehicle or treated rats (data not shown). Renal functional and blood analyses revealed no adverse effects of OPL-CCL2-LPM in the nephritis study. In all three cohorts, creatinine clearance estimates of GFR were variable but within the expected normal variation throughout the study. Basal values were in the normal range of around 1 ml/min. Basal urine protein measurements levels were negligible with values approximately 1·0 mg/24 h. Significant increases at day 5 in urine protein values were found in all rats, but the increase was substantially less in both treatment groups compared with vehicle (Fig. 3). A reduction of approximately 35% and 39% in urine protein was achieved in the 50 and 100 µg/kg-treated groups versus vehicle control rats respectively. Terminal (day 9) GFR and urine protein values decreased in all groups from day 5 values, reflecting the resolving nature of disease in this ATS model. There were no significant differences between vehicle and treatment groups at day 9.

image

Figure 3. Urine protein analysis. (a) Differences in urine protein measurements between vehicle and treated animals at days 0 and 5. Values are plotted as the average ± standard error of the mean.

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Histological and immunohistochemical analyses

Immunohistochemical staining analyses showed a reduction in the number of ED-1+ MΦ infiltrating the glomeruli with both doses of OPL-CCL2-LPM when compared with vehicle treated animals (Fig. 4a–c). Glomerular mean MΦ counts for vehicle, 50 and 100 µg/kg treatment groups were 4·4, 3·0 and 2·8 per glomeruli respectively (the higher dose treatment gave a P < 0·001; Table 1). The percentage reduction in mean MΦ number from vehicle values was 31 and 36% in the low- and high-dose groups respectively (Table 1).

image

Figure 4. Histological and immunohistochemical analyses. (a–c) Representative fields of ED-1-stained glomeruli from rats dosed with vehicle, 50 or 100 µg/kg of OPL-CCL2-leucocyte population modulator (LPM) respectively (n = 4 per group). The degree of glomerular injury, mesangial cell activation/proliferation and matrix synthesis was measured using haematoxylin and eosin (d–f), α-smooth muscle actin (SMA) (g–i) and fibronectin (j–l) staining. Representative fields of stained glomeruli (25 per rat) are shown with eight rats per cohort. Magnifications: 40×.

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Table 1.  ED-1+ macrophage cell counts and histological scores.
HistologyVehicleOPL-CCL2-LPM
50 µg/kg100 µg/kg
  1. The number of glomerular macrophages (G-ED-1+) in 25 glomeruli per rat and the interstitial macrophages (I-ED-1+) in five random fields per rat (n = 4 per group) were counted and presented as the average per glomeruli or field, respectively, ± standard error of the mean (s.e.m.). A total of 25 glomeruli for each rat (n = 8 per group) were scored in a blinded manner for each of the other staining analyses. Data are presented as the average score ± s.e.m. The calculated *P-values were P < 0·001. The values in parentheses represent the percentage decrease from vehicle. H&E, haematoxylin and eosin; LPM, leucocyte population modulator; MΦ, macrophages/monocytes; SMA, smooth muscle actin.

G-ED-1+4·40 ± 0·163·02 ± 0·23 (31)2·80 ± 0·23 (36)*
I-ED-1+31·70 ± 4·0025·50 ± 3·50 (22)22·21 ± 3·72 (33)
H&E2·21 ± 0·091·92 ± 0·14 (13)1·53 ± 0·12 (31)*
α-SMA2·10 ± 0·081·92 ± 0·14 (9)1·49 ± 0·10 (30)*
Fibronectin1·91 ± 0·101·75 ± 0·15 (9)1·46 ± 0·13 (24)*

The H&E analysis of treated rat glomeruli shows that there is less hypercellularity and a substantial degree of preservation of glomerular architecture after OPL-CCL2-LPM treatment when compared with vehicle control (Fig. 4d–f). For example, glomeruli of the vehicle group had the greatest degree of change showing an average score of 2·2 based on a hypercellular mesangium involving one-half to two-thirds of the glomerular area (Table 1). The 100 µg/kg OPL-CCL2-LPM treatment group scores averaged 1·5 (one-third to one-half glomerular area), representing 31% less severity. Similarly, mesangial cell activation, measured by α-SMA staining (Fig. 4g–i) as well as ECM protein synthesis by fibronectin staining (Fig. 4j–l) show clearly that glomeruli from OPL-CCL2-LPM-treated rats were less affected than vehicle treated animals (Table 1). The statistical significance of the data for the high-dose group compared with the vehicle group was P < 0·001 in all measured histological categories except interstitial MΦ counts, where values were highly variable within each cohort.

Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

We have previously reported that OPL-CCL2-LPM specifically targets and kills cells known to express the CCL2 cognate receptor CCR2 including human primary monocytes and human monocytic leukaemia cells [21]. Here we show that these cells express CCR2 and bind both CCL2 and the LPM. CCR2 expression and ligand binding on monocytes is of a heterogeneous nature. This is not surprising, as MΦ (monocyte and macrophage) subtypes are heterogeneous in nature and differentially express CCR2 [7,30,33–35]. Human rat, mouse and monkey monocytes have been divided into two main types based on the expression levels of several different antigens [30–36]. CD14highCD16- inflammatory cells express CCR2, while CD14lowCD16+ resident cells are CCR2-deficient. However, expression levels of CCR2 is variable on different on MΦ subtypes and ranges from high to low. CCR2 is an inducible receptor which is up-regulated under increasingly inflammatory microenvironments [30,33]. In addition, the activation state of CCR2 on primary monocytes is variable and is regulated in vivo by the specific nature of the cytokines present in the microenvironment [7,30,33–35].

The expression level of CCR2 on THP-1 cells appears low but binds both CCL2 and the LPM. In the case of CCL2, the binding appears less heterogeneous than is the case with primary monocytes. This is due probably to the fact that the cultured THP-1 cells are in a more synchronized state and express a more homogeneous population of activated CCR2 than freshly isolated peripheral monocytes. Ligand binding and internalization kinetic experiments are the subject of ongoing studies. For the sake of ease and reproducibility THP-1 cells were chosen to be used to develop a cytotoxicity bioassay which is being optimized. ID50 values calculated in this assay are routinely consistent between repeat-lot assays and between different lots of purified LPM.

Many human glomerular diseases including IgA nephropathy and lupus nephritis are characterized by MΦ infiltration, mesangial cell hypercellularity, ECM deposition, proteinuria and glomerulosclerosis. The ATS-GN model was chosen to assess the suitability of using OPL-CCL2-LPM as a therapeutic candidate for the treatment of human glomerulonephritides because this model exhibits most of the pathological markers exhibited in human diseases [37]. Briefly, the course of ATS-GN begins with rapid mesangiolysis followed by prominent mesangial cell activation and proliferation with mesangial ECM deposition. There is an up-regulation of CCL2 from tissue resident cells within minutes and is a powerful chemoattractant that is responsible for the glomerular and interstitial infiltration of activated CCR2+ MΦ over the first few days. Subsequently, MΦ produce chemokines including CCL2, cytokines and growth factors that contribute to the activation and proliferation of mesangial cells which up-regulate the production of α-SMA and synthesize ECM proteins including fibronectin and collagens, a prelude to fibrosis [37–42]. In several nephritis models including ATS-GN, neutralization of CCL2 has been found to attenuate the accumulation of infiltrating MΦ, mesangial cell activation, ECM protein production and sclerosis [40,43–46].

The major infiltrating inflammatory leucocytes in human and animal model glomerulonephritides include CCR2+[9,11–13]. OPL-CCL2-LPM treatment greatly reduced the numbers of activated ED-1+ MΦ in both the glomerular and interstitial compartments of diseased rats, although complete eradication of these cells was not achieved. One would not expect depletion of all MΦ because, as discussed, MΦ populations are heterogeneous and not all of them express CCR2. In addition, monocytes down-regulate CCR2 as they infiltrate inflammatory tissues and continue to differentiate into tissue macrophages and as the inflammatory microenvironment dissipates. The subtypes differentially express several antigenic markers that reflect different functional attributes [7,33–36]. This suggests that the LPM has the capacity to selectively eliminate inflammatory CCR2+ MΦ, while CCR2- subpopulations, which may play a role in tissue remodelling and repair, are spared. The decrease in numbers of MΦ following treatment with OPL-CCL2-LPM may represent the elimination of majority of CCR2 expressing cells that can be achieved at the doses administered. This was not possible to verify because of the lack of a suitable rat CCR2 antibody. Future studies will address this issue and should evaluate a broader range of doses in order to establish the minimum and maximum effective doses.

In vitro tissue culture reports suggest that cultured activated mesangial cells can express CCR2 [47–49]. In addition, both macrophages and the CCL2/CCR2 axis have been implicated in producing a prosclerotic effect on mesangial cells [50,51]. CCL2 up-regulation of mesangial ECM protein synthesis is transforming growth factor (TGF)-β and nuclear factor (NF)-κB-dependent [44]. Both resident kidney cells and infiltrating MΦ can instigate fibrosis directly via a CCL2/CCR2-dependent autocrine/paracrine loop [52,53]. Therefore, it is possible that in the mesangioproliferative phase of this model activated CCR2+ mesangial cells were also targeted by the fusion protein and treatment could have limited mesangial cell expansion. Glomerulosclerosis does not occur in this paradigm but fibrogenesis is detected by mesangial cell activation and proliferation (α-SMA) and ECM protein production (fibronectin). There was a significant diminution of these prefibrotic mesangial cell markers in the glomeruli of test article-treated versus vehicle-treated animals in this study. Presumably the elimination of activated MΦ by OPL-CCL2-LPM here not only attenuated MΦ inflammatory mediator production, including CCL2 and TGF-β which are responsible for the induction of mesangial cell hypercellularity, ECM protein synthesis and fibrogenesis, but also the direct deleterious tissue effects of MΦ-derived inflammatory mediators on kidney resident cells.

Proteinuria is a classical marker of renal dysfunction which, among other things, stimulates the expression of chemokines including CCL2, cytokines and other proinflammatory mediators by kidney resident cells, further exacerbating inflammation. In addition, it is thought that increased protein filtration may be directly toxic to tubular epithelial cells [54–56]. It has been reported that both in humans and rats there is a positive correlation between CCL2 expression, proteinuria and macrophage infiltration [15,54]. Proteinuria is not a prominent feature of this self-resolving disease model, and only moderate increases in urine protein are observed around the peak of disease activity (day 5) in this study. Nevertheless, urine protein was attenuated upon treatment with OPL-CCL2-LPM, suggesting a renal protective effect.

There is much need for novel and highly selective therapeutics for renal disease. We have chosen to develop an agent which exploits selectively the fact that the CCL2/CCR2 chemokine ligand/receptor axis is specifically up-regulated under inflammatory conditions and that there is a differential chemokine receptor expression on leucocyte subtypes. CCR2 expression is relatively restricted in the main to pathological leucocyte subpopulations in humans [10–12], which confers another level of selectivity for a CCR2 targeting agent.

Here we report that OPL-CCL2-LPM, a novel CCR2 targeting agent, demonstrates selectivity, efficacy and safety. Collectively, the data from the rat model of ATS-induced mesangioproliferative glomerulonephritis indicate that treatment with OPL-CCL2-LPM leads to the depletion of activated infiltrating MΦ; preservation of glomerular architecture by reducing glomerular lesions and reduces protein in the urine; and limiting mesangial hypercellularity, ECM protein synthesis and attenuation of renal function decline. The elimination of activated MΦ should attenuate the direct deleterious tissue effects of MΦ-derived inflammatory mediators and the persistence of inflammation by limiting the further recruitment of infiltrating leucocytes. Treatment with the LPM could then potentially slow down and limit the downstream fibrotic events that occur in many glomerulonephritides and nephropathies.

The OPL-CCL2-LPM appeared to be well tolerated in the rat: renal functional, blood chemical and histopathological analyses of a variety of different organs revealed no toxicity. The safety, cell-targeting specificity and efficacy of the LPM are supported further by additional studies in a variety of animal models, including a murine experimental autoimmune encephalomyelitis model of multiple sclerosis [57].

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

We thank Liliana Perdomo for excellent technical assistance and Drs B. Finck, B. Rovin and N. Turner for critically reviewing this manuscript.

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References