Immunobiology 216 (2011) 854–861
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Reduced expression of arrestin beta 2 by graft monocytes during acute rejection of rat kidneys Anna Zakrzewicz a,∗ , Gabriela Krasteva b , Jochen Wilhelm c , Hartmut Dietrich d , Sigrid Wilker a , Winfried Padberg a , Malgorzata Wygrecka e , Veronika Grau a a
Laboratory of Experimental Surgery, Department of General and Thoracic Surgery, Justus-Liebig-University Giessen, Rudolf-Buchheim-Str. 7, D-35385 Giessen, Germany Institute for Anatomy and Cell Biology, Justus-Liebig-University Giessen, Aulweg 123, D-35385 Giessen, Germany Department of Pathology, Justus-Liebig-University Giessen, Langhans-Str. 10, D-35385 Giessen, Germany d Department of Internal Medicine, Justus-Liebig-University Giessen, Klinik-Str. 36, D-35385 Giessen, Germany e Department of Biochemistry, Justus-Liebig-University Giessen, Friedrich-Str. 24, D-35385 Giessen, Germany b c
a r t i c l e
i n f o
Article history: Received 5 January 2010 Received in revised form 23 November 2010 Accepted 26 November 2010 Keywords: Arrestin beta 2 Intravascular leukocytes Kidney transplantation NF-kappaB Organ rejection
a b s t r a c t During acute rejection, numerous pro-inflammatory and cytotoxic monocytes accumulate in the vasculature of experimental renal allografts. Arrestins (ARRBs) are cellular regulators of inflammation, but nothing is known about their expression during rejection. Intravascular mononuclear graft leukocytes were isolated 4 days after kidney transplantation. ARRB1 and ARRB2 mRNA expression was reduced in blood leukocytes from allografts undergoing acute rejection, whereas on the protein level only ARRB2 was changed. Flow cytometry and confocal microscopy revealed ARRB1 and ARRB2 expression by monocytes and T cells, with a selective decrease in ARRB2 expression in monocytes during acute rejection. I-B directly interacted with ARRB2 and the levels of both proteins strongly correlated. Concomitantly, the mRNA expression of NF-B targeted genes increased. Our results suggest that activation of blood monocytes in renal isografts is dampened by high ARRB2 levels. During acute rejection, ARRB2 levels are reduced and classical monocyte activation is enabled via NF-B activation. © 2010 Elsevier GmbH. All rights reserved.
Introduction Kidney transplantation is a common treatment for patients with end-stage renal failure. Despite the use of potent immunosuppressive drugs, acute rejection episodes still remain an important problem as a major risk factor for the development of chronic rejection, which limits the success of kidney transplantation in the long run (Joosten et al. 2003; Leichtman et al. 2008). During acute rejection, renal allografts are heavily infiltrated by leukocytes (Grau et al. 1998; Barry and Bleackley 2002; Jose et al. 2003). Additionally, numerous mononuclear leukocytes accumulate in the blood vessels of the graft (Grau et al. 2001). Several studies focus on tissue infiltrating activated T cells and macrophages, which are thought to damage renal grafts (Tipping and Holdsworth 2003; Kluth et al. 2004; Ferenbach et al. 2007) whereas much less is known about intravascular graft leukocytes. Blood leukocytes, depending on their intravascular localization, belong to the central or to the marginal pool of the blood (van Furth
Abbreviations: ARRB2, arrestin beta 2; I-B, inhibitor kappaB; NF-B, nuclear factor kappaB. ∗ Corresponding author. Tel.: +49 641 99 44791; fax: +49 641 99 44769. E-mail address:
[email protected] (A. Zakrzewicz). 0171-2985/$ – see front matter © 2010 Elsevier GmbH. All rights reserved. doi:10.1016/j.imbio.2010.11.005
and Sluiter 1986). Leukocytes of the central pool move with the blood stream and are not directly interacting with the transplant. Only these cells are accessible to venous puncture. In contrast, in the marginal pool, recipient leukocytes encounter allogenic endothelial cells for the first time. Alloantigen is detected in the context of MHC antigens and the decision is taken if leukocytes are going to infiltrate the tissue. Furthermore, interactions of host leukocytes and donor endothelium directly result in graft endothelial cell activation and acute destruction (Reinders et al. 2006; Al-Lamki et al. 2008). Cells of the marginal pool can be harvested by intensively perfusing the graft vasculature, an approach obviously restricted to experimental animals (Steiniger et al. 2001). Monocytes interacting with allograft endothelia probably play a pivotal role in all phases of allograft rejection (Steiniger et al. 2001; Jose et al. 2003; Stehling et al. 2003, 2004). They can phagocytose cell debris and apoptotic cells originating from graft damage during surgery. Monocytes are a source of pro-inflammatory cytokines and directly damage the graft during acute rejection (Grau et al. 2001; Qi et al. 2008). In this study, we investigate intravascular mononuclear leukocytes in a fully allogenic rat model of acute renal allograft rejection. Transplantation in the Dark Agouti (DA) to Lewis (LEW) rat strain combination leads to a strong accumulation of leukocytes in the blood vessels of the graft until day 4 post-transplantation and to
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irreversible destruction within 5 days (Grau et al. 1998, 2001). About 73% of the intravascular allograft leukocytes are monocytes and about 14% are T cells (Grau et al. 2001). Allograft monocytes have been extensively characterized in this experimental model. They express a pattern of cell surface molecules compatible with strong activation and also pro-inflammatory cytokines like IL-1, IL-6, IL-12, and TNF-␣ (Grau et al. 2001). In addition, allograft monocytes express high levels of iNOS, exert direct cytotoxicity, and express tissue factor (TF), which can cause intravascular microthrombi (Grau et al. 2001; Stehling et al. 2003, 2004; Qi et al. 2008). The specific molecular and cellular mechanisms regulating monocyte function during allograft rejection are unknown, but their identification may provide novel therapeutic targets. Activation of genes involved in graft rejection such as IL-1, IL-6, TNF-␣, iNOS, and TF is predominantly regulated by the family of NF-B transcription factors (Blackwell and Christman 1997). In the cytoplasm, NF-B forms a complex with the inhibitor of kappa B (I-B), which prevents its translocation to the nucleus. Upon I-B phosphorylation and its subsequent degradation, the NF-B complex translocates to the nucleus where it binds to specific DNA sequences in the promoter regions of several genes and regulates their transcription (Kapahi et al. 2000; Karin and Ben-Neriah 2000; Bonizzi and Karin 2004). Inhibiting NF-B activation has been proposed as a strategy to promote allograft survival (Tergaonkar 2006). Recently, arrestin beta 1 (ARRB1) and ARRB2, multifunctional signaling molecules with well established functions in the desensitization and internalization of diverse cell surface receptors, have been reported to interact with I-B␣ (Gao et al. 2004; Witherow et al. 2004). Their interaction prevents phosphorylation and degradation of I-B and thus attenuates activation of NF-B. This observation was made in a range of cell lines including HEK293, HeLa, COS-7, Jurkat, and importantly also in THP-1, a human monocytic cell line (Gao et al. 2004; Witherow et al. 2004). Over-expression of ARRB2 in HEK293 cells inhibited TNF-␣-induced NF-B activation, reduced nuclear translocation of NF-B p65, and significantly inhibited NF-B DNA binding activity (Gao et al. 2004; Witherow et al. 2004). Consistent with this observation, silencing of ARRB2 by siRNA in vitro enhanced NF-B DNA binding activity, induced nuclear translocation, and mRNA expression of the NF-B target genes in response to pro-inflammatory stimuli (Gao et al. 2004). Differential expression of these regulatory proteins has been suggested to contribute to inflammatory diseases (Lattin et al. 2007). This hypothesis was supported in experiments using ARRB1 or ARRB2 deficient animals (Walker et al. 2003; Shi et al. 2007). However, little is known about differential ARRB expression and its impact on the I-B/NF-B system in vivo. During inflammation, an increased expression of ARRBs was observed in spleens and in inflamed peripheral tissue (Lattin et al. 2007). It is, however, unclear if this was due to an influx of leukocytes strongly expressing ARRBs. In this study, we demonstrate for the first time that ARRB1 and ARRB2 are expressed by intravascular renal graft leukocytes in vivo, and that ARRB2 expression is strongly reduced in monocytes but not in T cells during rejection. Furthermore, we demonstrate that ARRB2 interacts with I-B in vivo and that diminished expression of ARRB2 correlates with reduced levels of I-B.
Materials and methods Experimental animals and kidney transplantation LEW (RT11 ), DA (RT1av1 ) male rats weighing 260–300 g (Harlan Winkelmann, Borchen, Germany) were kept under conventional conditions. All animals received humane care following the German
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Law on the Protection of Animals, the “Principles of Laboratory Animal Care” formulated by the National Society for Medical Research as well as the NIH “Guide for the Care and Use of Laboratory Animals”. Kidneys were transplanted orthotopically to totally nephrectomized LEW recipients as described (Fabre et al. 1971), except that the ureter was anastomosed end to end. For allogenic transplantation DA rats and for isogenic transplantation LEW rats were used as donors. Total ischemic times remained below 30 min. Allograft recipients die 7.4 ± 0.7 days (mean ± SD, n = 10) after transplantation, whereas isograft recipients survive in good health (n = 10) (Grau et al. 1998). Isolation of mononuclear cells from renal vasculature To isolate mononuclear graft blood leukocytes, recipients were anaesthetized, heparinized, and the kidney was extensively perfused with cold Ca2+ and Mg2+ free PBS (PAA, Pasching, Austria), 2.7 mM EDTA, 0.1% BSA (Serva, Heidelberg, Germany) (Grau et al. 2001; Holler et al. 2008). To deplete erythrocytes, granulocytes, and platelets, perfusates were purified by Percoll density centrifugation (Scriba et al. 1996) and stored under liquid nitrogen until use. RNA isolation and cDNA synthesis Total cellular RNA was extracted from 5 × 106 intravascular mononuclear leukocytes harvested from kidney isografts or allografts on day 4 post-transplantation. RNA isolation was performed using the RNeasy Mini Kit (Qiagen, Hilden, Germany) and 1 g of total RNA was reversely transcribed using the M-MLV H− Reverse Transcriptase and 1 g of random hexamer primers (Promega, Mannheim, Germany). The reaction was carried out at 40 ◦ C for 1 h. Quantitative RT-PCR Quantitative RT-PCR (qRT-PCR) was used to assess the mRNA gene expression of ARRB1, ARRB2, TNF-␣, and IL-1. Reactions were performed in an ABI 7700 Sequence Detection System (Applied Biosystems, Foster City, CA) using Platinum SYBR green qPCR Super Mix-UDG (Invitrogen, Karlsruhe, Germany). Quantitative changes were analyzed by comparing Ct values of the genes of interest (n = 4 per experimental group, each sample was assessed in duplicates) to the house keeping gene. Pseudogene-free porphobilinogen deaminase (PBGD) gene was selected as reference gene as it was reported not to be regulated in monocytes under various culture conditions (Moosig et al. 2006). All primers for qRT-PCR were synthesized by MWG Biotech (Ebersberg, Germany). Sequences are indicated in Table 1. PCR included initial denaturation for 5 min at 95 ◦ C, followed by 45 cycles of 20 s at 95 ◦ C, 20 s at 60 ◦ C, and 10 s at 72 ◦ C. Protein isolation and immunoblotting Protein extracts from mononuclear cells were prepared as described previously (Laemmli 1970). Protein concentrations were determined using Micro BCATM protein Assay kit (Pierce Biotechnology, Rockford, IL). Equal amounts of protein (8 g) were resolved on 10 or 12% reducing SDS-polyacrylamide gels, and transferred onto polyvinylidene difluoride membranes (Millipore, Billerica, MA). Membranes were blocked with 5% non fat milk powder in 50 mM Tris–HCl, pH 7.6, 0.9% NaCl. Mouse monoclonal Abs (mAbs) to ARRB1 (Invitrogen, Carlsbad, CA) and to I-B (Cell Signaling Technology, Danvers, MA) were diluted 1:1000, and a mAb to ARRB2 (Abnova, Taipei, Taiwan) was diluted 1:2000. To ensure equal protein loading, membranes were incubated with a mAb to
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Table 1 List of primers employed for quantitative RT-PCR. GeneBankTM (accession number) ARRB1 (NM 012910) ARRB2 (NM 012911) TNF-␣ (NM 012675) IL-1 (NM 031512) PBGD (NM 013168)
Forward primer
5 5 5 5 5
Reverse primer
TCA CCG TCT ACC TGG GAA AG 3 AAG TCG AGC CCT AAC TGC AA 3 GGT CCC AAC AAG GAG GAG A 3 CCT GTT CTT TGA GGC TGA CA 3 GGC GCA GCT ACA GAG AAA GT 3
5 5 5 5 5
Amplicon size (bp)
CGC AGG TCA GTG TCA CGT AG 3 CAC AAA CAC TTT CCG GTC CT 3 GGG CTT GTC ACT CGA GTT TT 3 GCT GTG AGA TTT GAA GCT GGA 3 AGC CAG GAT AAT GGC ACT GA 3
126 134 96 101 115
ARRB1, arrestin 1; ARRB2, arrestin 2; PBGD, porphobilinogen deaminase.
GAPDH (Novus Biologicals, Littleton, CO), 1:20,000. Bound antibodies were visualized with horseradish peroxidase-conjugated secondary antibodies (Dako, Glostrup, Denmark), 1:5000, using the chemiluminescent reagent Lumi-Light Western blotting substrate (Roche, Mannheim, Germany). Densitometric analyses were performed using a digital gel documentation system (Biozym, Hessisch Oldendorf, Germany). All data of individual samples were divided by the values obtained for GAPDH on the same blot.
the laser (488 nm) power was set at 20%, for Cy3 the laser (543 nm) power was set at 100%, and for Cy5 the laser (633 nm) power was set at 50%. The following controls were always included: 1) single immunofluorescence staining for each antibody, 2) double immunofluorescence for ARRB1 or ARRB2 with either ED1 or R73, 3) omission of anti-ARRB antibodies, and 4) incubation with irrelevant mouse IgG labeled with Alexa 488 or Alexa 647. Co-immunoprecipitation
Flow cytometry To identify ARRB1 and ARRB2 expressing mononuclear leukocytes, cells were stained with a FITC-labeled mAb to CD5 (OX19) to stain T cells or with FITC-labeled mAb ED9 (BD Bioscience, San Diego, CA) directed to a member of the signal regulatory protein family (CD172a) to detect monocytes (Damoiseaux et al. 1989a,b; Adams et al. 1998). In rat blood ED9 binds to monocytes but also to granulocytes, which were depleted by Percoll density gradient centrifugation prior to flow cytometry (Scriba et al. 1996). After fixation and permeabilization with Cytofix/CytopermTM solution (BD Bioscience), a second staining with a PE-labeled mAb directed to ARRB1 or ARRB2 was performed. Antibody labeling was performed using Zenon Mouse IgG labeling kit (Invitrogen, Karlsruhe, Germany). To determine autofluorescence and unspecific antibody binding, PE-labeled irrelevant IgG1 (Clone X40 ) or IgG2a (Clone X39 ) antibodies (BD Bioscience) were used. Immuno-positive cell populations were defined as cells with higher immunofluorescence than the appropriate IgG control. A minimum of 2.5 × 105 cells per sample were analyzed using a fluorescent-activated cell sorter FACS Calibur (BD Bioscience) and Cell Quest software 3.2.1 package (BD Biosciences). Gates based on forward and side scatters were set to eliminate cellular debris and cell clusters.
7 × 106 intravascular mononuclear leukocytes harvested from kidney isografts were lysed in 100 l ice-cold lysis buffer (50 mM Tris–HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% (v/v) Triton-X100, 10% (v/v) glycerol, 0.1% SDS) containing a protease inhibitor cocktail (Roche, Mannheim, Germany). Lysates were centrifuged at 10,000 rpm, 10 min, 4 ◦ C, and incubated over-night at 4 ◦ C with 5 g of goat polyclonal anti-ARRB2 antibodies (Everest Biotech, Oxfordshire, UK) or 5 g of goat irrelevant control IgG (Santa Cruz Biotechnology). Immune complexes were precipitated by incubation with 50 l protein A-SepharoseTM CL-4B beads (Amersham Biosciences, Freiburg, Germany) for 1 h at 4 ◦ C. After extensive washing in ice-cold lysis buffer, the beads were resuspended in sample buffer and boiled. Statistics Data were analyzed by non-parametric Kruskal–Wallis test followed by Mann–Whitney rank sum test using SPSS software (Munich, Germany) with p ≤ 0.05 regarded as significant. Correlation was tested 2-sided using the Welch t-test, with p ≤ 0.05 regarded as significant. Results
Immunofluorescence microscopy Graft recipients were killed by inhalation of an overdose of isoflurane (Abbott, Wiesbaden, Germany). Kidneys were sliced, snap-frozen, and stored at −80 ◦ C until use. Cryostat sections (10 m) were fixed for 10 min in isopropanol (4 ◦ C) and air-dried. Sections were incubated for 1 h in 50% horse serum in PBS, followed by over-night incubation with mAbs to ARRB1 (1:50), and ARRB2 (1:25), in 5 mM phosphate buffer, 0.01% NaN3 , 4.48 g/l NaCl, at room temperature. Bound antibodies were detected with donkey anti-mouse IgG conjugated to Cy3 (Dianova, Hamburg, Germany), 1:1000 in PBS, for 1 h followed by washing in PBS. Thereafter, sections were fixed in 4% paraformaldehyde solubilized in 0.1 M phosphate buffer, pH 7.2. MAbs ED1 (directed to a rat CD68-like antigen; Serotec, Düsseldorf, Germany) and R73 (directed to the chain of the rat ␣/ T cell receptor; Serotec) were directly labeled using the Alexa 488 or Alexa 647 Zenon labeling kit (Invitrogen) and simultaneously bound to the sections, 1 h, room temperature. Thereafter, the slides were fixed again and cover-slipped in carbonate-buffered glycerol (pH 8.6). Sections were evaluated using a confocal laser scanning microscope (CLSM, Leica-TCS SP2 AOBS, Leica, Mannheim, Germany). For the detection of Alexa 488
Reduced mRNA expression of ARRB1 and ARRB2 during acute rejection We first investigated the expression of ARRB1 and ARRB2 mRNA during acute rejection of renal transplants by qRT-PCR and focused on the population of leukocytes accumulating in the lumina of graft blood vessels on day 4 post-transplantation. These cells are composed of about 50% monocytes in isografts and of about 73% monocytes in allografts and have been thoroughly characterized before (Grau et al. 2001). Expression of ARRB1 (Fig. 1A) and ARRB2 (Fig. 1B) was significantly reduced (p ≤ 0.05) at the mRNA level in intravascular leukocytes obtained from allogenic transplants (n = 4), compared with cells collected after isogenic transplantation (n = 4). The difference in Ct values of approximately 2.5 (Fig. 1) corresponds to an about six-fold reduction in ARRB mRNA expression. Reduced protein expression of ARRB2 during acute rejection To answer the question if ARRB expression is also changed at the protein level, immunoblots were performed and quan-
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Fig. 1. ARRB1 and ARRB2 mRNA expression by intravascular leukocytes isolated from renal isografts and allografts 4 days after transplantation. Changes in the expression levels of ARRB1 (A) and ARRB2 (B) were assessed by qRT-PCR. Porphobilinogen deaminase (PBGD) served as a reference gene. Gene expression levels are presented as Ct. *p ≤ 0.05 relative to leukocytes isolated after isogenic kidney transplantation (n = 4, per group). Box plots indicate median and percentiles 0, 25, 75, and 100.
tified by densitometry. Consistent with the qRT-PCR data, significantly (p ≤ 0.01; n = 5 each) reduced protein expression of ARRB2 was observed during acute rejection (Fig. 2C and D). In contrast, no difference in the ARRB1 protein expression was seen between leukocytes from allografts vs. isografts (Fig. 2A and B). Expression of ARRB1 and ARRB2 by leukocyte subpopulations So far, the experiments were performed on the entire intravascular population of mononuclear leukocytes. To examine ARRB1 and ARRB2 expression by monocytes and T cells separately, we stained frozen sections of kidneys on day 4 after allogenic transplantation. Intravascular monocytes and T lymphocytes were labeled with mAbs to ARRB1 (Fig. 3A). A similar expression pattern was seen for ARRB2 (Fig. 3B). In addition, we detected few ARRB1 and ARRB2 positive cells, negative for monocyte and T cell markers. Due to their low number, these cells were not identified. The results were confirmed on cytospots, prepared from cells isolated by perfusion of the graft vasculature (data not shown). In addition, two color flow cytometry was performed on mononuclear cells from day 4 grafts (Fig. 4). ARRB1 expression by monocytes (Fig. 4A and B) and T cells (Fig. 4C and D) was identical for isografts and allografts: about 80% of the monocytes and 40% of the T cells were ARRB1 immunoreactive. In contrast, the proportion of ARRB2 immunoreactive monocytes was reduced (p ≤ 0.01) in allografts (about 50%, n = 6) in comparison to isografts (about 80%, n = 6) (Fig. 4E and F). The proportion of ARRB2 positive T cells, however,
remained at about 40% in both, isograft and allograft perfusates (Fig. 4G and H).
Interaction of ARRB2 with I-B Recent studies demonstrated that ARRBs interact with I-B (Gao et al. 2004; Witherow et al. 2004) but this interaction has never been evidenced in primary leukocytes. Therefore, we immunoprecipitated ARRB2 from lysates of intravascular mononuclear isograft leukocytes. Indeed, ARRB2 and co-immunoprecipitated IB were detected by immunoblotting, confirming an interaction of both proteins in vivo. In control experiments, where antibodies to ARRB2 were replaced by irrelevant antibodies from the same species, neither ARRB2 nor I-B were detected. In the cell lysates prior to immunoprecipitation both, ARRB2 and I-B, were present (Fig. 5A).
I-B in intravascular leukocytes from renal iso- and allografts Given the role of the interactions between ARRB2 and I-B on I-B stability (Gao et al. 2004; Kizaki et al. 2008), we investigated IB levels in intravascular leukocytes on day 4 post-transplantation by immunoblotting (Fig. 5B and C). As expected, I-B levels were strongly reduced (p ≤ 0.01) in leukocytes from allografts in comparison to isografts (n = 5 each). Statistical evaluation revealed a positive linear correlation of I-B and ARRB2 protein expression levels (r = 0.977, 95%-CI = 0.903–0.995, p ≤ 0.001) (Fig. 5D).
Fig. 2. ARRB1 and ARRB2 protein expression by intravascular leukocytes isolated from renal isografts and allografts 4 days after transplantation. Expression of ARRB1 (A) and ARRB2 (C) was assessed by immunoblotting. Detection of GAPDH was performed to ensure equal protein loading. Densitometric quantification of the blots is depicted in Fig. (B) and (D) for ARRB1 and ARRB2, respectively. *p ≤ 0.01 relative to leukocytes isolated after isogenic kidney transplantation (n = 5, per group).
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Fig. 3. Localization of ARRB1 and ABBB2 in intravascular leukocytes. Frozen sections of renal allografts on day 4 after transplantation were triple-stained with fluorescently labeled antibodies and analyzed by confocal laser scanning microscopy. The micrographs depict details of graft veins with leukocyte accumulations in situ. ARRB1 (A) and ARRB2 (B) were visualized in red color. MAbs to a CD68-like antigen (ED1, green) and ␣/ T cell receptor positive T cells were detected with mAb R73 (blue). Representative sections out of three individual experiments are shown.
Expression of NF-B target genes As the activity of the NF-B transcription factor family strongly depends on the levels of endogenous I-B, we measured the mRNA expression of the classical pro-inflammatory monokines IL-1 and TNF-␣, which are regulated by NF-B. As predicted, the mRNA expression of IL-1 (Fig. 5E) and TNF-␣ (Fig. 5F) was increased in intravascular leukocytes obtained from allografts, compared with isograft cells (p < 0.05, n = 5). Discussion We demonstrate for the first time a reduction in ARRB2 expression by intravascular graft leukocytes during acute rejection of kidney allografts. Interestingly, the decrease in ARRB2 protein expression is only observed in monocytes but not in T cells. Reduced ARRB2 expression correlate with reduced levels of I-B protein and concomitantly, expression of NF-B targeted genes increases. Furthermore, we directly demonstrate that ARRB2 interacts with I-B in blood leukocytes freshly isolated from renal isografts. Systematic analysis of proteins interacting with ARRB1 and ARRB2 in the HEK293 cell line revealed more than 300 partially overlapping interaction partners (Xiao et al. 2007), suggesting that ARRBs exert numerous functions, which are just beginning to be understood. Interactions of ARRBs regulate a variety of signaling cascades and mediate the crosstalk between different signaling pathways. ARRB1 and ARRB2 were initially described as regulators of GPCR signaling. Upon ligand binding and phosphorylation of the receptors by GPCR kinases (GRKs), ARRBs form complexes causing receptor desensitization and internalization (Luttrell and Lefkowitz 2002; Shenoy and Lefkowitz 2003). Several additional functions of ARRBs have been discovered (Lefkowitz et al. 2006; Xiao et al. 2007). ARRBs serve as scaffold proteins that interact with cytosolic partners such as Src family kinases (Luttrell et al. 1999) and with components of the ERK1/2 and JNK3 MAP kinase cascades (McDonald et al. 2000; Tohgo et al. 2003). Additionally, ARRB1 was detected in the nucleus associated with transcription factors such as p300 and cAMP-response element-binding protein (Ma and Pei 2007). Here, we focus on the influence of ARRBs on the NF-B/I-B system because of its outstanding role in the activation of monocytes/macrophages (Wei and Zheng 2003; Bonizzi and Karin 2004; Gao et al. 2004; Witherow et al. 2004; Qi et al. 2008). In primary leukocytes freshly isolated from the blood vessels of renal isografts, we demonstrate a direct interaction of ARRB2 with I-B. This inter-
action was shown to prevent phosphorylation and degradation of I-B, resulting in an attenuation of NF-B activation and transcription of NF-B targeted genes in several cell lines (Gao et al. 2004; Witherow et al. 2004). Intravascular graft leukocytes from renal allografts down-regulated the expression of both ARRB2 mRNA and protein. Hence, ARRB2 protein levels are at least in part regulated on the mRNA level. We postulated that the observed decrease in ARRB2 expression by allograft monocytes reduces I-B stability and results in NF-B activation. Accordingly, I-B protein levels were lower in allograft leukocytes compared to isograft leukocytes and the mRNA expression of the NF-B target genes IL-1 and TNF␣ was up-regulated. These results are in line with our previous conventional PCR data on the same experimental model, which indicated that the NF-B target genes TNF-␣, iNOS and TF are upregulated during acute rejection (Grau et al. 2001). Our in vivo study does not directly indicate a link between the decrease in ARRB2 expression and I-B destabilization, resulting in NF-B activation, however, previous in vitro data strongly suggest such a causal connection (Gao et al. 2004; Witherow et al. 2004). In contrast to ARRB2, leukocytic ARRB1 protein expression was not affected during rejection, whereas decreased ARRB1 mRNA levels were seen. As ARRB1 is highly homologous to ARRB2, it may also interact with I-B and compensate for the decreased expression of ARRB2. However, ARRB1 and ARRB2 proteins differ in crucial aspects. They have different binding affinities to GPCRs (Oakley et al. 2000), by association to different binding partners (Lefkowitz and Shenoy 2005), as well as by differences in their subcellular localization (Wang et al. 2003). Furthermore, selective over-expression of ARRB1 or ARRB2, leads to a marked inhibition of NF-B signaling (Gao et al. 2004; Witherow et al. 2004) and selective suppression of ARRB1 or ARRB2 expression leads to an induction of NF-B activity (Gao et al. 2004; Witherow et al. 2004). These results suggest that both, ARRB1 and ARRB2, can stabilize IB protein, but a decreased expression of one of them is sufficient to affect NF-B signaling. ARRB1 may be essential for other aspects of inflammation and is therefore not down-regulated during acute rejection: It promotes CD4+ T cell survival and consequently animals deficient in ARRB1 are less prone to develop experimental encephalomyelitis (Shi et al. 2007). Interestingly, the decrease in ARRB2 protein levels is only observed in intravascular allograft monocytes but not in T lymphocytes, suggesting that the expression of ARRB2 is regulated in a cell specific manner. However, almost nothing is known on the mechanisms regulating ARRB2 expression. Kizaki et al. reported a strong correlation between ARRB2 expression and beta adrener-
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Fig. 4. ARRB1 and ARRB2 expression by intravascular leukocytes subpopulations. Percentage of ARRB1 (B and D) and ARRB2 (F and H) positive monocytes and T cells was obtained by two-color flow cytometry analyses. Anti-ARRB1 and anti-ARRB2 antibodies labeled with PE were used to detect intracellular ARRBs levels, whereas PE labeled IgG1 and IgG2a antibodies of irrelevant specificity served as isotype controls. FITC labeled mAb anti-CD172 (ED9) was used to identify monocytes, whereas a FITC labeled mAb to CD5 (OX19) served as a marker for T cells. *p ≤ 0.01 relative to leukocytes isolated after isogenic kidney transplantation (n = 6, per group). Representative results out of six independent experiments are illustrated (A, C, E, and G). Box plots indicate median and percentiles 0, 25, 75, and 100.
gic 2 receptor level (2AR) in a macrophages cell line. According to their observation, reduction of 2AR levels upon LPS stimulation results in a down-regulation of ARRB2 by a yet unknown mechanism (Kizaki et al. 2008). Endogenous ligands of toll-like receptors such as high-mobility group box 1 have been described to be involved in acute rejection of heart allografts (Duan et al. 2010) and are probably also released by renal allografts. Hence activation of toll-like receptors might also happen in monocytes during allograft rejection, because the mRNA level of 2AR in allograft blood leukocytes is significantly reduced in comparison to cells obtained from isografts (data not shown). In our experimental setting we cannot decide if the reduction in monocytic ARRB2 expression in allografts in comparison to isografts is due to gene
regulation or due to a change in frequencies of different monocyte subpopulations. Differential regulation of ARRB2 in monocytes and T cells is probably of functional importance. T cells in ARRB2 deficient mice exhibit migration defects in models for asthma and metastatic tumor growth (Walker et al. 2003; Raghuwanshi et al. 2008). Accordingly, migration of T cells does not seem to be impaired in our model of acute rejection. In contrast, in experimental endotoxemia, which involves pro-inflammatory monocytes/macrophages, ARRB2 deficiency results in higher expression of pro-inflammatory cytokines and in an increased sensitivity to endotoxin (Wang et al. 2006). This is in line with our data, implying a link between decreased ARRB2 expression and increased expression
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Fig. 5. Interaction of ARRB2 with the I-B/NF-B system. Interaction between ARRB2 and I-B was analyzed by immunoprecipitation (A). Cell lysate obtained from intravascular leukocytes 4 days after isogenic transplantation was immunoprecipitated with goat anti-ARRB2 antibody. Irrelevant goat antibodies were used as negative control. Precipitates were separated by SDS-polyacrylamide gel electrophoresis. Immunoblots (IB) with anti-ARRB2 antibodies were performed to verify efficient immunoprecipitation, and with anti-I-B antibodies to detect interaction. Lysate used for immunoprecipitation was analyzed to control for the presence of both proteins. Representative results of three independent experiments are shown. I-B protein level was analyzed by immunoblotting (B). Protein extracts of mononuclear leukocytes isolated from renal isografts and allografts on day 4 post-transplantation were resolved on 12% SDS-polyacrylamide gel and immunoblotting was performed with anti-I-B mAb. Detection of GAPDH served as a loading control. The intensity of the I-B signal was quantified by densitometry analysis (C). *p ≤ 0.01 relative to leukocytes isolated after isogenic kidney transplantation (n = 5, per group). Correlation between normalized OD values of I-B and ARRB2 was assessed by Welch t-test (D), (r = 0.977, 95%-CI = 0.903–0.995*, p ≤ 0.001, *asymptotic CI based on Fisher’s Z-transform). The mRNA expression of NF-B regulated genes IL-1 (E) and TNF-␣ (F) were assessed by quantitative PCR. Porphobilinogen deaminase (PBGD) served as a reference gene. Changes in gene expression are presented as Ct. *p ≤ 0.05 relative to leukocytes isolated after isogenic kidney transplantation (n = 4, per group). Box plots indicate median and percentiles 0, 25, 75, and 100.
of pro-inflammatory monokines. To elucidate the roles of ARRB2 and ARRB1 in organ rejection, more studies are needed including organ transplantation in mice over-expressing ARRBs or in mice with targeted ARRB deficiencies in different leukocyte subpopulations. In conclusion, we demonstrate for the first time differential ARRB2 expression in vivo during acute allograft rejection. The current data strongly support the hypothesis that decreased expression of ARRB2 by cells of the innate immune system is involved in the pathogenesis of acute inflammatory diseases. Reduced expression of ARRB2 might play a critical role in activation of monocytes during rejection. Strategies aiming at an induction of ARRB2 expression by allograft monocytes could improve transplant outcome.
Acknowledgments The authors wish to thank Sabine Stumpf, Petra Freitag, Renate Plaß, and Kathrin Petri for excellent technical assistance, Sandra Iffländer for experimental animal care, as well as Ulrike Berges and Dariusz Zakrzewicz for help with the art work. These studies were supported by a Junior Research Grant of the Medical Faculty of the University of Giessen (to A.Z.). References Adams, S., van der Laan, L.J., Vernon-Wilson, E., Renardel de Lavalette, C., Dopp, E.A., Dijkstra, C.D., Simmons, D.L., van den Berg, T.K., 1998. Signal-regulatory protein is selectively expressed by myeloid and neuronal cells. J. Immunol. 161, 1853–1859.
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