Down-Regulated during Inflammation Mononuclear Blood Leukocytes ...

The Journal of Immunology

Neuropeptide Y Is Expressed by Rat Mononuclear Blood Leukocytes and Strongly Down-Regulated during Inflammation1 Julia Holler,2* Anna Zakrzewicz,2* Andreas Kaufmann,‡ Jochen Wilhelm,§ Gabriele Fuchs-Moll,* Hartmut Dietrich,¶ Winfried Padberg,* Jitka Kuncova´,储 Wolfgang Kummer,† and Veronika Grau3* Neuropeptide Y (NPY), a classical sympathetic comediator, regulates immunological functions including T cell activation and migration of blood leukocytes. A NPY-mediated neuroimmune cross-talk is well conceivable in sympathetically innervated tissues. In denervated, e.g., transplanted organs, however, leukocyte function is not fundamentally disturbed. Thus, we hypothesized that NPY is expressed by blood leukocytes themselves and regulated during inflammation. NPY mRNA and peptide expression were analyzed in mononuclear leukocytes isolated from the blood vessels of healthy rat kidneys, as well as from the blood vessels of isogeneic and allogeneic renal grafts transplanted in the Dark Agouti to Lewis or in the Fischer 344 to Lewis rat strain combination. Depending on the donor strain, acute allograft rejection is either fatal or reversible but both experimental models are characterized by massive accumulation of intravascular leukocytes. Leukocytes, predominantly monocytes, isolated from the blood vessels of untreated kidneys and isografts expressed high amounts of NPY mRNA and peptide, similar to expression levels in sympathetic ganglia. During acute allograft rejection, leukocytic NPY expression drastically dropped to ⬃1% of control levels in both rat strain combinations. In conclusion, NPY is an abundantly produced and tightly regulated cytokine of mononuclear blood leukocytes. The Journal of Immunology, 2008, 181: 6906 – 6912.

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europeptide Y (NPY)4 links the sympathetic nervous system to the immune system, as NPY-releasing sympathetic nerves innervate lymphoid organs and NPY receptors are expressed by leukocytes (1–3). Among other functions, NPY strongly regulates T cell function, Ag presentation, blood leukocyte adhesion to endothelia, and leukocyte transmigration (2–5). Whereas a sympathetic modulation of leukocytes in innervated organs represents a widely accepted concept, it cannot be involved in the local regulation of leukocyte adhesion and migration seen in denervated organs such as grafts during the first days after transplantation. This raises the question for other regulated sources of *Department of General and Thoracic Surgery, Laboratory of Experimental Surgery, and †Institute for Anatomy and Cell Biology, University of Giessen Lung Center, Justus-Liebig-University Giessen, Giessen, Germany; ‡Institute of Immunology, Philipps University Marburg, Marburg, Germany; §Department of Pathology, and ¶Department of Internal Medicine (Nephrology), Justus-Liebig-University Giessen, Giessen, Germany; and 储Department of Physiology, Charles University Medical School Plzenˇ, Plzenˇ, Czech Republic Received for publication October 9, 2007. Accepted for publication September 10, 2008. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1

A part of this study was supported by the Czech Republic (Grant MSM 0021620819).

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J.H. and A.Z. equally contributed to this study.

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Address correspondence and reprint requests to Prof. Dr. Veronika Grau, Department of General and Thoracic Surgery, Laboratory of Experimental Surgery, University of Giessen Lung Center, Justus-Liebig-University Giessen, Rudolf-Buchheim Strasse 7, D-35385 Giessen, Germany. E-mail address: Veronika.Grau@chiru. med.uni-giessen.de

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Abbreviations used in this paper: NPY, neuropeptide Y; iNOS, inducible NO synthase; CT, cycle threshold.

Copyright © 2008 by The American Association of Immunologists, Inc. 0022-1767/08/$2.00 www.jimmunol.org

NPY. Reports on NPY expression by blood leukocytes are scarce and no peptide data exist (6, 7). Platelets secrete NPY (6, 8) and NPY mRNA expression can be induced in vitro in human leukocytes by stimulation with T cell mitogens or nerve growth factor (7, 9). In this study, we determined whether NPY is expressed by mononuclear blood leukocytes in vivo and, if so, whether its expression is changed during inflammation. Healthy rat kidneys and recently transplanted renal grafts are ideal experimental models for this purpose because 1) intravascular blood leukocytes, the population interacting with endothelial cells (the so-called marginal pool) and the population of leukocytes moving with the central blood stream, can be isolated by vascular perfusion; 2) a huge number of intravascular and infiltrating mononuclear leukocytes accumulates during acute rejection, one of the most vigorous immune reactions known (10 –13); and 3) since renal grafts are denervated during surgery, neurons can be excluded as a source of NPY. In this study, we use two different experimental models of acute renal allograft rejection, a fatal and a reversible one: 1) after transplantation in the Dark agouti (DA) to Lewis (LEW) rat strain combination, the kidneys are irreversibly destroyed by fulminant acute rejection within 5 days after transplantation. Accumulating intravascular graft leukocytes isolated on day 4 during the phase of graft destruction have been extensively characterized by our group (11, 12). As evidenced by flow cytometry, ⬃14% of the intravascular allograft leukocytes are T cells and 73% are monocytes. In contrast to isograft leukocytes, allograft leukocytes express high levels of inducible NO synthase (iNOS), TNF-␣, and tissue factor. Furthermore, allograft monocytes are cytotoxic and express a pattern of cell surface marker proteins typical for monocyte activation (11, 12). 2) In the second experimental model, Fischer 344 (F344) kidneys transplanted to LEW recipients survived for at least 6 mo but developed lesions typical for chronic


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Table I. Characteristics of primers used for RT-PCR Gene

Primer

Sequence

Fragment Size (bp)

GAPDH

Sense Antisense Sense Antisense Sense Antisense

5⬘-CGTCTTCACCACCATGGAGA-3⬘ 5⬘-CGGCCATCACGCCACAGTTT-3⬘ 5⬘-GCTCTATCCCTGCTCGTGTGTT-3⬘ 5⬘-GTAGTGTCGCAGAGCGGAGTA-3⬘ 5⬘-GGATGTGGCTACCACTTTGAA-3⬘ 5⬘-AAAAGACCGCACCGAAGA-3⬘

300

NPY iNOS

rejection. We investigated a reversible early acute rejection episode peaking at day 9 posttransplantation and demonstrate that even more leukocytes accumulate compared with the DA to LEW model. In this study, we demonstrate for the first time that normal blood leukocytes strongly express NPY in vivo and that this NPY expression is drastically down-regulated during inflammation.

Materials and Methods Renal transplantation, tissue sampling, and isolation of mononuclear leukocytes Male LEW(RT1l), DA(RT1av1), and F344(RT1lv1) rats were provided by Harlan Winkelmann. Surgery was performed in LEW recipients weighing 250 –300 g. For allogeneic transplantation, DA or F344 rats and for isogeneic transplantations LEW rats were used as donors. Animal care and animal experiments were performed following the current version of the German Law on the Protection of Animals as well as the National Institutes of Health principles of laboratory animal care. Kidneys were transplanted orthotopically to totally nephrectomized recipients mainly according to the technique described by Fabre et al. (14), except that the ureter was anastomosed end-to-end. Total ischemic times remained below 30 min. After transplantation in the fully allogeneic DA to LEW rat strain combination, allograft recipients died within 7.4 ⫾ 0.7 days (mean ⫾ SD, n ⫽ 10) after transplantation, whereas isograft recipients survived in good health (n ⫽ 10) (11). F344 allografts in LEW recipients passed through a reversible acute rejection episode but retained live sustaining function for more than 6 mo (15, 16). This rat strain combination is characterized by a minor difference in the MHC class I locus. Control rats and graft recipients were sacrificed by inhalation of an overdose of isoflurane (Forene; Abbott Laboratories) for tissue sampling. Kidneys were removed, cut in pieces, and either snap frozen in liquid nitrogen for RNA isolation or fixed for paraffin histology. Stellate ganglia were excised for RNA isolation under a microscope and snap frozen as well. Mononuclear leukocytes were isolated by vascular perfusion and density gradient centrifugation from normal LEW kidneys and from grafts as described in detail previously (12). The animals were anesthetized by i.p. application of 60 mg/kg sodium pentobarbital (Narcoren; Merial). In the vicinity of the renal blood vessels, the inferior vena cava and the abdominal aorta were dissected and all local lumbar vessels were ligated and the rats received an i.v. injection of 200 U of heparin (Liquemin N 5000; Roche). The vena cava and the aorta were ligated cranial to the left renal vessels. A 1.4 ⫻ 2.1-mm vein catheter (Braun) was inserted into the vena cava inferior and a 0.5 ⫻ 0.9-mm vein catheter (Braun) was inserted into the abdominal aorta. The experimental animals were killed by opening the pleural cavity, blood from the central blood stream was obtained by heart puncture, and, finally, the thoracic aorta and the vena cava were transected. Kidneys were perfused with cold Ca2⫹- and Mg2⫹-free PBS (PAA) containing EDTA (2.7 mM) and 0.1% BSA (Serva), and 100 ml of perfusate were collected. Percoll (Pharmacia Biotech) isopycnic density gradient centrifugation was conducted to deplete erythrocytes, granulocytes, and platelets (9). Cell recovery ranged from 63 to 94%. Perfused grafts were fixed in freshly dissolved buffered paraformaldehyde for conventional paraffin histology and hemalum-eosin staining.

Flow cytometry Primary Abs to rat cell surface Ags were purchased from Serotec. Ab staining and flow cytometry were mainly performed as described previously (12). In brief, to analyze the leukocyte composition of perfusate cells and mononuclear leukocytes purified by density gradient centrifugation, the cells were resuspended in an appropriate volume of PBS/0.1% BSA

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(PBS/BSA) and subjected to flow cytometry using FITC-labeled mAbs ED9 (CD172a), Ox19 (CD5), Ox33 (CD45R, specific for rat B cells), and 10/78 (CD161). CD172a-positive cells were further differentiated according to their physical parameters in monocytes (low granularity) and granulocytes (high granularity). Flow cytometry was performed on a FACSCalibur flow cytometer (BD Biosciences) using the CellQuest software 3.2.1 (BBD Biosciences). Linear amplification was used for forward scatter and side scatter and logarithmic amplification for fluorescence. Leukocytes were separated from debris, cell aggregates, and erythrocytes applying a broad life gate. Thirty thousand events were measured in each sample.

Cell culture Mononuclear leukocytes were isolated from the blood vessels of normal LEW kidneys. Cells were transferred to 24-well plates (Greiner bio-one) in RPMI 1640 (PAA) supplemented with 10% heat-inactivated FBS (PAA), 2 mM L-glutamine, penicillin, and streptomycin (PAA). Per well, 0.7– 0.8 million cells were cultured in 1 ml of medium for 1, 2, 6, 12, or 24 h at 37°C in 5% CO2. Thereafter, RNA was isolated from adhering and nonadhering leukocytes together.

Quantitative RT-PCR RNA was isolated with the RNeasy Mini kit (Qiagen) from 0.7 to 5 ⫻ 106 mononuclear leukocytes each, from sympathetic stellate ganglia that served as reference for NPY expression and from pieces of kidneys (both from healthy LEW rats). Contaminating DNA was destroyed (RNase free DNase kit; Qiagen), cDNA was synthesized (Omniscript reverse transcriptase; Qiagen), and real-time quantitative PCR was performed in triplicates with an iCycler (Bio-Rad) using the QuantiTec SYBR Green PCR kit (Qiagen). Primer pairs specific for rat GAPDH, NPY, and iNOS (MWG Biotech) (Table I) were used at concentrations of 0.6 ␮M. PCR conditions were 15 min at 94°C, 45 cycles of 30 s at 94°C, 30 s at 60°C, and 30 s at 72°C. To assess the homogeneity and identity of the PCR products, the melting curves were analyzed, the products were separated on agarose gels, and sequenced (Applied Biosystems Prism 310 Sequencer, Applied Biosystems cycle-sequencing protocol). Controls omitting the reverse transcription step or the template were always performed. The relative gene expression values were calculated by the equation 2⫺⌬CT, where ⌬CT is the difference in CT values between the housekeeping gene GAPDH and the gene of interest. Data for each experimental group are expressed in relation to the expression in the control which was set to 1 arbitrary unit.

Radioimmunoassay Peptides were extracted from 5 ⫻ 106 mononuclear leukocytes in 0.1 M acetic acid at 95°C for 15 min. Thereafter, the samples were chilled, homogenized, and centrifuged. The supernatant was neutralized with Tris base, centrifuged, and stored at ⫺70°C until RIA. Plasma was collected by heart puncture. Peptides were extracted from the plasma with ice-cold ethanol. After centrifugation, the pellet was extracted again with 70% ethanol and the supernatants of both extraction steps were pooled, evaporated, and stored at ⫺70°C. NPY concentrations were assayed in duplicates using a commercial RIA kit (Phoenix Pharmaceuticals).

Statistics Data are reported as means ⫾ SD and were analyzed by the Kruskal-Wallis test followed by the Mann-Whitney U rank sum test (SPSS software) with p ⱕ 0.05 set as level for significance.

Immunhistochemistry Kidneys were fixed in 4% buffered paraformaldehyde and embedded in paraffin. Sections of 6 ␮m were dewaxed and rehydrated. To unmask Ags in the fixed tissue, Ag retrieval was performed in 0.01 M sodium citrate buffer (pH 6.0) for 15 min at 120°C, 1.1 bar, followed by 1% H2O2 in PBS

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NPY EXPRESSION BY BLOOD LEUKOCYTES Table II. Percentage of leukocytes obtained from the vasculature of isogeneic LEW to LEW and allogeneic F344 to LEW kidneys on day 9 after transplantation analyzed by flow cytometrya Leukocyte No. (Million)

Isogeneic (n ⫽ 5) Perfusate Mononuclear leukocytes Allogeneic (n ⫽ 5) Perfusate Mononuclear leukocytes

Monocytes

10 ⫾ 6 6.5 ⫾ 4.7

49.1 ⫾ 7.4 58.8 ⫾ 4.3

152 ⫾ 17 123 ⫾ 23

69.8 ⫾ 4.0** 71.5 ⫾ 1.7**

Granulocytes

T Cells

15.8 ⫾ 10.2 0.2 ⫾ 0.1 2.0 ⫾ 1.0** 0.2 ⫾ 0.1

B Cells

NK Cells

19.1 ⫾ 4.2 23.6 ⫾ 3.8

7.3 ⫾ 1.1 7.5 ⫾ 0.8

8.0 ⫾ 2.9 8.8 ⫾ 1.3

19.1 ⫾ 1.2 17.9 ⫾ 1.5*

3.6 ⫾ 1.7* 3.3 ⫾ 1.8**

4.8 ⫾ 0.3** 4.9 ⫾ 0.6**

a The perfusates contain all leukocytes harvested and the mononuclear leukocytes were subsequently purified by Percoll density gradient centrifugation. The data are expressed as means ⫾ SD; significant difference between isotransplanted and allotransplanted animals, ⴱ, p ⱕ 0.05 and ⴱⴱ, p ⱕ 0.01, respectively.

(pH 7.2), 1% BSA (Serva), and 0.1% NaN3 (Merck) in PBS for 30 min each. For single staining, polyclonal rabbit Abs to porcine NPY (Sigma-Aldrich) were diluted 1/2500 in PBS/BSA/NaN3. These Abs cross-react with rat NPY because porcine (accession no. ABD66744) and rat (accession no. NP_036746) NPY only differ in a single amino acid. Bound Abs were detected using the anti-rabbit Ig EnVision peroxidase system (DakoCytomation) and 3,3⬘-diaminobenzidine (Sigma-Aldrich) as chromogen. As a control, the primary Ab was omitted. Selected sections were mildly counterstained with hemalum. ED1 (Serotec) was detected after Ag retrieval using the anti-mouse/ rabbit Ig EnVision alkaline phosphatase (DakoCytomation) containing 5% heat-inactivated normal rat serum and the chromogen Fast Blue. Antimouse/rabbit Ig EnVision alkaline phosphatase binds to mouse and to rabbit Abs. HIS52 (Serotec) was detected without prior Ag retrieval with a two-layer detection system consisting of rabbit anti-mouse Ig (DakoCytomation), followed by anti-mouse/rabbit Ig EnVision alkaline phosphatase and Fast Blue. After the first staining step, the sections were treated with citrate buffer (pH 6.0) for 15 min at 120°C, 1.1 bar, to remove Abs and enzymes. NPY was detected with 3,3⬘-diaminobenzidine in the second staining step. Three kinds of controls were performed: 1) omission of primary Abs in both staining steps, 2) omission of ED1 or HIS52, and 3) omission of NPY Abs. Sections were evaluated with an Olympus BX51 microscope.

transplantation, 10 ⫾ 6 million leukocytes were harvested per isograft (n ⫽ 5). In comparison to isografts, cell numbers were dramatically increased ( p ⱕ 0.01) in the blood vessels of allografts from which 152 ⫾ 17 million leukocytes were harvested (n ⫽ 5). The cellular composition of this cell population was analyzed by flow cytometry and is summarized in Table II. To study NPY expression on the mRNA and on the peptide level, mononuclear leukocytes were purified by density gradient centrifugation to eliminate erythrocytes, granulocytes, and platelets from the samples (Table II). As revealed by paraffin histology, the vessels of perfused renal grafts were devoid of blood cells, indicating that the perfusates

Results Leukocyte isolation from renal blood vessels In this study, we focused on the population of leukocytes in the lumina of blood vessels of normal rat kidneys and transplants. These cells include leukocytes floating with the central blood stream and the so-called marginal pool of blood leukocytes which interact with endothelial cells. After transplantation in the fully allogeneic DA to LEW rat strain combination, 90.6 ⫾ 17.3 million leukocytes (n ⫽ 10) were isolated on day 4 posttransplantation, 8.9 ⫾ 2.8 million leukocytes were obtained from the blood vessels of day 4 isografts (n ⫽ 10), and 5.1 ⫾ 0.3 million leukocytes from untreated LEW kidneys (n ⫽ 5). These cell numbers are in good accordance with published data on this experimental model (12, 13). The cellular composition of the intravascular graft leukocytes has been characterized before by flow cytometry (12). In short, monocytes were most numerous in the cell population isolated from untreated LEW kidneys (⬃62%), LEW to LEW isografts (50%), and DA to LEW allografts (73%) followed by T cells which amounted to ⬃20% in untreated kidneys, 31% in isografts and 14% in allografts (12). The percentage of B cells, NK cells, and granulocytes remained below 10% throughout (12). To identify the maximum of intravascular leukocyte accumulation during reversible acute rejection in orientating experiments, intravascular leukocytes were isolated from F344 to LEW renal allografts at intervals of 2–3 days until day 20 posttransplantation (data not shown). This rat strain combination is characterized by a minor MHC class I mismatch. Intravascular leukocyte accumulation peaked around day 9 after transplantation: On day 9 post

FIGURE 1. Real-time quantitative RT-PCR analysis of NPY mRNA expression by stellate ganglia (SG; n ⫽ 5) as well as intravascular mononuclear leukocytes isolated from normal control kidneys (C), renal isografts (iso; LEW3 LEW), and allografts (allo; DA3 LEW and F3443 LEW) on days 4 and 9 after transplantation (n ⫽ 4 each). The box plots (A) show median and percentiles 0, 25, 75, and 100; ⴱ, p ⱕ 0.05. Data are expressed as arbitrary units which are normalized to 1 U in controls. The PCR product was separated in agarose gels to confirm product size and homogeneity (B). M, Marker; K, kidney.


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FIGURE 2. Analysis of NPY peptide expression by RIA (A and B) and immunohistochemistry (C–H). NPY was measured in mononuclear leukocytes (A) isolated from normal kidneys (LEW), renal isografts (iso; LEW3 LEW), and allografts (allo; DA3 LEW) on day 4 posttransplantation. The box plots (A) show median and percentiles 0, 25, 75, and 100; ⴱⴱ, p ⱕ 0.01. NPY immunoreactivity was measured in plasma samples obtained by heart puncture (B). The small asterisk represents data beyond ⫾ three times SD. NPY-immunopositive perivascular nerve endings (arrows) are stained in brown on paraffin sections of normal LEW kidneys (C). These structures are missing in day 4 isografts (D). NPY immunoreactivity is abundant in small blood vessels (arrows) of isografts (E), whereas allograft (F) single cells are only faintly stained (arrows). The sections (E and F) were mildly counterstained with hemalum. Double-staining experiments were performed in isografts (G and H) with mAb HIS52 (G) to stain endothelial cells (blue). NPY-expressing cells (brown) are detected in the lumina of blood vessels. ED1 was used to detect monocytes (blue) which coexpress NPY (brown).

contained both the population of the central blood stream and the so-called marginal cell pool. Expression of NPY by intravascular graft leukocytes NPY mRNA was readily detected by real-time RT-PCR in mononuclear leukocytes from the blood vessels of normal rat kidneys (n ⫽ 4; Fig. 1A). Leukocytic NPY mRNA expression did not change 4 days after isogeneic transplantation (n ⫽ 4) in the LEW rat, whereas a drastic down-regulation to ⬃1% of control levels

was obvious in day 4 allografts (n ⫽ 4, p ⱕ 0.05; Fig. 1A) which were transplanted in the DA to LEW rat strain combination. In the DA to LEW strain combination, renal allografts are irreversibly destroyed between days 4 and 5, whereas isografts were accepted (11). Day 4 was chosen because intravascular mononuclear leukocytes are most numerous at this point in time (12, 13). To exclude the possibility that the reduction of NPY mRNA just results from organ destruction, F344 to LEW allografts were investigated. In our laboratory, LEW recipients of F344 allografts

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NPY EXPRESSION BY BLOOD LEUKOCYTES

survived for at least 6 mo after transplantation. The NPY mRNA content of mononuclear leukocytes from day 9 F344 to LEW allografts (n ⫽ 4) was also drastically reduced in comparison to controls and to day 9 isografts (n ⫽ 4, p ⱕ 0.05; Fig. 1A). Leukocytic NPY mRNA expression was unexpectedly high in leukocytes from healthy kidneys and isografts. Compared with the sympathetic stellate ganglion, an abundant source of neuronal NPY (17), leukocytic mRNA expression was in the same range (Fig. 1). Because NPY mRNA expression may be restricted to the socalled marginal pool of leukocytes which is harvested by renal perfusion, we additionally isolated mononuclear leukocytes from the central blood stream of healthy rats. NPY mRNA levels in these leukocytes were even higher compared with the corresponding mononuclear cells isolated by perfusion of the renal blood vessels (n ⫽ 5 each, p ⫽ 0.009). It should, however, be kept in mind that the proportion of the leukocyte subpopulations considerably differs among regular blood samples and renal perfusates (12). Gel electrophoresis of RT-PCR products resulted in a single band of the expected size (Fig. 1B) and product identity was confirmed by sequencing. Next, we tested by RIA whether NPY peptide was present. Mononuclear leukocytes isolated from the blood vessels of normal kidneys contained immunoreactivity corresponding to 578.5 ⫾ 192.8 pg of NPY per million cells (n ⫽ 5). In day 4 isografts, 178.4 ⫾ 93.3 pg/million cells were detected (n ⫽ 6; p ⱕ 0.01) and in allograft leukocytes the NPY content drastically dropped to 6.5 ⫾ 1.9 pg (DA to LEW, n ⫽ 6; p ⱕ 0.01) (Fig. 2A). Local changes in the NPY production by graft leukocytes were not mirrored by systemic NPY levels: NPY-like immunoreactivity in the plasma of graft recipients which was collected by heart puncture was not significantly changed during rejection. There was, however, a tendency toward higher levels in graft recipients compared with controls ( p ⱕ 0.1, n ⫽ 6 each; Fig. 2B). Histological localization of NPY immunoreactivity in renal grafts Finally, NPY expression was assessed on histological sections. NPY-immunoreactive nerve fibers innervated arteries in normal kidneys (n ⫽ 4; Fig. 2C). These structures were absent in LEW to LEW isografts and DA to LEW allografts on day 4 after transplantation (n ⫽ 4 each), reflecting denervation during surgery (Fig. 2D). In renal isografts, strong NPY immunoreactivity was obvious in peritubular regions (Fig. 2E), most probably inside small blood vessels. Immunoreactivity was weaker although still detectable in allografts (Fig. 2F). Day 9 isografts and F344 to LEW allografts exhibited a similar distribution of NPY immunoreactivity (n ⫽ 4 each). In leukocytic infiltrates which are abundant in DA to LEW and in F344 to LEW allografts, almost no immunoreactivity was present. Control sections where the primary Ab was omitted remained unstained. To demonstrate the intravascular localization of NPY-expressing cells, double-staining experiments were performed with mAb HIS52. HIS52 recognizes RECA-A which is specifically expressed by endothelial cells. Indeed, most NPY immunoreactivity was localized inside the lumina of blood vessels (Fig. 2G). Since monocytes are the dominating cell population among intravascular graft leukocytes (12) we performed double-staining experiments with mAb ED1 (CD68-like) specific for monocytes and macrophages. Most NPY-immunoreactive leukocytes residing in the blood vessel of renal transplants were identified as ED1-positive monocytes (Fig. 2H). No double staining was seen on control sections.

FIGURE 3. NPY (A) and iNOS (B) mRNA expression in the tissue of renal grafts transplanted in the isogeneic (iso) LEW3 LEW (n ⫽ 5) and in the allogeneic (allo) DA3 LEW (n ⫽ 6) rat strain combination on day 4 posttransplantation. Kidneys from healthy LEW rats (n ⫽ 4) were used a control (C) The box plots show median and percentiles 0, 25, 75, and 100; ⴱ, p ⱕ 0.05. Data are expressed as arbitrary units which are normalized to 1 U in controls. Open circles indicate values above three times SD.

Expression of NPY mRNA in the graft tissue Immunohistochemistry of renal allografts suggested that graft-infiltrating leukocytes express low amounts of NPY. To corroborate this observation, we detected NPY mRNA by quantitative RTPCR in the tissue. As expected, NPY mRNA levels were low in the tissue and did not differ among untreated control kidneys (n ⫽ 4), isografts (n ⫽ 5), and allografts (n ⫽ 6; Fig. 3A). To exclude the possibility that mRNA originating from infiltrating leukocytes could not be detected by RT-PCR in the renal tissue, we analyzed iNOS mRNA which is predominantly expressed by activated macrophages, in the same samples. The mRNA of iNOS was significantly more abundant in allografts compared with untreated kidneys ( p ⫽ 0.014) and to isografts ( p ⫽ 0.016 Fig. 3B).


FIGURE 4. Time course of leukocytic NPY expression in vitro. Mononuclear leukocytes were isolated from healthy kidneys, cultivated for 1–24 h, and the level of NPY mRNA was analyzed by quantitative RT-PCR (n ⫽ 4). The box plots show median and percentiles 0, 25, 75, and 100; ⴱ, p ⱕ 0.05 different from leukocytes after 1 h of cell culture. Data are expressed as arbitrary units which are normalized to 1 U in the samples harvested after 1 h of cell culture.

Expression of NPY mRNA by mononuclear leukocytes in vitro Mononuclear cells isolated from the blood vessels of normal LEW rats (n ⫽ 4) were cultured and NPY mRNA levels were measured in the cells by quantitative RT-PCR after 1, 2, 6, 12, and 24 h in vitro (Fig. 4). After 12 h in culture, NPY mRNA levels significantly ( p ⫽ 0.021) decreased to ⬃25% of the levels measured after 1 h, and after 1 day in culture NPY mRNA levels went down to 4% of the initial values ( p ⫽ 0.021). The decrease in the NPY mRNA concentrations was not due to a general decrease in the mRNA levels, since the CT values for the housekeeping gene were not changed after cell culture for 1 day (data not shown).

Discussion Our study is the first to demonstrate that blood leukocytes. including monocytes. express large amounts of NPY mRNA and peptide under resting and mildly activated conditions. RNA expression levels are in the range of those in stellate ganglia which are an abundant source of NPY (17). Moreover, the NPY content of leukocytes is similar to that of the rat CNS (18). High NPY expression levels are seen in leukocytes isolated from normal kidneys and from renal isografts. As grafts are denervated during transplantation, leukocytic NPY cannot originate from the nervous system. Furthermore, we demonstrate a drastic down-regulation of local leukocytic NPY expression in the blood vessels of the graft during acute rejection. The decrease in NPY expression is not related to graft destruction since it is also seen during reversible acute rejection. Systemic NPY peptide levels, however, tend to increase after isogeneic and allogeneic transplantation. This is in accordance with data on human kidney transplant recipients that had higher plasma NPY levels compared with healthy controls (19, 20). Systemic plasma levels might mirror neuronal NPY release in response to perioperative stress or impaired renal NPY elimination. Our results suggesting strong NPY expression by resting rat blood leukocytes seem to be in contrast to data on human leukocytes where NPY mRNA was absent under resting conditions but induced by proinflammatory stimuli (7). At first sight, this discrep-

6911 ancy might be due to the fact that human blood samples exclusively contain leukocytes from the central blood stream and no cells belonging to the so-called marginal pool, whereas renal perfusate cells contain both leukocyte populations. However, we detected high NPY mRNA levels in leukocytes of the central blood stream of the rat as well, so that high NPY expression by leukocytes of the marginal pool cannot account for the observed differences. Instead, handling of cells before NPY mRNA measurement critically differed in these two studies. Human blood leukocytes were not analyzed directly after harvest but taken into culture. Monocytes were differentiated in vitro to macrophages and lymphocytes were purified by an E-rosetting technique which also involves incubation in vitro (7). Hence, absence of NPY expression in these cells is in line with our present data demonstrating a drastic decrease in NPY mRNA levels after 1 day of leukocyte culture. Monocytes are the dominating cell population in renal perfusates (12) and most NPY immunoreactivity was localized in these cells. Therefore, it is likely that the observed decrease in NPY expression is due to this cell population. The intensity of NPY expression inversely correlates with the activation and differentiation of the monocytes/macrophages: 1) perfusate cells from control kidneys and isografts that contain predominantly resting monocytes (12) express high NPY mRNA and peptide. 2) Low NPY levels were measured in blood leukocytes from renal allografts that contain highly activated monocytes (12). 3) Renal allograft tissue, which is strongly infiltrated by macrophages (11), expresses low levels of NPY mRNA. 4) During culture of leukocytes harvested from the blood vessels of normal rat kidneys, the levels of NPY mRNA drastically decreased, whereas no changes in the mRNA expression of the housekeeping gene were seen. It is well known that monocytes quickly differentiate in vitro to become macrophages. At present, we do not know the molecular mechanisms leading to the drastic decrease in intravascular NPY expression during acute rejection. A simple explanation may be that leukocytes expressing high levels of NPY infiltrate into the transplanted kidneys, leaving the leukocytes expressing low levels in the blood vessels. We can exclude this possibility because NPY mRNA did not increase in the allograft tissue, whereas iNOS mRNA, a typical product of infiltrating proinflammatory macrophages, strongly increased during rejection. Cell adhesion or proinflammatory mediators may be involved in down-regulating NPY. We therefore cultured leukocytes under nonadhering conditions but observed a similar down-regulation of NPY as in regular cell culture plates (data not shown). In orientating experiments, addition of pro- and anti-inflammatory recombinant rat cytokines (IFN-␥, IL-1␤, TNF-␣, IL-10) also neither accelerated nor decelerated the decrease in NPY expression in vitro. More studies are needed to elucidate the molecular pathways leading to the down-regulation of leukocytic NPY which may involve promising targets for the development of novel therapeutics. Numerous publications already underline the pivotal role of NPY in the regulation of the innate and the adaptive immune system (3, 4, 21–23). Intravenous application of NPY to rats quickly increases the number of CD4pos T cells, monocytes, and NK cells in the central blood stream (4). NPY appears to inhibit the interaction of these leukocytes with endothelial cells, resulting in their mobilizing from the so-called marginal pool. Furthermore, exogenous NPY application reduces tissue immigration of blood monocytes (21) and appears to regulate the phagocytic activity of monocytes/macrophages (22). Wheway et al. (3) recently demonstrated that the Y1 receptor for NPY is essential for the capability of APCs

6912 to prime T cells. At the same time, Th1 cell-mediated inflammatory responses are strongly down-regulated by NPY (3). This bimodal function of NPY on APCs and Th1 cells appears to be the reason why application of NPY in vivo blunts experimental immune diseases like septic shock or experimental autoimmune encephalomyelitis (21, 23) but promotes experimental colitis (24). In view of these data, it is attractive to hypothesize that NPY released from blood leukocytes under resting conditions silences T cells and prevents leukocyte accumulation inside the lumina of blood vessels as well as monocyte extravasation. At the same time, T cell priming by APCs might be enabled. During acute allograft rejection, NPY production is down-regulated in intravascular graft leukocytes in response to yet unknown signals. Accordingly, Th1 cell function, leukocyte accumulation in the marginal pool of allograft blood vessels, and graft infiltration are enabled. These are typical hallmarks of acute renal allograft rejection. APCs, however, might loose their ability to prime T cells when NPY levels decrease during rejection, which might eventually limit the immune reaction. In conclusion, this is the first report demonstrating NPY mRNA and peptide expression by blood leukocytes in vivo and its drastic down-regulation during inflammation. Thus, NPY shall be considered as a versatile cytokine abundantly produced and tightly regulated by mononuclear leukocytes rather than being interpreted solely as a neuronal one-way signal modulating immune functions.

Acknowledgments We thank Sigrid Wilker, Petra Hartmann, Kathrin Petri, Sandra Iffla¨nder, and Renate Plass for excellent technical assistance, Ulrike Berges for help with the art work as well as Klaus Steger for providing the iCycler.

Disclosures The authors have no financial conflict of interest.

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