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Klinik für Anaesthesiologie und Operative Intensivmedizin, Charité-Universitätsmedizin Berlin, Campus Benjamin Franklin (S.A.M., N.S., M.S., C.S.), D-12200 Berlin, Germany; and Institut für Anatomie, Charité-Universitätsmedizin Berlin, Campus Benjamin Franklin (M.S.), D-14195 Berlin, Germany
Address all correspondence and requests for reprints to: Shaaban A. Mousa, Ph.D., Klinik für Anaesthesiologie und Operative Intensivmedizin, Charité-Universitätsmedizin Berlin, Campus Benjamin Franklin, Hindenburgdamm 30, D-12200 Berlin, Germany. E-mail: shaaban.mousa{at}charite.de.
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Introduction |
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POMC is synthesized mainly in the neurointermediate lobe of the pituitary gland (8) and is the precursor of END and other peptides (e.g. corticotropin, ACTH). In contrast to the extensively studied classical posttranslational processing of POMC in the pituitary gland, little is known regarding END processing and pathways of release from immunocytes under inflammatory conditions. In the pituitary gland of adult mammals, POMC processing begins as the nascent polypeptide chain enters the endoplasmic reticulum (ER) directed by the signal peptide (9), and POMC cleavage begins in the trans-Golgi network (TGN) (10, 11). The POMC prohormone is directed to the regulated secretory pathway at the TGN by binding to a sorting receptor, identified as (membrane-bound) carboxypeptidase E (CPE) (12). Two mammalian prohormone convertases, PC1 (also called PC1/3) and PC2, cleave POMC and other peptide precursors within the TGN (13, 14). PC1/3 mediates the initial cleavage at paired basic residues into ACTH and ß-lipotropic hormone (ßLPH) (13, 15). The inactive pro-PC2 is bound to 7B2 (a chaperone-like binding protein) and is transported from the ER to later compartments of the secretory pathway, where it matures to active PC2 (16); thereafter, PC2 converts ßLPH into ß-MSH and END (17). The subsequent secretion of END requires secretory granules deriving from the Golgi network for transport to the cell membrane (18). In this study we examine whether the components of this classical secretory pathway are also present and functional within inflammatory cells. To this end we studied 1) POMC, CPE, PC1, PC2, 7B2, and END proteins by Western blotting; 2) the distribution of POMC, CPE, PC1, PC2, 7B2, and END immunoreactivity within cells of inflamed sc paw tissue and blood by single and double immunohistochemical techniques; 3) the subcellular distribution of END using immunoelectron microscopy; and 4) END release from immune cells upon incubation with noradrenaline in vitro.
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Subjects and Methods |
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Reagents
The following polyclonal rabbit antirat antibodies were used: anti-END with no cross-reactivity against ACTH (supplier’s information; Peninsula Laboratories, Belmont, CA; and ProGen Biotechnik GmbH, Heidelberg, Germany), anti-DP4 against a common epitope of ACTH and POMC (provided by Dr. Y. Peng Loh, NIH, Bethesda, MD), anti-POMC antiserum directed against the N-terminal amino acid sequence 27–52 (Phoenix Pharmaceuticals, Inc., Karlsruhe, Germany), anti-PC1/3 and anti-PC2 (provided by Drs. N. G. Seidah, Clinical Research Institute of Montréal, Québec, Canada; D. F. Steiner, University of Chicago, IL; and N. Birch, University of Auckland, Auckland, New Zealand), anti-CPE against the C or N terminal, respectively (provided by Drs. Y. Peng Loh and L. Fricker, Albert Einstein College of Medicine, Bronx, NY), and anti-7B2 (provided by Drs. N. G. Seidah, Clinical Research Institute of Montreal; P. Collini, Department of Pathology, University of Milan, Milan, Italy). Further materials included the Vectastain Elite Kit (Vector Laboratories, Inc., Burlingame, CA), paraformaldehyde and glutaraldehyde (Sigma-Aldrich Corp., St. Louis, MO), Freund’s complete adjuvant (FCA) (Calbiochem, La Jolla, CA), and halothane (Halocarbon Laboratories, Willy Rüsch GmbH, Boblingen, Germany).
Induction of inflammation
Male Wistar rats were sedated by brief halothane anesthesia and received an intraplantar injection of 0.15 ml FCA into the right hind paw. This treatment produces a localized inflammation of the inoculated paw characterized by increased susceptibility to painful stimuli (hyperalgesia) and increases in paw volume, paw temperature, and infiltration of various types of immune cells (3, 4, 19).
Western blot analysis of POMC, CPE, PC1, PC2, 7B2, and END in circulating leukocytes
The isolation of circulating leukocytes was accomplished from venous blood of animals without (control) and with paw inflammation (4 d after FCA treatment) using the dextran sedimentation method. Briefly, rats (n = 6/experiment) were anesthetized with halothane; blood samples (8 ml) were obtained by direct parasternal cardiac puncture and kept in heparin-coated tubes. NaCl solution (0.9%) containing 3% dextran was pipetted into a tube, and an equal volume of blood was layered on top and kept for 45 min at room temperature. The white blood cell phase was transferred to a new tube and centrifuged for 10 min at 350 x g at room temperature. Erythrocytes contaminating the white blood cell fraction were lysed by hypotonic shock, and the purified blood leukocytes were isolated by repeated centrifugation (20). To this end, the supernatant was centrifuged (10 min, 350 x g), the cell pellet was suspended in 10 ml ice-cold 0.2% NaCl for 30 sec, 10 ml 1.6% NaCl were added, and then the tube was centrifuged (10 min, 350 x g). White blood cells (1.2 x 108) were homogenized using Ultraturrax at 4 C in a lysis buffer (0.01 M PBS, pH 7.4, containing 0.5% sodium deoxycholate and 0.5% Nonidet P-40) in the presence of 1 µM pepstatin, 1 µM leupeptin, 2 µM phenylmethylsulfonylfluoride, 130 µM bestatin, and 10 µg/ml aprotinin (all from Sigma-Aldrich Corp.). After 15 min of standing on ice, lysates were centrifuged for 10 min at 27,000 x g at 4 C. Protein (80 µg) from the supernatant was precipitated by trichloroacetic acid and solubilized in modified SDS-PAGE sample buffer consisting of 5 M urea, 0.17 M sodium dodecyl sulfate, 50 mM Tris, and 5% mercaptoethanol and loaded onto a 12% polyacrylamide gel for detection of PC1, PC2, and CPE and on 15% polyacrylamide gel for detection of 7B2 and POMC. For detection of END, aliquots of centrifuged lysates (80 µg protein) were diluted in 4x SDS-PAGE sample buffer and subjected to electrophoresis on a 20% polyacrylamide gel. Proteins were electrophoresed according to the method of Laemmli (21) and were transferred to nitrocellulose membrane (Bio-Rad Laboratories, Munich, Germany) using a Bio-Rad Trans-Blot apparatus. Membranes were blocked with 5% milk in PBS/Tween 20 (0.5%). Blots were probed with anti-END (ProGen Biotechnik GmbH, Heidelberg, Germany), anti-POMC (Phoenix Pharmaceuticals, Inc.), anti-7B2, anti-PC1 (directed against N terminal), anti-PC2 (directed against C terminal; diluted at 1:1,000), or anti-CPE (directed against N terminal; diluted at 1:2,000) for 2 h at room temperature. After incubation with the secondary antibody (peroxidase-conjugated antirabbit IgG; 1:1,500; Vector Laboratories, Inc.) for 2 h at room temperature, detection of immunoreactive bands was carried out using an enhanced chemiluminescence (ECL) system (Amersham Pharmacia Biotech, Freiburg, Germany), and the blot was immediately exposed to autoradiograph film for 30 sec to 3 min. Each experiment was repeated three times.
Quantification of immunoblotting
Western blots of circulating leukocytes from animals with and without paw inflammation were scanned. The same amount of protein from leukocytes of treated and control animals was loaded onto each lane for each antigen under investigation, and the Image-Pro Analysis package (Media Cybernetics, Analytical Imaging Solutions Group, Gleichen, Germany) was used to quantify changes in immunodensities. The upper and lower threshold density ranges were adjusted to encompass and match the immunoreactivity (ECL reaction product) to provide an image with immunoreactive material appearing in color (black) pixels and nonimmunoreactive material appearing in white pixels. A standardized box was positioned over each band. In the case of PC1, the measurement was performed on the 66-kDa band. The area and density of pixels within the threshold values representing immunoreactivity were measured, and the integrated density (the product of the area and density) was calculated. Integrated densities of controls and treated groups were compared and statistically analyzed. A percent change (treated/control) was then calculated to demonstrate differences between the two groups. As Western blot ECL reactions can vary despite a standard protocol, comparison of both groups was performed within the same blot.
Immunohistochemistry
Four days after FCA inoculation, rats were deeply anesthetized with halothane and perfused transcardially with 100 ml 0.1 M PBS (pH 7.4) and 300 ml cold PBS containing 4% paraformaldehyde and 0.2% picric acid (pH 7.4; fixative solution) for light microscopic immunohistochemistry and with 4% paraformaldehyde/0.1% glutaraldehyde/0.2% picric acid solution (pH 7.4) for electron microscopy, respectively. The skin with adjacent sc tissue was removed, postfixed for 90 min at 4 C in the fixative solution, and cryoprotected overnight at 4 C in PBS containing 10% sucrose. The tissues were then embedded in Tissue-Tek compound (OCT, Miles, Inc., Elkhart, IN) and frozen. Consecutive sections (6 µm thick) prepared on cryostat were mounted onto gelatin-coated slides. The isolation of leukocytes was accomplished from venous blood of controls and animals with paw inflammation (4 d after FCA treatment) using the dextran sedimentation method (as mentioned above). Cell pellets were reconstituted in 5 ml 0.1 M PBS, and 50,000 leukocytes in suspension were then centrifuged by a Shandon Cytospin 3 (Thermo Shandon, Pittsburgh, PA) at 20 x g for 3 min on glass slides. The circulating leukocytes were fixed for 30 min in the fixative solution. Each experiment was repeated three times.
Single staining procedures
Immunohistochemical staining of the serial sections and circulating leukocytes was performed with a Vectastain avidin-biotin peroxidase complex (ABC; Vectastain Elite Kit, Vector Laboratories, Inc.), as described previously (22). Unless otherwise stated, all incubations were performed at room temperature, and PBS was used for washing (three times for 10 min each time) after each step. The sections and circulating leukocytes were incubated with PBS, 0.3% H2O2, and 10% methanol for 45 min to block endogenous peroxidase. To prevent nonspecific binding, the sections were incubated for 60 min in PBS containing 0.3% Triton X-100, 1% BSA, 4% goat serum, and 4% horse serum (block solution). The sections were then incubated overnight at 4 C with rabbit polyclonal antibodies against END, PC1/3, PC2, CPE, POMC, or 7B2 (1:1000 dilution). Thereafter, the sections were incubated for 1 h with a goat antirabbit biotinylated secondary antibody (Vector Laboratories, Inc.) and then with ABC for 45 min. Finally, the sections were washed and stained with 3',3'-diaminobenzidine tetrahydrochloride (DAB; Sigma-Aldrich) containing 0.01% H2O2 in 0.05 M Tris-buffered saline (pH 7.6) for 3–5 min. After the enzymatic reaction, the sections and circulating leukocytes were washed in tap water, counterstained with thionin, then dehydrated in alcohol, cleared in xylene, and mounted in DPX (Merck & Co., Darmstadt, Germany).
Immunoreactive cells were identified by the following morphological criteria: 1) macrophages/monocytes in inflamed sc tissue by large cell bodies, vacuolated cytoplasm, and irregular-shaped nuclei; 2) monocytes in circulation by large cell bodies and kidney-shaped nuclei; 3) lymphocytes by small cell bodies, large nuclei, and small amounts of cytoplasm; and 4) polymorphonuclear leukocytes by large cell bodies and multisegmented nuclei.
Double staining procedures
Immunohistochemical double staining of tissue sections and circulating leukocytes was performed as described previously (23, 24, 25). Briefly, sections and circulating leukocytes stained in the first sequence with antibody against END, POMC, or PC2 (as described above) were treated with 0.3% H2O2 for 30 min to inactivate peroxidase in ABC, washed in several changes of PBS, incubated with blocking solution, and then processed as follows in a second sequence: 1) sections and circulating leukocytes stained for END were incubated with a second primary antibody against POMC or PC2; 2) sections and circulating leukocytes stained for POMC were incubated with a second primary antibody against CPE, PC1, or PC2; and 3) sections and circulating leukocytes stained for PC2 were incubated with a second primary antibody against 7B2. All incubations were performed overnight at 4 C, and slides were then washed in PBS and exposed to the biotinylated secondary antibody (Vector Laboratories) for 1 h and to ABC for 45 min. Finally, the sections were washed and stained using a HistoGreen Peroxidase-Substrate Kit (Linaris, Wertheim-Bettingen, Germany). The chromogen DAB used for the first primary antiserum appeared brown, whereas Histogreen used for the second primary antiserum appeared green. After the enzymatic reaction, the sections and circulating leukocytes were washed in distilled water, dehydrated in alcohol, cleared in xylene, and mounted in DPX.
Specificity controls
To demonstrate specificity of staining, the following controls were included. 1) Preabsorption of diluted antibody against END, POMC, CPE, PC1, PC2, or 7B2 with 5 µg/ml purified END (Peninsula Laboratories, Belmont, CA), POMC (Phoenix Pharmaceuticals), CPE (provided by Dr. Y. Peng Loh), recombinant PC1 or PC2 antigens (provided by Drs. N. G. Seidah and D. F. Steiner, respectively), or 7B2 (provided by Dr. N. G. Seidah), respectively, was performed for 24 h at 4 C. The control incubation of diluted POMC, CPE, PC1, PC2, and 7B2 antisera with END did not block their immunoreactivity, indicating that these antibodies have no cross-reactivity with END. Additionally, preincubation of POMC and PC2 antisera with END had no effect on POMC or PC2 immunoreactivity in either circulating immune cells or inflamed sc paw tissue. 2) Omission of the primary antisera, the secondary antibodies, or ABC did not show any staining. 3) Either the first or second primary antibody and either the first or second secondary antibody were omitted. 4) The primary antisera were reversed between the first and second sequences of the immunostaining procedure. 5) The percentages of immunoreactive cells in the single immunostaining experiments were compared with those in the second sequence of double immunostaining.
Quantification of immunostaining
In sc paw tissue and circulating leukocytes, immunoreactive cells were counted by a blinded experimenter in three tissue sections or three cytospin slides per animal. Five squares (38.4 mm2 each) per section were analyzed using a Zeiss microscope (objective, x40 x10; Carl Zeiss, Oberkochen, Germany). The percentage of immunostained cells was determined by the formula: immunostained cells/total number of immune cells x 100. In double labeling experiments, the percentage of POMC- or PC2-positive cells expressing END was calculated as the number of POMC- or PC2-immunoreactive cells double-stained against END, divided by the number of all POMC- or PC2-immunoreactive cells, respectively. The percentage of CPE- or PC1-positive cells expressing POMC and the percentage of 7B2-positive cells expressing PC2 were calculated analogously. Five rats per group were used for analysis. Values are the mean ± SEM.
Immunoelectron microscopy
Free-floating sc paw sections (40 µm) were incubated with antibody against END. The immunostaining was performed in the same way as for light microscopy. The immunoreaction was then visualized by incubation with nickel chloride-enhanced DAB (DAB containing 0.01% H2O2 and 0.08% nickel chloride in 0.05 M Tris-buffered saline, pH 7.6) for 3–5 min (26). The sections were postfixed in 1% tannic acid (in 0.1 M phosphate buffer) and 1% osmium tetroxide solution (in 0.1 M PBS), dehydrated in ethanol, and embedded in Epon. Semithin and ultrathin sections were cut on a Reichert Ultracut (Leica, Nussloch, Germany), followed by contrasting with 2% uranyl acetate/lead citrate. Finally, the ultrathin sections were examined under a transmission electron microscope (TEM 10, Zeiss).
Tissue preparation and release experiments
Four days after FCA treatment, rats were euthanized with halothane, and popliteal lymph nodes were collected from inflamed hind limbs (n = 20 rats). Lymph nodes were minced, homogenized, and filtered through a 70-µm pore size sieve (BD Biosciences, Franklin Lakes, NJ). Cells were washed in Hanks’ balanced salt solution (HBSS; Sigma-Aldrich) and centrifuged at 300 x g for 10 min at 20 C using a swinging bucket rotor of a Heraeus centrifuge (Heraeus-Christ GmbH, Osterode, Germany). After three washing procedures, cells were reconstituted in HBSS containing 5 µg/ml bestatin (Sigma-Aldrich) and 40 µg/ml aprotinin (Sigma-Aldrich), aiming at a concentration of 1.3 x 108 cells/ml. Cell viability determined by the trypan blue exclusion method was greater than 97%. Cell suspension (300 µl) was preincubated with either HBSS or with a combination of phentolamine (50–250 ng; Sigma-Aldrich) and propranolol (50–250 ng; Sigma) at 37 C in a shaking water bath. After 5 min, either HBSS or noradrenaline (100 ng; Sigma) was added. Five minutes later, the suspension (total volume, 400 µl) was centrifuged at 300 x g at 4 C for 10 min. Three-hundred-microliter aliquots of the supernatants and the cell pellets were stored at -20 C until further processing.
RIA
Cell pellets were thawed and reconstituted in 0.3 ml RIA buffer (0.1 M sodium phosphate, 0.05 M NaCl, 0.01% NaN3, 0.1% BSA, 0.1% Triton X-100, 5 µg/ml bestatin, and 40 µg/ml aprotinin) using an RIA kit (Peninsula Laboratories). Afterward, cell pellets were lysed by a freezing/thawing procedure and sonicated for 5 min using an ultrasonic water bath (RK 52H, Sonorex, Hamburg, Germany). Lysates were centrifuged for 10 min at 14,000 x g at 4 C. Using the RIA kit (Peninsula Laboratories) the END content was determined in the supernatants of the centrifuged lysates and in the thawed supernatants of the release experiments supplemented with serum albumin (0.1%; Sigma-Aldrich) and Triton X-100 (0.1%; Sigma-Aldrich), respectively. The content of END in the samples was calculated by extrapolation from the standard curve.
Statistical analysis
Data are represented as the mean ± SEM and were analyzed by t test for parametric data and by Mann-Whitney U test for nonparametric data. Differences were considered significant at P < 0.05. All tests were performed using SigmaStat 2.03 statistical software (Jandel Corp., San Ramon, CA).
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). The anti-PC1 antibody detected a double band of 66 and 60 kDa. The anti-PC2 and anti-7B2 antibodies recognized only 65- and 23-kDa bands for PC2 and 7B2, respectively (Fig. 1). There were no differences in CPE and 7B2, but there was a significant increase in the density of POMC- (36.7 ± 3.1%), PC1- (63.3 ± 5.9%), PC2- (38.5 ± 4.7%), and END - (29.9 ± 3.9%)-immunoreactive bands of leukocytes from treated vs. untreated rats (P < 0.05; Fig. 1).
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, A–F). In FCA-treated animals, a higher proportion of leukocytes was immunoreactive for END (51.0 ± 4.6%), POMC (55.3 ± 2.6%), CPE (50.3 ± 6.8%), PC1 (49.7 ± 2.2%), PC2 (46.9 ± 2.7%), and 7B2 (52.6 ± 3.7%; Fig. 3, A–F). These differences were significant for POMC, PC1, PC2, and END (P < 0.05), but not for CPE or 7B2. The immunoreactivity could be detected in cells morphologically identified as macrophages/monocytes, polymorphonuclear leukocytes (granulocytes), and lymphocytes from FCA-treated animals (Fig. 3, A–F) and as macrophages/monocytes and granulocytes from nontreated animals (Fig. 2, A–F). Preabsorption of all antibodies with 5 µg/ml of their respective antigen completely abolished immunostaining (data not shown).
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). These cells occurred in the inflammatory foci within the plantar sc tissue and had morphological appearances consistent with macrophages/monocytes, granulocytes, and lymphocytes (Fig. 4, A, D, F, H, J, and L). END-, POMC-, CPE-, PC1-, PC2-, and 7B2-immunoreactive immune cells were undetectable in noninflamed sc paw tissue (Fig. 2L
). When immunostained serial sections for END, POMC, CPE, PC1, PC2, or 7B2 were compared and matched, an almost identical distribution pattern around the inflammatory foci was observed. Preabsorption of all antibodies with 5 µg/ml of their respective antigen completely abolished immunostaining (data not shown).
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, G and H). Also, these cells showed a colocalization of POMC with CPE (86.4 ± 3.9%), PC1 (94.8 ± 3.8%), and PC2 (83.6 ± 7.3%; Fig. 2, I–K). Circulating leukocytes of FCA-treated animals revealed colocalization of END with both POMC (77.0 ± 6.9%) and PC2 (93.1 ± 4.3%; Fig. 3, G and H). POMC was colocalized in CPE- (89.8 ± 4.3%), PC1- (96.7 ± 3.3%), and PC2-(86.5 ± 6.3%) immunoreactive immune cells. Few cells were immunolabeled for CPE (10.1 ± 4.3%), PC1 (3.6 ± 0.5%), or PC2 (13.5 ± 6.3%) alone (Fig. 3, I–K). Almost all cells positive for CPE, PC1, or PC2 were also immunoreactive for POMC. PC2 was colocalized in 7B2 (78.3 ± 2.6%)-immunoreactive immune cells. Some cells (21.7 ± 2.7%) showed immunoreactivity of 7B2 alone (Fig. 3L). There was no statistical difference (P > 0.05) in these percentages between control and FCA-treated animals. Omission of either the first or second primary antibody and omission of either the first or second secondary antibody did not produce the first or second (double) color, respectively. Reversing the primary antisera between the first and second sequences of the immunostaining procedure yielded the same results. There was no significant difference in the percentages of POMC- (55.3 ± 2.6), CPE- (50.3 ± 6.8), PC1- (49.7 ± 2.2), PC2- (46.9 ± 2.7), and 7B2 (52.6 ± 3.7)-immunoreactive circulating leukocytes obtained by single immunostaining compared with those of POMC- (56.2 ± 3.2), CPE- (49.2 ± 4.3), PC1- (46.8 ± 2.4), PC2- (42.7 ± 2.9), and 7B- (54.9 ± 3.8)-immunoreactive circulating leukocytes obtained by the second sequence of double immunostaining (P > 0.05 for each comparison).
Double immunohistochemistry in inflamed sc paw tissue
POMC was colocalized in CPE- (88.6 ± 1.0%), PC1- (94.4 ± 0.5%), and PC2 (97.0 ± 0.4%)-immunoreactive immune cells. Few cells were immunolabeled for CPE (11.4 ± 1.0%), PC1 (3.6 ± 0.5%), or PC2 (3.0 ± 0.4%) alone (Fig. 5). Almost all cells positive for CPE, PC1, or PC2 were also immunoreactive for POMC (Fig. 5). END was colocalized in POMC- (91.0 ± 1.2%) and PC2 (98.4 ± 0.5%)-immunoreactive immune cells. Few cells were labeled for POMC (7.0 ± 1.5%) or PC2 (1.6 ± 1.1%) alone (Fig. 6). Almost all cells immunoreactive for PC2 were also immunostained for 7B2 (90.2 ± 1.6%). Some cells showed immunoreactivity of 7B2 (9.8 ± 1.6%) alone (Fig. 6, G–I).
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Immunoelectron microscopy of END in inflamed sc tissue
END-immunoreactive cells were morphologically identified as macrophages, monocytes, granulocytes, and lymphocytes (Fig. 7). These cells showed a highly developed rough ER and an extensive Golgi apparatus (Fig. 8, A and B). Immunoreactive cells contained numerous secretory granules packed in small or large membranous structures. Immunostaining was confined to secretory granules, which were grouped in small or large membranous vesicular structures (Fig. 8, E and F). The smaller membranous structures containing END-immunoreactive granules were found within the deep cytoplasm, and the larger ones were arranged at the cell periphery and in extended processes (Fig. 8, E and F). Some secretory granules located at the trans-side of Golgi stacks showed immunostaining (Fig. 8B). Some secretory granules were packed in membranous compartments extending from Golgi cysternae (Fig. 8, C and D). No immunoreactivity was found in association with the plasma membrane or the nucleus. Preabsorption of antibody against END with 5 µg/ml purified END completely abolished immunostaining (Fig. 8A).
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The POMC gene has three exons and two introns. Exon 2 encodes a signal peptide essential for classical protein processing and secretion (27), and exon 3 encodes various bioactive peptides, such as ACTH, ßLPH, MSH, and END. There have been numerous reports of POMC mRNA in immune cells, but some studies report a lack of full-length transcripts (28, 29, 30). A lack of exon 2 encoding the signal sequence necessary for ER membrane translocation suggests that the propeptide products are neither processed nor secreted and therefore are nonfunctional (31). Recently, Lyons and Blalock (32) detected the full-length POMC mRNA in immune cells by rapid amplification of cDNA ends-PCR. The researchers suggested that the apparent differences from previous results may be due to differences in PCR techniques, primer extension, and/or ribonuclease (18). Furthermore, they reported that in nonstimulated mononuclear cells the amount of full-length POMC mRNA is low, but that mitogenic stimulation enhanced the abundance of this mRNA (32). Our model involves a strong inflammatory stimulus in vivo, which is conceivably similar to the in vitro stimulation used in the aforementioned studies. Thus, it was our objective to extend those studies and to examine the enzymatic machinery required for POMC sorting, processing, storage, and vesicular release of END in inflammatory cells.
In the present studies we provide evidence for the expression of the POMC precursor, POMC-derived peptides (END), the sorting receptor (CPE), proteolytic enzymes (PC1 and PC2), and the binding protein 7B2 in circulating and resident leukocytes. Our Western blot analysis of CPE, POMC, and END in circulating leukocytes shows bands of approximately 53, 31, and 3.5 kDa, respectively, comparable to those previously reported in neuroendocrine cells (13, 33). Our anti-PC1 antibody detected a double band of 66 and 60 kDa. The 66-kDa band corresponds to the form previously observed in rat alveolar macrophages and spleen mononuclear cells (20) and in a human monocytic leukemia cell line (34). The 60-kDa form may represent degradation products of PC1, as the activated isoform has been found to be unstable (35). The anti-PC2 and anti-7B2 antibodies recognized bands of approximately 65 and 23 kDa, respectively. These values correspond to those previously estimated for PC2 and 7B2 in pituitary cell lines and pancreatic islets (36, 37). Thus, all of the antibodies we used are characterized by their respective recognition of specific bands with different molecular masses similar to those detected in neuroendocrine cells (12, 13, 33, 36, 38). Importantly, these results demonstrate that cross-reactivity is excluded. We also found an increase in POMC, PC2, and END in circulating leukocytes from treated vs. untreated rats, indicating an up-regulation of these proteins under conditions of inflammatory pain. Since circulating cells of both treated and untreated animals displayed immunoreactive POMC, PC1, PC2, and END, painful inflammation appears to increase, but not induce, gene expression of these proteins.
Our immunohistochemistry shows that END, POMC, CPE, PC1, PC2, and 7B2 are expressed within immune cells in blood and in inflamed, but not in noninflamed, sc tissue. In inflamed tissue, immune cells expressing these compounds are found mainly in the periphery of inflammatory foci. Their morphological appearances are consistent with macrophages/monocytes, granulocytes, and lymphocytes. In the blood of untreated animals, immunoreactive cells can be differentiated into macrophages/monocytes and granulocytes. Consistent with our findings in Western blots, there was a significant increase in POMC-, PC1-, PC2-, and END-immunoreactive leukocytes within the circulation after induction of inflammation. Thus, our single staining results in this model of inflammatory pain agree with previous studies showing PC2 in peripheral and liver-infiltrating granulocytes, PC1 in alveolar macrophages and spleen mononuclear cells from lipopolysaccharide-treated rats (20), as well as PC1, PC2, and POMC in spleen macrophages/monocytes and lymphocytes from diabetic (39) and untreated (40) rats.
To date there has been no evidence for colocalization of POMC or END together with these processing enzymes in immune cells. Our double-staining experiments show that 7B2 and PC2 are clearly coexpressed within immune cells in circulation and inflamed sc tissue. Furthermore, we observed that PC1, PC2, and CPE are colocalized with POMC immunoreactivity. Our results agree with those of Nakashima et al. (39), who reported that POMC is colocalized with PC1 and PC2 in the spleen of diabetic rats. The present observations suggest that the two convertases and CPE are active in the processing of POMC into END under the pathological condition of inflammatory pain. This is further supported by the expression of both POMC and PC2 by END-positive immune cells. In mice lacking CPE, POMC was not only poorly processed, but was secreted in an unregulated manner (i.e. constitutively) from pituitary cells (12, 38, 41). Thus, the colocalization of POMC with CPE and END in our model may be consistent with the idea that CPE targets POMC to the regulated secretory pathway in immune cells, similar to its action in neuroendocrine cells (12).
Our previous studies showed that END release from immune cells is calcium dependent and stimulated by potassium, consistent with vesicular release (6). The secretory granule is responsible for the storage and transport of material from the Golgi apparatus to the cell membrane, where secretion takes place (42). Our ultrastructural observations show that END-immunoreactive inflammatory cells, morphologically definable as macrophages, monocytes, granulocytes, and lymphocytes, contain a highly developed rough ER and an extensive Golgi apparatus, similar to pituitary cells. Immunostaining of END was confined to secretory granules, which were grouped in small or large membranous vesicular structures. The smaller END-immunoreactive secretory granules were localized within cytoplasm, and the larger ones were arranged at the cell periphery and within extended processes ready for the exocytosis process, similar to the pituitary (43). Intense stimulation of such cells, which can be provided by persistent inflammatory pain and by environmental stress in our model (3, 4, 7), conceivably leads to END secretion.
As stress is typically associated with catecholamine release, we studied the liberation of END from an immune cell suspension by noradrenaline in vitro. We found that noradrenaline induced the release of immunoreactive END into the supernatant. This release was reversed by the adrenergic antagonists phentolamine and propranolol, indicating that it was mediated by the activation of adrenergic receptors on the cells, consistent with earlier indirect evidence (5). These results support and extend our previous studies showing receptor-specific END release from immune cells by CRH and IL-1ß (6).
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Abbreviations: ABC, Avidin-biotin peroxidase complex; CPE, carboxypeptidase E; DAB, 3',3'-diaminobenzidine tetrahydrochloride; ECL, enhanced chemiluminescence; END, ß-endorphin; ER, endoplasmic reticulum; FCA, Freund’s complete adjuvant; HBSS, Hanks’ balanced salt solution; LPH, ß-lipotropic hormone; PC, prohormone convertase; POMC, proopiomelanocortin; TGN, trans-Golgi network.
Received September 25, 2003.
Accepted for publication November 12, 2003.
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-melanotropin. Proc Natl Acad Sci USA 90:4922–4926) distribution in hamster and sheep brain: colocalization in gonadotropin-releasing hormone and other identified neurons. Endocrinology 138:5039–5047This article has been cited by other articles:
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J. P. Williams, J. P. Thompson, J. McDonald, T. A. Barnes, T. Cote, D. J. Rowbotham, and D. G. Lambert Human Peripheral Blood Mononuclear Cells Express Nociceptin/Orphanin FQ, but Not {micro}, {delta}, or {kappa} Opioid Receptors Anesth. Analg., October 1, 2007; 105(4): 998 - 1005. [Abstract] [Full Text] [PDF]
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 S. A Mousa, R. H Straub, M. Schafer, and C. Stein {beta}-Endorphin, Met-enkephalin and corresponding opioid receptors within synovium of patients with joint trauma, osteoarthritis and rheumatoid arthritis Ann Rheum Dis, July 1, 2007; 66(7): 871 - 879. [Abstract] [Full Text] [PDF]
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 S. A. Mousa, B. P. Cheppudira, M. Shaqura, O. Fischer, J. Hofmann, R. Hellweg, and M. Schafer Nerve growth factor governs the enhanced ability of opioids to suppress inflammatory pain Brain, February 1, 2007; 130(2): 502 - 513. [Abstract] [Full Text] [PDF]
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 H. L. Rittner, D. Labuz, M. Schaefer, S. A. Mousa, S. Schulz, M. Schafer, C. Stein, and A. Brack Pain control by CXCR2 ligands through Ca2+-regulated release of opioid peptides from polymorphonuclear cells FASEB J, December 1, 2006; 20(14): 2627 - 2629. [Abstract] [Full Text] [PDF]
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 H. L. Rittner, H. Machelska, M. Schafer, C. Stein, and A. Brack Comment on "Neutrophils: are they hyperalgesic or anti-hyperalgesic?" J. Leukoc. Biol., October 1, 2006; 80(4): 729 - 730. [Full Text] [PDF]
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 H. Shen, H. Sprott, A. Aeschlimann, R. E. Gay, B. A. Michel, S. Gay, and H. Sprott Analgesic action of acetaminophen in symptomatic osteoarthritis of the knee Rheumatology, June 1, 2006; 45(6): 765 - 770. [Abstract] [Full Text] [PDF]
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H. L. Rittner, S. A. Mousa, D. Labuz, K. Beschmann, M. Schafer, C. Stein, and A. Brack Selective local PMN recruitment by CXCL1 or CXCL2/3 injection does not cause inflammatory pain J. Leukoc. Biol., May 1, 2006; 79(5): 1022 - 1032. [Abstract] [Full Text] [PDF]
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 D. Labuz, S. Berger, S. A. Mousa, C. Zollner, H. L. Rittner, M. A. Shaqura, T. Segovia-Silvestre, B. Przewlocka, C. Stein, and H. Machelska Peripheral antinociceptive effects of exogenous and immune cell-derived endomorphins in prolonged inflammatory pain. J. Neurosci., April 19, 2006; 26(16): 4350 - 4358. [Abstract] [Full Text] [PDF]
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H. L. Rittner, H. Machelska, and C. Stein Leukocytes in the regulation of pain and analgesia J. Leukoc. Biol., December 1, 2005; 78(6): 1215 - 1222. [Abstract] [Full Text] [PDF]
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 J. P. Williams and D. G. Lambert Editorial II: Opioids and the neuroimmune axis Br. J. Anaesth., January 1, 2005; 94(1): 3 - 6. [Full Text] [PDF]
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