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Endocrinology Vol. 144, No. 12 5578-5584
Copyright © 2003 by The Endocrine Society

In Vitro and in Vivo Calcitonin I Gene Expression in Parenchymal Cells: A Novel Product of Human Adipose Tissue

Philippe Linscheid, Dalma Seboek, Eric S. Nylen, Igor Langer, Mirjam Schlatter, Kenneth L. Becker, Ulrich Keller and Beat Müller

Department of Research (P.L., D.S.), Division of Endocrinology, Diabetology and Clinical Nutrition (U.K., B.M.), Department of Visceral Surgery (I.L.), and Department of Plastic Surgery (M.S.), University Hospitals, CH-4031 Basel, Switzerland; and Department of Medicine, George Washington University and Veterans Affairs Medical Center (E.S.N., K.L.B.), Washington, D.C. 20422

Address all correspondence and requests for reprints to: Philippe Linscheid, Ph.D., Department of Research, University Hospitals, Hebelstrasse 20, 4031 Basel, Switzerland. E-mail: philippe.linscheid{at}unibas.ch.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Circulating levels of calcitonin precursors (CTpr), including procalcitonin (ProCT), increase up to several thousand-fold in human sepsis, and immunoneutralization improves survival in two animal models of this disease. Herein, we analyzed inflammation-mediated calcitonin I gene (CALC I) expression in human adipocyte primary cultures and in adipose tissue samples from infected and noninfected patients with different levels of serum ProCT. In ex vivo differentiated adipocytes, the expression of CT mRNA increased 24-fold (P < 0.05) after the administration of Escherichia coli endotoxin (lipopolysaccharide) and 37-fold (P < 0.05) after IL-1ß administration by 6 h. ProCT protein secretion into culture supernatant increased 13.5-fold (P < 0.01) with lipopolysaccharide treatment and 15.2-fold (P < 0.01) with IL-1ß after 48 h. In coculture experiments, adipocyte CT mRNA expression was evoked by E. coli-activated macrophages in which CT mRNA was undetectable. The marked IL-1ß-mediated ProCT release was inhibited by 89% during coadministration with interferon-{gamma} (IFN{gamma}). In patients with infection and markedly increased serum ProCT, CT mRNA was detected in adipose tissue biopsies. Hence, we demonstrate that ProCT, which is suspected to mediate deleterious effects in sepsis and inflammation, is a novel product of adipose tissue secretion. The inhibiting effect of IFN{gamma} on IL-1ß-induced CT mRNA expression and on ProCT secretion might explain previous observations that serum ProCT concentrations increase less in systemic viral compared with bacterial infections.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
ADIPOSE TISSUE IS increasingly recognized as a major endocrine organ in humans. The numerous peptide hormones released by adipocytes have been proposed to affect energy homeostasis, glucose and lipid metabolism, immune response, and reproduction (1). Most of these signaling molecules appear to be deregulated when mass is markedly altered, being increased in the obese state or decreased in lipoatrophy.

In systemic microbial infections, circulating levels of calcitonin (CT) precursors (CTpr), including procalcitonin (ProCT), increase up to several thousand-fold, and this increase correlates with the severity of the illness and with mortality (2, 3, 4). Furthermore, CTpr may contribute to the deleterious effects of systemic infection as shown in experimental animals (5, 6, 7).

CTpr originate from the calcitonin I (CALC I) gene on chromosome 11. Similar to many peptide hormones, mature CT is initially biosynthesized as a larger prohormone, ProCT, which is subsequently processed into smaller peptides, including CT (8, 9). The classical neuroendocrine paradigm limits the expression of CALC I exclusively to neuroendocrine cells, mainly the C cells of the thyroid. However, increased plasma ProCT levels have been reported in thyroidectomized patients with inflammation (10, 11). We recently documented the generalized, tissue-wide, nonneuroendocrine expression of CT-mRNA in animal models of sepsis (12, 13). To elucidate the source of ProCT in human sepsis, we studied the effects of cytokines and lipopolysaccharide (LPS) on CALC I induction and ProCT secretion in human adipocytes. In addition, we examined CT-mRNA expression in adipose tissue samples obtained from infected and noninfected patients.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Adipocyte cultures
For ex vivo stimulation, after informed consent was granted, 50–500 g adipose tissue were obtained from noninfected patients undergoing plastic surgery. Primary cultures of human adipocytes were performed as previously described (14, 15) with modifications. Briefly, adipose tissue was minced, digested in 1 mg/ml collagenase 2 (Worthington Biochemical Corp., Freehold, NJ), filtered (150-µm pore size nylon mesh) and centrifuged at 200 x g. The cell pellet was resuspended twice in erythrocyte lysis buffer, washed, and seeded at a density of approximately 33,000 cells/cm2 in 6- or 12-well plates. After 18-h incubation in DMEM/Ham’s F-12 with 10% fetal calf serum allowing attachment, cells were washed in PBS and cultured in serum-free medium supplemented with agents (isobutylmethylxanthine, dexamethasone, insulin, transferrin, and T3) that induce differentiation of preadipocytes to adipocytes. During the first 2 d, 1 µM rosiglitazone (provided by GlaxoSmithKline, Worthing, UK) was also present. Triglyceride-storing adipocytes, representing 40–80% of cultured cells, are visible within 5–10 d. Differentiation was confirmed by RT-PCR analysis for adipocyte-specific peroxisome proliferator-activated receptor {gamma}2 expression (16). Adipocytes were maintained for an additional 4 d in DMEM/Ham’s F-12 with 10% FCS before experiments.

In addition, floating mature adipocytes obtained after the centrifugation step were washed, inoculated into 50-ml flasks (BD Biosciences, Franklin Lakes, NJ) completely filled with medium (DMEM/Ham’s F-12 with 10% FCS), and allowed to attach to the upper surface for 72 h at 37 C (17, 18). Flasks were subsequently turned around, and the attached purified, triglyceride-storing adipocytes were cultured in 5 ml medium for experiments.

Adipocytes were stimulated for time periods ranging from 2–60 h with the following agents: 1 µg/ml lipopolysaccharide (LPS), 100 U/ml interferon-{gamma} (IFN{gamma}), 10 ng/ml TNF{alpha}, and 20 U/ml IL-1ß. Human-specific cytokines were purchased from PeproTech (London, UK), and LPS (Escherichia coli 026:B6) was obtained from Sigma-Chemie (Buchs, Switzerland).

The viability of adipocytes after stimulation was assessed via trypan blue staining: viable cells exclude trypan blue; dead cells stain blue.

Adipocytes and macrophages in cocultures
White blood cells were isolated by Ficoll-Plaque Plus (Amersham Pharmacia Biotech, Uppsala, Sweden) and washed four times with Hanks’ buffered salt solution (Invitrogen, Basel, Switzerland) supplemented with 0.5% human albumin (Blutspendedienst SRK, Bern, Switzerland). Cells were resuspended in Iscove’s Modified Dulbecco’s Medium (IMDM) with 20% human serum and seeded in cell culture inserts with 0.4-µm pore size (BD Biosciences). After 1-h incubation to allow attachment of monocytes, inserts were washed four times with Hanks’ buffered salt solution supplemented with 0.5% human albumin. Fresh IMDM with 20% human serum was supplied, and monocytes were cultured for 5 d, allowing differentiation to macrophages. For experiments, inserts with macrophages were added to wells containing ex vivo differentiated adipocytes, which were previously kept for 2 d in IMDM with 20% human serum. Upper chambers containing macrophages were supplemented for 2 h with live E. coli and kept in coculture with adipocytes for an additional 22 h.

RT-PCR
Total RNA from homogenized tissues or adipocyte cultures was extracted by the single step guanidinium isothiocyanate method with a commercial reagent (Tri-Reagent, Molecular Research Center, Inc., Cincinnati, OH) according to the manufacturer’s protocol. Extracted RNA was quantified spectrophotometrically, and the quality was assessed by gel electrophoresis. Equal amounts of RNA per tissue or in vitro treatment were subjected to RT (Omniscript RT kit, Qiagen, Basel, Switzerland). PCR was performed on a conventional thermal cycler (TGradient, Biometra, Gottingen, Germany) using the PCR Taq core kit (Qiagen) and the following intron border-spanning oligonucleotides: CT (232-bp product; GenBank accession no. X00356), 5'-TGAGCTGGAGCAGGAGCAAG-3' (sense) and 5'-GTTGGCATTCTGGGGCATGCTAA-3' (antisense); IL-6 (284-bp product; GenBank accession no. NM_000600), 5'-GCAAAGAGGCACTGGCAGAAA-3' (sense) and 5'-CAGGCTGGCATTTGTGGTTG-3' (antisense); TNF{alpha} (310-bp product; GenBank accession no. M10988), 5'-GGCCCAGGCAGTCAGATCAT-3' (sense) and 5'-GGGGCTCTTGATGGCAGAGA-3' (antisense); and ß-actin (198-bp product; GenBank accession no. AF076191), 5'-TTCTGACCCATGCCCACCAT-3' (sense) and 5'-ATGGATGATGATATCGCCGCGCTC-3' (antisense). The annealing temperature was 65 C, except for CT (67 C). Thirty-five cycles of PCR were used for CT and TNF{alpha} detection. Cycles were reduced to 28 for IL-6 and ß-actin to stop the reaction in the linear phase of amplification. IL-6 and TNF{alpha} were used as controls of inflammatory stimulation, and ß-actin was used to verify equal quantities of RNA loading in each reaction. PCR products were separated and visualized on 1.5% agarose gels containing 0.5 µg/ml ethidium bromide. PCR product identity was confirmed by direct nucleotide sequencing of the PCR products by dye deoxy terminator cycle sequencing.

Quantitative analyses of CT-mRNA expression
cDNA obtained as described above was subjected to quantitative real-time PCR analysis using the ABI 7000 sequence detection system (PerkinElmer, Branchburg, NJ). Specific primers yielding short PCR products suitable for SYBR-Green detection were designed using Primer Express software (version 1.0, PE Applied Biosystems, Foster City, CA). Sequences of primers were as follows: CT (92-bp product; GenBank accession no. X00356), 3'-GTGCAGATGAAGGCCAGTGA-5' (sense) and 3'-TCAGATTACCACACCGCTTAGATC-5' (antisense); and hypoxanthine-guanine phosphoribosyltransferase (HPRT; 85-bp product; GenBank accession no. M26434), 3'-TCAGGCAGTATAATCCAAAGATGGT-5' (sense) and 3'-AGTCTGGCTTATATCCAACACTTCG-5' (antisense). The reaction volume was 22 µl, and the conditions were set as suggested by the manufacturer. Each cDNA sample tested for quantitative CT mRNA expression was also subjected to HPRT mRNA analysis. Results were expressed as the ratio of the respective CT mRNA and HPRT mRNA threshold values. Product identity was confirmed by sequence analysis and electrophoresis on a 2.5% agarose gel containing ethidium bromide.

CT precursor concentrations
ProCT concentrations were determined in supernatants by an ultrasensitive chemiluminometric assay with a functional sensitivity of 6 pg/ml (ProCa-S Assay, B.R.A.H.M.S. GmbH, Hennigsdorf-Berlin, Germany).

Patients
Adipose tissue samples were obtained from four infected patients with elevated serum ProCT requiring laparotomy (mean age, 44 yr; range, 19–65 yr). The septicemia was due to peritonitis because of perforated sigmoid diverticulitis, perforated appendicitis, ischemic colitis of the sigmoid colon, and necrotizing proctocolitis with perforation of the rectum and descending colon. Also, adipose tissue was collected from noninfected patients requiring elective surgery (mean age, 53 yr; range, 29–71 yr). Informed consent was obtained. Harvested tissues were immediately incubated in RNA-later (Ambion, Inc., Austin, TX) to prevent RNA degradation. The samples were snap-frozen and stored at -70 C. Tissues were powdered under liquid nitrogen before RNA extraction using Tri-Reagent.

Statistical analysis
Results are presented as the mean ± SEM. Groups of experiments were compared statistically using t tests. In addition, two group comparisons corrected for multiple testing, i.e. one-way ANOVA with post hoc analysis for least square difference, were performed.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
CALC I gene induction in vitro
We first analyzed the effects of inflammatory mediators on CT mRNA expression in adipose tissue-derived cells. In ex vivo differentiated adipocytes and in mature explanted adipocytes obtained from noninfected patients undergoing plastic surgery, CT mRNA was not detected by conventional RT-PCR analysis using 35 amplification cycles (Fig. 1Go). After 45 cycles of real-time PCR using specifically designed primers, trace amounts of CT mRNA were detected on a 2.5% agarose gel (Fig. 2AGo). Accordingly, analysis of ProCT content in supernatants of ex vivo differentiated unstimulated, control adipocytes was below or around the detection limit of 5 pg/ml (Fig. 2BGo). After 6-h exposure to a combination of LPS and inflammatory cytokines (IFN{gamma}, TNF{alpha}, and IL-1ß), both the ex vivo differentiated as well as the mature adipocytes revealed induced CT mRNA expression (Fig. 1Go). CT mRNA induction was also observed in adipocytes kept in coculture with E. coli activated macrophages (Fig. 1Go). In contrast, CT mRNA induction was not observed in macrophages stimulated with E. coli.



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  		FIG. 1. Induction of the CALC I gene in cultured adipocytes. RT-PCR analysis was performed with RNA obtained from ex vivo differentiated adipocytes and from mature adipocytes kept in so-called ceiling cultures after 6-h stimulation with or without cytokines (IFN{gamma}, 100 U/ml; TNF{alpha}, 10 ng/ml; IL-1ß, 20 U/ml) and LPS (1 µg/ml), denoted as mix. Alternatively, ex vivo differentiated adipocytes were kept in cocultures with macrophages activated with E. coli (Ec). Presented data are one representative from at least five independent experiments.

 


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  		FIG. 2. Quantitative analysis of CT mRNA expression and ProCT release by ex vivo differentiated adipocytes. A, Quantitative real-time PCR analysis was performed with cDNA obtained from ex vivo differentiated adipocytes using SYBR-Green detection. Incubation time was 6 h, and the following concentrations were used: IFN{gamma}, 100 U/ml; TNF{alpha}, 10 ng/ml; IL-1ß, 20 U/ml; and LPS, 1 µg/ml. CT mRNA threshold values were normalized with HPRT mRNA values and amplification products were visualized on 2.5% agarose gels. B, After 48-h cytokine treatments, supernatants of ex vivo differentiated adipocytes were subjected to chemiluminometric ProCT protein analysis. Random values between 0 and 5 were generated for measurements below the detection limit of 5 pg/ml. The data shown are the mean ± SEM from three (A) or four (B) independent experiments. *, P < 0.05 vs. control. {dagger}, P < 0.01 for the comparison of LPS and IL-1ß vs. control, respectively. §, P < 0.01 for the comparison of IL-1ß plus IFN{gamma} vs. IL-1ß alone.

 
cDNAs obtained from adipocytes treated with LPS or single cytokines were subjected to quantitative real-time PCR analysis. Threshold values of CT mRNA were normalized using the HPRT mRNA value obtained from the respective cDNA preparation during the same PCR run. IFN{gamma} alone had no effect on CT mRNA induction (Fig. 2AGo), whereas TNF{alpha} provoked a 13.2-fold increase compared with nonstimulated control adipocytes. After treatments with mixed cytokines or LPS alone, the increases in CT mRNA induction were 23.2- and 24-fold (P < 0.01), respectively. Interestingly, the strongest induction of CT mRNA was observed after treatment with IL-1ß, resulting in a 37-fold (P < 0.01) increase. The marked induction of CT mRNA by IL-1ß was confirmed in explanted mature adipocytes (Fig. 1Go).

Subjecting undifferentiated preadipocytes to cytokine treatment did not result in CT mRNA expression (not shown)

ProCT secretion in vitro
In supernatants of control or IFN{gamma}-treated adipocytes, the ProCT protein concentration was below or at the detection limit of 5 pg/ml after 48-h incubation (Fig. 2BGo). In contrast, ProCT protein secretion was increased to 13 ± 6.0 pg/ml in supernatants of TNF{alpha}-treated cells (Fig. 2BGo). Administration of LPS or IL-1ß alone or of combined cytokines led to average ProCT protein concentrations of 53.6 ± 17.3 pg/ml (P < 0.01), 61.0 ± 20.5 pg/ml (P < 0.01), and 28.4 ± 14.9 pg/ml, respectively (n = 4 for each agent). IL-1ß induced ProCT secretion at concentrations as low as 0.2 U/ml (not shown). The viability of adipocytes after 48-h exposure to LPS, IFN{gamma}, TNF{alpha}, and IL-1ß, alone or in combination, was unchanged as assessed by trypan blue staining (not shown).

Interestingly, in ex vivo differentiated adipocytes, antagonistic effects of IFN{gamma} on IL-1ß activity were noted. Administration of 100 U/ml IFN{gamma} for 48 h reduced IL-1ß-mediated ProCT secretion by 89% (Fig. 2BGo). Accordingly, mRNA analysis by both conventional RT-PCR and quantitative real-time PCR revealed a strong transcriptional inhibition of CT mRNA expression by IFN{gamma} over time periods ranging up to 60 h (Fig. 3AGo). ProCT release, which was measurable starting from 10 h of stimulation, was strongly inhibited in the presence of IFN{gamma} at all time points (Fig. 3BGo).



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  		FIG. 3. Time course of CALC I expression and ProCT secretion. Ex vivo differentiated adipocytes were treated with 20 U/ml IL-1ß alone or together with 100 U/ml IFN{gamma}. After 2-, 4-, 6-, 8-, 10-, 24-, 48-, or 60-h incubation periods, total RNA was extracted and analyzed for CT mRNA abundance with quantitative real-time PCR and conventional RT-PCR (A). ProCT secretion into culture supernatant was analyzed by chemiluminometric assay (B). Results are presented as the mean ± SD of two independent experiments.

 
CT mRNA expression in adipose tissue obtained from septic and nonseptic humans
In several patients with infection and elevated serum ProCT we found CT mRNA expression in sc and omental fat depots (Fig. 4Go). Control experiments confirmed that, as expected, the CALC I gene was not expressed in adipose tissues from noninfected control patients. Average CT mRNA expression in adipose tissue was enhanced 1962-fold in RNAs obtained from infected vs. noninfected patients, as assessed by real-time PCR.



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  		FIG. 4. Extrathyroidal nonneuroendocrine expression of CT mRNA in humans with infection. Subcutaneous (s) and omental (o) adipose tissue biopsies were obtained intraoperatively from patients with infection and markedly increased circulating ProCT levels as indicated. Noninfected tissues were obtained from patients with normal levels of serum ProCT as controls. Total RNA extractions were subjected to CT mRNA analysis by RT-PCR. Amplification products were visualized on agarose gels containing 0.5 µg/ml ethidium bromide. Verification of mRNA as the source of amplification template was obtained by omitting the RT in reactions for pooled infected samples (rt-), resulting in no bands after PCR. RNA extracted from a medullary thyroid carcinoma cell line was taken as a positive control (c+). The results of quantitative real-time PCR analysis are presented in arbitrary units as the mean ± SEM. The data shown are from four infected and four control patients, respectively. n.d., ProCT concentration less than 0.5 ng/ml.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present studies are the first demonstration of ProCT production and secretion by human adipocytes in the presence of inflammatory mediators. It also confirms the extrathyroidal production of human ProCT previously shown in animal studies (12). Our initial adipocyte model consisted of ex vivo differentiated preadipocytes, in which other cell types (e.g. endothelial cells) were potentially present in the cultures; these could provide a nonadipose source of CT mRNA expression and ProCT release. Hence, the experiments were successfully repeated using mature and purified adipose cells cultured in adipocyte-selecting ceiling cultures (17, 18). The density of adherent adipocytes obtained by this technique is relatively low, but suitable for RT-PCR analysis. The experiments using adipocytes and macrophages in coculture demonstrated that molecules of endogenous origin have the capacity to induce the CALC I gene in adipocytes. Interestingly, CT mRNA was not detectable in activated macrophages. Furthermore, CT mRNA could not be induced in nondifferentiated preadipocytes and in numerous human cell lines. This suggests that infection-mediated CALC I gene expression is limited to differentiated parenchymal cells, here exemplified by adipocytes.

Among the inflammatory cytokines tested in the present report, IL-1ß acted as a potent stimulator of CT mRNA expression and ProCT synthesis. TNF{alpha} moderately stimulated CT mRNA expression and ProCT release. Both IL-1ß and TNF{alpha} have been ascribed significant roles in the cytokine mediation of sepsis and septic shock (19). Interestingly, parenterally administered recombinant TNF{alpha} was reported to increase serum ProCT levels into the septic range in non-infected humans, and ProCT could be measured in supernatants from TNF{alpha} and IL-6 stimulated liver slices, tissue which is composed of various cell types (20). However, in these studies the cellular source and mechanisms could not be determined, because no further molecular analyses had been performed. Presumably, the increase in CT mRNA gene transcription is mediated by one or several microbial-specific response elements in the CALC I gene promoter (21). During bacterial infections, a combined stimulation by microbial products (e.g. LPS) and of proinflammatory mediators of the host response (e.g. TNF{alpha} and IL-1ß) results in a generalized tissue-wide induction of CT mRNA and a consequent secretion of CTpr, including ProCT. LPS treatment alone also strongly induced ProCT synthesis. Hence, infection-related CALC I gene expression in adipocytes appears not to depend on inflammatory mediators from other cell types. This is in accordance with CD14 expression in human adipocytes (22) as well as LPS activity mediated via Toll-like receptors in murine adipocytes (23).

In several adipose tissue biopsies from infected subjects with high circulating ProCT we demonstrated in vivo extrathyroidal expression of CT mRNA. As expected, in fat samples from noninfected control patients CT mRNA was not present. Due to the large mass of adipose tissue in the human organism, we postulate that adipocytes contribute substantially to the systemic elevation of circulating ProCT in infected patients. Increased morbidity and mortality were recently reported in critically ill morbidly obese patients compared with nonobese patients (24). It is tempting to speculate that adipose tissue-derived ProCT contributes to the complications reported in obese intensive care units patients. In this context it is notable that the administration of human ProCT worsened the outcome, whereas immunoneutralization of endogenous ProCT improved survival in septic hamsters (5). In septic pigs, iv immunoneutralization of ProCT reduced mortality and improved physiologic and metabolic parameters even when administered after the animals were moribund (6). However, at present in humans no clinical data are available on this issue. Mortality rates in sepsis are very dependent on multiple host- and pathogen-related factors (e.g. comorbidities of the patient, virulence of the bacteria, among others). Hence, a presumed harmful effect of the additional ProCT secretion by the abundant adipose tissue mass in obese patients might be masked by these powerful infection-related factors. Possible roles of other cell types and tissues in humans are currently under investigation in our laboratory.

The present finding of CT mRNA in stimulated adipocytes and adipose tissue from septic patients is in contrast to the conventional endocrine concept of a tissue-specific CALC I expression restricted mostly to thyroidal C cells. Previously, the hypothesis was advanced, that CALC I gene products are a prototype of hormokine mediators (12). As such, they may follow either a classical hormonal expression or, alternatively, a cytokine-like expression pathway (Fig. 5Go). In sepsis, the predominance of serum ProCT as opposed to serum mature CT is indicative of a constitutive pathway within cells lacking secretory granules and, hence, a bypassing of much of the classic neuroendocrine enzymatic processing. Consequently, as is the case for most cytokines, in sepsis there is little to no intracellular storage of ProCT within nonneuroendocrine cells (12).



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  		FIG. 5. Schematic diagram of CALC I expression in adipocytes and thyroidal C cells. In the classical neuroendocrine paradigm, the expression of CT mRNA is restricted to neuroendocrine cells, mainly C cells of the thyroid. Initially, the 116-amino acid prohormone ProCT is synthesized and subsequently processed to the considerably smaller mature CT. In sepsis and inflammation, proinflammatory mediators induce CT mRNA. In contrast to thyroidal cells, adipocytes and other parenchymal cells lack secretory granules, and hence, unprocessed ProCT is released in a nonregulated, constitutive manner.

 
Our findings provide a molecular basis for the utility of circulating ProCT in the clinical diagnosis of systemic bacterial infections. Costimulation experiments using the strongest CT mRNA inducer, IL-1ß, revealed that IFN{gamma} acts as a potent inhibitor of IL-1ß-mediated CALC I gene induction. IFNs, including IFN{gamma}, play a pivotal role in early antiviral defense mechanisms (25). In contrast to bacterial infection, viral infections usually induce only a modest increase in circulating ProCT (2, 10). This phenomenon is clinically relevant; for example, one can expedite the diagnosis and treatment of a meningitis of bacterial origin and distinguish it from viral meningitis (26, 27). Our finding of an antagonistic effect of IL-1ß and IFN{gamma} on CT-mRNA induction and ProCT release might explain this clinically important phenomenon. During the host response to a viral infection, IFN{gamma} secretion might inhibit CT mRNA induction, thereby reducing the increase in ProCT levels in response to an inflammatory stimulus (Fig. 5Go). Other studies previously described opposing effects of IL-1ß and IFN{gamma} (28). Activation of nuclear factor-{kappa}B plays a central role in inflammatory signaling (29), but cytokine-specific activation of upstream factors, including signal transducers and activators of transcription and Janus kinases, are possible mediators of the antagonistic activities described herein.

In conclusion, the present report is the first demonstration of human extrathyroidal CT mRNA expression and ProCT release from parenchymal tissue. Furthermore, we have demonstrated that adipose tissue-derived cell cultures are useful for the investigation of inflammatory CT mRNA expression and ProCT release. It is hoped that this new experimental model will provide a tool to further study mechanisms and action of sepsis-related extrathyroidal ProCT production.


   Acknowledgments
 
We are grateful to Gerhard Pierer and Jon C. White for providing adipose tissue, and to Richard H. Snider, Jr., and Dominik Schaer for helpful discussions and most valuable conceptual advice. We thank Peter Huber for his kind collaboration, and Ursula Schmieder, Kaethi Dembinski, and Susanne Vosmeer for their excellent technical assistance. The ultrasensitive assays for CTpr determination were kindly provided by B.R.A.H.M.S. GmbH (Hennigsdorf, Germany).


   Footnotes
 
This work was supported by grants from the Swiss National Science Foundation (32-59012.99 and 32-068209.02), the Sonderprogramm zur Förderung des akademischen Nachwuchses der Universität Basel, the Nora van Meeuwen-Häfliger Foundation, the Krokus Foundation, and unconditional research grants from Novartis AG and B.R.A.H.M.S. GmbH (all to B.M.).

Abbreviations: CT, Calcitonin; CTpr, calcitonin precursors; HPRT, hypoxanthine-guanine phosphoribosyltransferase; IFN{gamma}, interferon-{gamma}; IMDM, Iscove’s modified Dulbecco’s medium; LPS, lipopolysaccharide; ProCT, procalcitonin.

Received July 10, 2003.

Accepted for publication August 12, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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