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Original Studies |
Laboratory of Molecular Endocrinology, Massachusetts General Hospital, Howard Hughes Medical Institute, Harvard Medical School (B.M., J.F.H.), Boston, Massachusetts 02114; and Department of Surgery and Medicine, George Washington University and the Veterans Affairs Medical Center (J.C.W., E.S.N., R.S.S., K.L.B.), Washington, DC 20422
Address all correspondence and requests for reprints to: Beat Müller, Division of Endocrinology, Diabetology and Clinical Nutrition, Department of Internal Medicine, University Hospitals, Petersgraben 4, CH-4031 Basel, Switzerland. E-mail: happymiller{at}bigfoot.com
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and interleukin-6). Our
findings, which describe a potentially new mechanism of host response
to a microbial infection mediated by CTpr, introduce a new
pathophysiological role for the CT-I gene. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Male, Golden Syrian hamsters, 90–120 g (Harlan Animals, Indianapolis, IN), were individually housed in a controlled environment with 12-h dark, 12-h light cycles and were given water and rodent chow ad libitum. The experiments adhered to the NIH guidelines on the use of experimental animals and were approved by the Institutional Review Board at the Veterans Affairs Medical Center before initiating the experiments. Escherichia coli (E. coli, O18:K1:H7) were obtained from the Walter Reed Army Institute of Research, Washington, DC. The bacteria were grown in 100 mL Miller LB (Luria-Bertani) broth. The optical density of the suspension was measured at 600 nm on a Stasar III spectrophotometer (Gilford Instruments, Oberlin, OH) and quantified by interpolation on a previously constructed curve of optical density plotted against colony forming units (cfu). Additional specimens were taken from the stock solution, diluted, and plated to confirm the counts. Suspensions of 1.66 x 109 cfu/mL E. coli were pipetted in 0.5-mL aliquots into 8-mm embedding molds (Shandon-Upshaw, Warrington, PA). Pellets were made by adding 0.5 mL sterile molten agar to each mold, resulting in a final bacterial dose of 8.3 x 108 cfu (LD50 at 72 h in this model of sepsis). All animals were fasted the night before surgery. Each animal was anesthetized with pentobarbital (50 mg/kg, ip), and the E. coli-impregnated pellet was implanted into the right lower quadrant of the peritoneal cavity via midline laparotomy. The incisions were closed in one layer with a nonabsorbable suture. Hamsters were individually caged and given unrestricted access to water and chow. Septic animals were killed by exsanguination via cardiac puncture, after pentobarbital anesthesia, 12 h later. Control hamsters implanted with noninfected pellets were killed similarly.
Peptide RIA and chromatography by gel filtration and high-performance liquid chromatography (HPLC)
The RIA design was similar to that previously reported (13). Hamster serum total immunoreactive CT (iCT) was determined with an antiserum (R1B4) raised to the midregion of mature human CT (11- to 18-amino acid fragment). This antiserum cross-reacts with CT (both human and hamster), in its mature amidated form, or within its precursor propeptides (i.e. ProCT, the conjoined CT:CCP-I, or the free unamidated CT). R1B4 was specifically chosen for this study because it cross-reacted well with the precursor peptides as well as mature CT. Other midregion-directed antisera that we tested cross-reacted poorly with all forms of hamster, most likely because of differences in the hamster sequence in this region. Although antisera directed against the carboxylterminus of CT, such as our Ab4 (13), cross-react well with mature hamster CT, they cross-react poorly (5–20%) with CTpr (i.e. CT:CCP-I and ProCT). R1B4 was preincubated with standards or unknowns (20–100 µL) and 50 µL goat antirabbit IgG bound to iron particles in 0.2 mL 0.2% gelatin-borate buffer (0.13 mol/L H3BO3 containing 9 g NaCl, 2 g gelatin, 1 mL Triton-X 100, and 0.1 g merthiolate/L, at pH = 7.5) at 4 C for 4 days. After addition of 50 µL I125-hCT, incubation was continued for 2 days. Bound and free hormones were separated with magnetic tube racks (maximum bound, 37%; sensitivity, 2 pg; 50% B/B0, 50 pg). Selected tissues were weighed, homogenized, and defatted with diethyl ether and chloroform. Peptide was extracted with NH4OH:CH3CN and assayed as described (14). The ratio was calculated from values expressed as pg iCT/g wet weight of control and of septic tissues.
The Sephadex G-75 superfine gel filtration and C18 reverse-phase HPLC techniques have been described previously (7). For HPLC, extracts from septic blood and control and septic tissue were injected in 200-µL aliquots (800–8000 fmol iCT), and 60 1-min fractions were collected for RIA with R1B4 or, in some cases, R1B4 and carboxylterminal antiserum Ab4.
Tissue extraction, RNA preparation, and cloning of hamster CT
Tissues harvested from anesthetized hamsters were incubated in RNAlater (Ambion, Inc., Austin, TX) to prevent RNA degradation. Sedimentation of red blood cells was achieved by the addition of an equal volume of 6% (wt/vol) Dextran (Sigma D-7265) in 0.9% NaCl, pH 7.2, to heparinized blood. A pellet of white blood cells was obtained by centrifugation (400 x g for 3 min) of the upper plasma phase. A pellet of peritoneal macrophages was obtained by rinsing the peritoneal cavity with 10 mL 0.9% NaCl (pH 7.2) and centrifugation at 400 x g for 3 min. Total RNA was extracted from homogenized tissues, by the single-step quanidinium-isothiocyanate method, with a commercial reagent (TRIzol reagent, Gibco BRL, Life Technologies, Inc., Gaithersburg, MD) according to the manufacturer’s protocol. Extracted RNA was quantified spectrophotometrically, and the quality was assessed by gel electrophoresis. Cross-species-specific synthetic oligonucleotides were used for PCR amplification of complementary DNA (cDNA) prepared from control and septic thyroid and septic spleen. Rapid amplification of cDNA ends was performed using a commercially available Kit (Gibco BRL, Life Technologies, Inc.). PCR was performed using a proofreading thermostable DNA polymerase (Advantage cDNA Polymerase Mix, CLONTECH Laboratories, Inc., Palo Alto, CA). For sequencing purposes, PCR fragments were blunt ligated into the plasmid vector pCR II (Zero Blunt TOPO PCR cloning kit, Invitrogen, San Diego, CA). The cDNA sequencing was done by Dye Deoxy Cycle Sequencing (Perkin-Elmer Corp., PE Applied Biosystems, Foster City, CA) according to the manufacturer’s instructions, and sequences were analyzed on model ABI 377 PRISM DNA Sequencing System (PE Applied Biosystems).
RT-PCR
Equal amounts of RNA per tissue were subjected to RT by oligo-dT
priming with Moloney murine leukemia virus reverse transcriptase
(Superscript, Gibco BRL, Life Technologies, Inc.). PCR was performed using introns border-spanning
oligonucleotides for CT (sense primer:
5'-GCAGCCTCCATGCAGTACCTTTCAGG-3', antisense primer:
5'-AGGCCTGCCCTTGCCCTCTG-3'), interleukin (IL)-6 (sense primer:
5'-TGGAACTTCCGGTGATACAAATAAATGA-3', antisense primer:
5'-CATTGTTCGTCACAAACTCCAGGTAGAT-3'), tumor necrosis factor (TNF)-
(sense primer: 5'-GCCTCTTCTCCTTCCTGCTTGTG-3', antisense primer:
5'-CATGGAGCCGATGATAGGGTTGG-3'). ß-actin was used as an internal
control to verify similar quantities of RNA loading in each reaction
(sense primer: 5'-GCACAGGCCTTTCGCAGCTCTTTCTTC-3', antisense primer:
5'-CGTCATCCATGGCGAACTGGTG-3'). PCR reaction was performed on a
conventional thermal cycler (Crocodile III Appligene Oncor, Watford,
UK) under the following conditions: CT: 10 min at 95 C (1 cycle), 15
sec at 95 C, 20 sec at 58 C, 60 sec at 72 C (34 cycles); for all other
genes: 60 sec at 95 C (1 cycle), 15 sec at 95 C, 60 sec at 64 C (IL-6,
34 cycles), 62 C (TNF, 34 cycles), 60 C (b-Actin, 34 cycles). PCR
products were separated and visualized on agarose gels containing 0.5
µg/mL ethidium bromide. All tissues of one PCR product were
electrophoresed on a single gel. Verification of RNA as the source of
amplification template was obtained by omitting the RT reaction for
pooled septic and control samples, respectively, and using a negative
control that contained no RNA and resulted in no bands after PCR. PCR
product identity was confirmed by direct nucleotide sequencing of the
PCR products of at least four randomly selected tissues by dye deoxy
terminator cycle sequencing, as described above. In addition, for CT,
specificity was verified for all tissues by Southern blotting after
hybridization and labeling with a
32P-radiolabeled oligoprobe covering a CT
specific area in exon IV (5'-GCCCAGCATGCAGGTACTCAGATTCC-3').
Quantitative analyses of CT-messenger RNA (CT-mRNA) expression (Taq-man technology)
The cDNA of hamster tissues was amplified with a PCR method using real-time detection and the 5' nuclease assay (Perkin-Elmer Corp. PE Applied Biosystems). Briefly, a nonextendable oligonucleotide hybridization probe is labeled with a reporter fluorescent dye at the 5' end and a quencher-fluorescent dye at the 3' end. During the extension phase of the PCR cycle, the 5' nuclease activity of the Taq DNA polymerase cleaves the hybridization probe to release the reporter dye from the probe, separating reporter dye from quencher dye. The relative increase in reporter dye emission is proportional to the amount of PCR product accumulated and monitored in real time during PCR amplification using the ABIPRISM 7700 Sequence detector (PE Applied Biosystems). A computer algorithm calculates the threshold cycle at which each PCR amplification reaches a fixed threshold, the CT value, representing a quantitative measurement of the copies of the target found in any sample. Each sample was assayed in quadruplicate, and the median CT CT and ßActin CT values were used to calculate the CT/ßActin ratio using the formula for the comparative CT method (provided by PE Applied Biosystems), 2CT-ßActin/2 CT-CT. Finally, the increase of CT-mRNA in sepsis was expressed as the CT/ßActin ratio of the septic tissues in relation to the CT/ßActin ratio of the respective control tissues.
In situ hybridization
Cryosections were stained using antisense 35S-radiolabeled antisense riboprobes and visualized as silver grains. An increased expression of CT-mRNA was visualized in septic lung, liver, and kidney. Tissue was harvested and quick frozen in liquid nitrogen. After embedding in optimum cutting temperature compound (O.C.T.-compound, Tissue-Tek, Sakuraus Finetek U.S.A., Inc., Torrance, CA), 6-µm frozen sections were cut in a cryostat microtome at -20 C, thaw-mounted onto coated slides, fixed in 4% paraformaldehyde, washed in PBS, and kept at -70 C. In situ hybridization was performed as described (15). Briefly, after rehydration in PBS, the slides were permeabilized in 0.2 HCl, acetylated in 0.1 triethanolamine and 0.25% acetic anhydride, and delipidated and dehydrated in ethanol and chloroform before hybridization. If applicable, the slides were incubated in 10 µg/mL ribonuclease (Rnase) before dehydration. Sections were covered with heat-denatured hybridization buffer containing 10 mmol/L Tris-HCl (pH 7.6), 600 mmol/L NaCl, 50% (vol/vol) formamide, 10% dextran sulfate, 200 µg/mL yeast transfer RNA, 1 x Denhardt’s solution, 0.25% (vol/vol) SDS, 0.05% (vol/vol) dithiothreitol, and 35S-labeled (5 x 107 cpm/mL) antisense or sense riboprobe covering the CT-specific exon Iv of the CALC-I gene (plasmid kindly provided by Jo Yeakley, La Jolla, CA). Hybridization occurred overnight (16 h) at 55 C, and excess probe was removed by washing in 50% 2x SSC/formamide and incubation with Rnase A. After the final washes in 2 x and 0.2 x SSC, the sections were dehydrated in ethanol, dipped in NTB2 film emulsion (Eastman Kodak Co., Lowell, MA), and developed after 12 h (thyroid) or 72 h (for all other tissues) as suggested by autoradiography. Finally, the sections were counterstained with hematoxylin and eosin.
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Results |
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). HPLC (and gel filtration) of septic
and control tissues confirmed that the iCT in septic specimens
consisted mainly of CTpr (Fig. 2).
Interestingly, ProCT, CT:CCP-I, and N-ProCT are the predominant forms
of CTpr found in human septic sera (7). In hamster septic
sera, the predominant forms of iCT found by a CT assay are ProCT and
CT:CCP-I. We did not further analyze the hamster serum for other CTpr,
namely N-ProCT, which (in analogy to our human findings) are likely to
be present in substantial amounts. Interestingly, both control and
septic lung contained substantial iCT peaks corresponding to mature CT
and CT-dimer, whereas the major iCT increase in sepsis was ProCT. The
iCT concentration is higher for the thyroid than for any other control
or septic tissue. However, both control and septic thyroid extracts
contained mainly mature CT.
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). As expected, in control tissues, the
CT-mRNA expression was restricted to the thyroid and to neuroendocrine
cells in the lung. In contrast, in sepsis, we found a uniform and
ubiquitous expression of CT-mRNA. The specificity of the RT-PCR
reaction was ascertained by Southern hybridization, using a hamster
CT-specific radiolabeled oligonucleotide, and further confirmed by
direct sequencing of the PCR products. The levels of expression in
septic extrathyroidal tissues, however, seemed to be
severalthousand fold lower, compared with the expression in
control or septic thyroid (Fig. 3, lowest panels).
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). We found a significant induction of
CT-mRNA expression in all septic tissues examined, in contrast to
control tissues. Very dramatic increases were observed in classical
immune organs and cells (e.g. spleen and peritoneal
macrophages) but also in other tissues (e.g. fat, testes,
and brain).
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). A similar amplification pattern of
the housekeeping ß-actin gene in septic and control tissues was
maintained to ensure comparability. Importantly, the median increase of
CT-mRNA in sepsis, as calculated by Taq-Man quantitative
PCR, was significantly higher than the increase of TNF- and IL-6
mRNA (46 vs. 4 vs. 8; P = 0.004
by nonparametric Kruskal-Wallis ANOVA). Peritoneal macrophages had a
strong CT-mRNA signal, whereas white blood cells had only a relatively
weak signal, in comparison to the strong signal for TNF- mRNA.
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). Increased expression of CT-mRNA
was visualized ubiquitously in septic lung, liver, and kidney. As
control, C cells of the thyroid were positive both in control and
septic tissues, and no signal was seen with Rnase pretreatment or using
the sense probe. The CT-mRNA was diffusely distributed in septic
tissues and not restricted to specific areas or cell types. This
diffuse pattern was not seen in control thyroid, where the expression
was highly restricted to the parafollicular neuroendocrine C cells, as
shown by in situ hybridization (as negative controls, no
hybridization signal was seen in thyroid or septic tissues after Rnase
pretreatment or use of the sense probe). Importantly, CT-mRNA
expression was much higher in thyroidal C cells than in other cells,
concordant with the results of RT-PCR. To avoid overexposure, thyroid
sections had to be exposed to the film emulsion for a much shorter time
(12 h for thyroid, compared with 72 h for the other tissues).
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Discussion |
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CTpr correlate with the presence, course, and outcome of sepsis in
humans (11). Most classical proinflammatory cytokines
(e.g. TNF-, IL-1ß, or IL-6) are detected, if at all,
briefly or intermittently. These mediators are primarily found in local
pools after an inflammatory challenge. In contrast, CTpr circulate
widely and are specifically and persistently elevated by many orders of
magnitude in the systemic inflammatory response caused by a microbial
infection. To better understand the pathophysiologic background of CTpr
induction in sepsis, we used an animal model of sepsis analogous to
human sepsis. As in humans, serum CTpr levels are markedly increased in
septic hamsters and correlate with mortality (13).
Compared with controls, CTpr levels were also significantly increased
in most of the septic hamster tissues analyzed. HPLC (and gel
chromatography, not shown) confirmed the presence not only of ProCT but
also of large amounts of other CTpr in sepsis. Concordant with human
data, no increase of mature CT was detected in septic hamster tissues
(11). Interestingly, we did not find significant levels of
CTpr in either control or septic thyroid.
The high CTpr protein content of specific septic tissues could be explained by local synthesis and/or by binding to receptors within those tissues. To evaluate the expression of mRNA during sepsis, we cloned the hamster CT-cDNA and analyzed a broad selection of control and septic tissues by RT-PCR. In control animals, as expected, the principal site of CT-mRNA expression is C cells of the thyroid. In septic animals, however, we observed a ubiquitous induction of CT-mRNA in all tissues examined. In sepsis, the relative increase of CT-mRNA was much more dramatic in extrathyroidal tissues than in the thyroid. However, the cellular extrathyroidal CT-mRNA expression in sepsis remained much lower than that in the C cells. Nonetheless, this was more than compensated for by the much larger mass of extrathyroidal tissue. Therefore, the extrathyroidal cells contributed almost exclusively to the elevated serum CTpr levels found in sepsis. Under these circumstances, the entire body could be viewed as an endocrine gland.
Importantly, CT-mRNA seemed more specifically increased in sepsis than
classical proinflammatory cytokines (i.e. TNF- and IL-6).
For example, high levels of TNF- mRNA expression were also found in
several tissues of control animals (e.g. adipose tissue). In
this respect, important nonimmune related functions have recently been
ascribed to TNF-, specifically, a role in the insulin resistance of
adipose tissue (16, 17). The more uniform up-regulation of
CT-mRNA in systemic microbial infection provides a molecular basis for
the superior diagnostic accuracy of plasma CTpr levels in patients with
sepsis, compared with classical markers of infection. The clinical
value of traditional markers, including cytokines (e.g.
C-reactive protein, IL-6, and TNF-), for the detection of systemic
infection is limited, mainly by the variability of results. These
markers reflect a nonspecific response of the organism to various
stimuli, all resulting in a relatively common systemic inflammatory
response, present in most critically ill patients (18, 19). Moreover, in contrast to the transient increases of
cytokines in experimental sepsis, the increases of serum CTpr are
sustained for longer than 24 h (20). Thus, the serum
CTpr increase seems to be more specifically associated with the
presence of a microbial infection.
The RNA for RT-PCR was obtained from homogenized tissues. However, parenchymal tissues consist of various different cell types. Thus, a positive signal could be interpreted as an expression of CT-mRNA by a cell type present in all septic tissues (e.g. macrophages, endothelial cells, or neuronal cells), and/or by various parenchymal cell types (e.g. hepatocytes in liver). However, as shown by in situ hybridization, in septic tissues, CT-mRNA was diffusely distributed and not restricted to specific areas or cell types. Clearly, in septic hamsters, CTpr are produced by multiple tissues and cell types throughout the body, in contrast to control hamsters, in which the CT-mRNA expression is largely restricted to the parafollicular neuroendocrine C cells.
The histological picture of the source of CTpr might reflect
infiltrating blood cells in parenchymal tissues. There are conflicting
data concerning white blood cells as a source of CTpr in sepsis
(21, 22). Our data show a relatively low expression of
CTpr in the white blood cells of septic animals (and also of humans;
unpublished data); this is in striking contrast to the high expression
of classical cytokines (e.g. TNF-) within these cells.
Moreover, evidence from case reports of septic patients with high serum
levels of CTpr, even after near-complete eradication of the leukocyte
population by chemotherapy, also suggests that white blood cells are
not a major source of the increased CTpr levels found in human sepsis
(23, 24).
Our data suggest a novel, specific mechanism of host response to a microbial infection that is reminiscent of cytokine expression. In this pathway, CTpr, acting as so-called hormokine mediators, challenge the current physiological paradigm of the CALC-I gene. In the absence of infection, the transcription of the CALC-I gene is suppressed and is restricted to a selective expression in neuroendocrine cells found mainly in thyroid and lung. In these neuroendocrine cells, the mature, yet relatively inactive, hormone is processed and stored in secretory granules. A microbial infection is now shown to induce a ubiquitous, yet specific, increase of CALC-I gene-expression and a release of CTpr from tissues and cell types throughout the body. This production of hormokines is mediated by as-yet-unknown factors and might be induced either directly via microbial toxins or indirectly via a humoral or cell-mediated host response. Interestingly, serum levels of mature CT are not substantially elevated in microbial infection (7, 11). The predominance of CTpr indicates a constitutive pathway in cells lacking secretion granules, and hence bypassing much of the enzymatic processing (25). A shift from a regulated pathway of secretion (which produces a mature peptide) to a nonstoring, bulk-flow, constitutive form of secretion has been induced experimentally (26, 27, 28). Similarly to most cytokines, there is no intracellular storage of CTpr in sepsis, explaining why little or no CTpr has been seen by immunohistochemistry performed on frozen sections (data not shown).
In contrast to the normal situation, in sepsis, the transcriptional
regulation of the CALC-I gene seems to be stimulus-related, rather than
tissue-specific. Our findings imply a novel role for CALC-I gene
products in the immune reaction, akin to classical cytokines
(e.g. TNF-, IL-1ß, or IL-6), in addition to their
classical endocrine functions. In contrast to other cytokines, however,
CTpr seem to be induced by a unique and more specific transcriptional
activation cascade.
Mortality rates for sepsis have not declined, despite advances in the understanding of its pathophysiology, the advent of new antibiotics, and improvements in intensive care medicine (29). Our CTpr findings could suggest novel approaches to the development of new therapies for improving the unfavorable prognosis of sepsis. The prior findings that ProCT is toxic only in septic animals, but not in controls, and that immunoneutralization of endogenous CTpr markedly reduces mortality in septic hamsters, point to CTpr as a potential target for therapeutic intervention (12). Elevated serum CTpr have been shown to be the most reliable markers of microbial infections in critically ill patients (11). The prolonged increase of serum CTpr levels in severe infections provides an equally sustained target for therapy. Last, further molecular investigation will be important to unravel the role of the hormokine and its prototype, the CALC-I gene, in microbial infections.
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Acknowledgments |
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Footnotes |
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2 An investigator with the Howard Hughes Medical Institute.
Received June 20, 2000.
Revised August 30, 2000.
Accepted September 13, 2000.
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