Procalcitonin and the Calcitonin Gene Family of Peptides in Inflammation, Infection, and Sepsis: A Journey from Calcitonin Back to Its Precursors

K. L. Becker, E. S. Nylén, J. C. White, B. Müller and R. H. Snider, Jr.

Veterans Affairs Medical Center and George Washington University (K.L.B., E.S.N., J.C.W., R.H.S.), Washington, D.C. 20422; and University Hospitals (B.M.), CH-4031 Basel, Switzerland

Address all correspondence and requests for reprints to: Dr. Kenneth L. Becker, Director of Endocrinology, Veterans Affairs Medical Center, 50 Irving Street NW, Washington, D.C. 20422. E-mail: klb1{at}erols.com.


    Introduction
 Top
 Introduction
 Conclusions
 References
 
Calcitonin (CT) is a hormone that received its name because of its secretion in response to induced hypercalcemia and its hypocalcemic effect (1). It was shown to originate from the thyroid gland (2). More specifically, the hormone was revealed to be located within the thyroidal parafollicular cells, interspersed within and about the follicular epithelium (3, 4, 5). Subsequently termed C cells, they occur primarily in the central region of each lobe of the human thyroid gland (6, 7). These cells, which have CT-containing secretion granules, are neuroendocrine. Embryologically, they originate from the neural crest and migrate to the ultimobranchial glands (8). In mammals, the ultimobranchial glands fuse with the thyroid gland.

It was the demonstration that medullary thyroid cancer (MTC) was a malignancy of the C cells (5, 9) that eventually led to the isolation of human CT from this tumor and the determination of its structure (10, 11). Simultaneously, the amino acid sequence of porcine CT was determined (12). Later, the development of immunoassays of serum CT in humans led to the observation that the level of this hormone was increased in the serum of patients with MTC (13, 14, 15) and to the demonstration that these levels were further augmented after iv calcium and/or pentagastrin administration (13, 16, 17). These findings had a great impact on the clinical diagnosis, the evaluation of efficacy of surgical extirpation, and the follow-up monitoring of MTC. Although RET germline mutation testing has replaced CT for the purpose of determining the presence of carriers of this tumor associated with multiple endocrine neoplasia type 2 (18, 19), the measurement of serum CT has become and has remained the classical clinical marker for MTC.

Immature and mature CT

It had been found that immunoreactive CT was present in multiple heterogeneous forms in MTC tissue as well as in the serum of patients with this tumor (20, 21, 22, 23, 24). Consequently, it became apparent that when this peptide was measured with antisera recognizant to different epitopes the values varied according to the antiserum and the immunochemical heterogeneity (25). The phenomenon of heterogeneity was then further clarified by a series of studies which demonstrated that CT is biosynthesized as part of a larger prohormone, procalcitonin (ProCT) (Fig. 1Go) (21, 23, 25, 26, 27).



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FIG. 1. Schematic representation of ProCT and the other CT precursors (CTpr) derived from this prohormone (i.e. NProCT, CT-CCP-I, and CCP-I). The mean concentrations of these peptides in normal serum is indicated. Note that there is appreciably more free NProCT in the serum than CT(1–32). In sepsis, the principal elevations involve the intact ProCT, free NProCT, and free conjoined CT-CCP-I peptide. Sequencing reveals that in sepsis, the ProCT may lack the first two amino acids of the aminoterminus of the molecule, presumably due to enzymatic hydrolytic aminoterminal truncation (110 ), and perhaps other cleavage forms are present as well (111 ). The comparative extent to which any one of these peptides is increased varies among patients. Levels of the free CCP-I peptide also increase but to a lesser extent. In sepsis, serum CT(1–32) concentrations are undetectable, normal, or only slightly to moderately elevated (data from Ref. 30 ).

 
The term "mature" hormone has been used to indicate a bioactive hormonal peptide that has been derived from a larger precursor prohormone. This prohormone may be less active, inactive, or characterized by an activity that differs entirely from the mature hormone. Not uncommonly, much of the bioactivity of a mature hormone may be linked to an amidation that occurs at its carboxyl end. Within ProCT, CT is in a nonamidated, immature 33-amino acid form, terminating with a glycine (28). It then undergoes posttranslational processing that results in production of several additional free peptides as well as mature CT (29, 30, 31).

All species of mature CT contain 32 amino acids, with a disulfide bridge at the amino terminal end (between amino acid positions 1 and 7) and a proline at the carboxyterminal end; hence, for the purpose of clarity in this manuscript, the term CT(1–32) will be used specifically to refer to this peptide. Among the various species of CT(1–32), the amino acid sequence of the peptide tends to be well conserved within the amino acid ring structure at the amino terminus, but there are differences elsewhere within the molecule (32, 33, 34). At the carboxyl terminus of the CT(1–32), the proline is amidated (35, 36). Importantly, both the ring structure and this amidated proline are essential for the full expression of the known bioactions of this hormone.

The accurate quantification of the free CT(1–32) peptide requires the selective detection of the amidated carboxyl terminal portion of the molecule, thus excluding the nonamidated 33-amino acid immature CT, which is found within some of the larger molecular weight precursors. Such commercially available assays were not developed until the late 1980s; they use a double-antibody method: one antibody reacts selectively with the amidated region and the other with a different portion of the molecule (most commonly the midportion). Thus, these assays do not cross-react with immature CT (37, 38, 39). In this regard, it is important to emphasize that most CT studies in the literature relating to physiopharmacologic manipulations as well as such influences as age, gender, pregnancy, and hormonal milieu were not documented with these specific assays.

Physiologic actions of CT

Hundreds of studies of the possible role of CT(1–32) have been performed. The great bulk of in vitro and in vivo investigations have involved laboratory animals, some with prior parathyroidectomy and some without. Often the species of CT(1–32) used in these experiments were other than human (e.g. salmon, porcine, eel), the amino acid sequences of which differ. Furthermore, pharmacologic, not physiologic, doses often were employed. As a result, many actions have been incorrectly imputed to this peptide. Known or alleged biologic actions of CT(1–32) have been reviewed elsewhere (31, 33, 40, 41).

Although seemingly relevant effects have been observed in blood, bone, kidneys, and the respiratory, gastrointestinal, embryogenic, and central nervous systems (40, 42, 43, 44, 45), the function of CT(1–32) in humans remains enigmatic (41). The hormone is not confined to the thyroid gland, and it is impossible to extirpate all cells producing this peptide (see below). However, the recent development of a knockout mouse in which the coding sequences for both CT(1–32) and CT gene-related peptide (CGRP)-I were deleted have provided important information (46). In these animals, no birth defects or difficulty with procreation were noted, and serum levels of basal calcium-related values were normal. However, these mice exhibited an increased calcemic response and a greater bone resorption in response to exogenous PTH, perhaps due to the absence of an otherwise inhibiting effect of CT(1–32) on bone resorption. Surprisingly, these knockout mice manifested a markedly increased bone formation; also, in contrast to wild-type mice that lose bone mass after ovariectomy, they maintained their bone mass. These findings suggest that the CT/CGRP-I gene product may somehow regulate bone formation, either directly or indirectly. Further studies of these interesting observations are needed to determine whether this action is related to CT(1–32), CGRP-I, or both acting conjointly, and also whether it is species specific. Additional studies should also determine whether the induced knockout results in a compensatory overexpression of the gene that gives rise to CGRP-II, which, as a result, may conceivably modulate or modify the resultant phenotype.

The classic and best-studied action of CT(1–32), which appears to occur generally throughout the mammalian species, is the action on the osteoclast (34, 47, 48). Acutely, this hormone alters the osteoclast sensitivity to ambient calcium and induces quiescence of osteoclast motility and a retraction of the pseudopods that is associated with a cessation of membrane ruffling. The peptide also inhibits the elaboration by the osteoclast of acid phosphatase, carbonic anhydrase II, focal adhesion kinase, and osteopontin. Possible anabolic effects of CT(1–32) on the osteoblast have been reported (49) but require further documentation. The overall impact of the osteoclastic inhibition is to decrease bone resorption (50). Nevertheless, neither the diminution of serum CT(1–32) occurring subsequent to thyroidectomy nor the marked excess of serum levels of CT(1–32) that occurs in patients with MTC, appear to be associated with alterations in serum calcium or noticeable decreases or increases of bone mass (51). Perhaps the major function of CT(1–32) is to combat acute hypercalcemia in emergency situations and/or to conserve calcium stores during growth, pregnancy, and lactation.

The osteoclast has a CT receptor, as do other cells elsewhere, e.g. monocytes, kidney, brain, pituitary, placenta, prostate, testis, lung, and liver (52, 53, 54, 55). The CT(1–32) receptor has been cloned (56, 57, 58). In the human, there is a polymorphism of this receptor (59), which may clinically influence bone density and quality (60), and these different isoforms may also have other functional implications. Stimulation of the CT(1–32) receptor induces increased cAMP and an increased cytosolic free calcium concentration, accompanied by activation of the MAPK pathway (61).

Therapeutic effects of CT(1–32)

Although the usage of CT(1–32) as a drug has greatly diminished, it continues to have a therapeutic role, principally in osteoporosis and Paget disease. The commercially used drug is the amidated, synthetic salmon preparation; in the United States, human CT(1–32) has become an orphan drug. For osteoporosis, it is commonly agreed that bisphosphonate therapy is more effective than CT(1–32) and is currently the preferable therapy (62). However, salmon CT(1–32) has been well demonstrated to increase bone density and decrease fracture rate, especially in the vertebrae (63, 64, 65); adverse effects are minimal. For osteoporosis, the nasal or sc route can be used. Bisphosphonates also have replaced CT(1–32) for the therapy of Paget disease (66, 67); nevertheless, sc salmon CT(1–32) still may be useful in the occasional patient who cannot tolerate high doses of bisphosphonates (68). In the United States, the nasal CT(1–32) preparation is not approved for Paget disease. As an acute or subacute therapy for hypercalcemia, the sc or im administration of salmon CT(1–32) is not a reliable procedure; the treatment of choice is adequate hydration and iv bisphosphonates (69). Various possible antinociceptive effects of CT(1–32) have been described (70). For example, in patients with painful osteolytic metastases, symptomatic therapy may be beneficial (71); however, the limited evidence in the literature does not support its use for this purpose (72).

ProCT and CT precursors

CT(1–32) is biosynthesized from the polypeptide precursor, ProCT. This 116-amino acid prohormone is comprised of three constituent peptides: a 57-amino acid sequence at the amino terminus (NProCT); the centrally positioned immature CT that contains a terminal glycine; and a 21-amino acid CT carboxyterminus peptide I (CCP-I) (Fig. 1Go) (28). Subsequent enzymatic posttranslational processing yields several peptides (31, 73, 74); in addition to CT(1–32), the serum of normal persons contains intact ProCT, free NProCT, free CCP-I, and the free conjoined CT-CCP-I peptide. Interestingly, the normal molar concentration of circulating NProCT is 2-fold higher than that of CT(1–32) (4.15 fmol/ml vs. 1.84 fmol/ml) (30). Because these peptides are relevant to and precede the biosynthesis of CT(1–32), they have been given the collective appellation CT precursors (CTpr) (75, 76). The extent to which CTpr may have physiologic functions is under study (see below).

CTpr in MTC

An appreciable number of conditions are associated with increased serum levels of CTpr (Table 1Go). Although it has been known for many years that normal thyroid gland, MTC tissue, and the serum of MTC patients contain large amounts of ProCT as well as its component peptides (22, 77), the potential clinical utility of CTpr as a serum marker for MTC has very rarely received attention. In this respect, initially it was found that the carboxyterminal region of ProCT (corresponding to CCP-I, also termed katacalcin) was secreted into the medium of MTC cultures as well as into the serum of patients with this tumor in a calcium-dependent manner (78). Subsequently an assay for CCP-I was evaluated as a serum marker for MTC (79). It was later reported that the NProCT cleavage product of ProCT also was a potential marker for this tumor (80). Recently, in a study of MTC patients (81), a sensitive and rapid (3-h) two-antibody sandwich assay that quantifies both intact ProCT and CT-CCP-I (82) was compared with an assay that is specific for CT(1–32). CTpr were found to be universally present in the serum of patients with MTC. They were increased whenever CT(1–32) was increased and, on a pg/ml basis, exceeded CT(1–32) levels by about 10-fold. Also, both markers responded to pentagastrin. Whether CTpr are more sensitive markers for the presence of MTC or might be more useful prognostically remains to be determined.


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TABLE 1. Clinical conditions in which serum CTprs are increased1

 
CTpr in several other clinical disorders

Other neuroendocrine tumors, such as small cell cancer of the lung (SCLC), carcinoid, pheochromocytoma, and pancreatic islet tumors may exhibit increased serum CTpr. In contrast to MTC, in these conditions, the serum CTpr/ CT(1–32) ratio is further increased, probably because these lesions lack sufficient posttranslational enzymatic capability (30, 83, 84). In the case of SCLC and carcinoid tumor, the occurrence of increased serum CTpr is very frequent. Using an immunoassay that is reactive to the midportion of CT [and hence detects the presence of the ProCT and free CT-CCP-I molecules as well as CT(1–32)], there was found to be an association between the levels obtained and the clinical course of the tumor (85). Similarly, levels usually decreased concomitantly with radiotherapy and/or chemotherapy. Also, decreased levels corresponded to clinical remissions. Interestingly, SCLC is thought to originate from the same precursor cell as does the normal pulmonary neuroendocrine (PNE) cell, a cell that contains large amounts immunoreactive CT (42, 86, 87). However, lung cancers other than SCLC may contain and secrete immunoreactive CT. In such patients, these peptides may originate from the tumor, admixed SCLC cells, or adjacent noncancerous PNE cells that are known to become hyperplastic in response to chronic cigarette smoking (88, 89). Heterogeneity studies that have been performed in such cancer patients also reveal a large CTpr/CT(1–32) ratio (83).

Some noncancerous conditions that are associated with increased levels of serum CTpr appear to be explicable, all or in part, on the basis of hypersecretion or hyperplasia of the PNE cells [i.e. chronic bronchitis (e.g. smoking, occupational, cystic fibrosis), chronic obstructive pulmonary disease, acute inhalational burn injury, acute chemical pneumonitis, and tuberculosis] (84, 90). Furthermore, the kidney plays an important role in the metabolism of CT(1–32) (91), and renal disease often is associated with increased serum immunoreactive CT levels, much of it consisting of CT-precursor peptides (24, 92, 93).

Lastly, as emphasized in the present review, extraordinary increases of serum CTpr occur in patients with severe inflammation, systemic infection, and sepsis. Indeed, CTpr serum levels can be used as markers for the presence and severity of these conditions. Furthermore, in these conditions, high levels of CTpr appear to play a harmful role, and their immunoneutralization offers the potential of effective therapy.

CTpr in inflammation, systemic infection, and sepsis

Pathophysiology. Inflammation, a highly complex phenomenon that may be beneficial and/or detrimental to the host, is a reaction to a large variety of injuries. Inflammation can be local or systemic. It is characterized by vasodilation, attraction of polymorphonuclear cells and lymphocytes, activation of macrophages, altered capillary function, transudation of serum into the tissues, and the release of various humoral substances (Table 2Go).


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TABLE 2. Schematic illustration of events and humoral factors following exposure to a variety of insults that trigger an inflammatory response1

 
There are a variety of distinct conditions that manifest systemic inflammation (e.g. severe burns, pneumonitis or other marked local infections, bacteremia, endotoxemia, trauma, heat stroke, and pancreatitis). Typically any of these conditions may lead to a clinical syndrome that has been termed the systemic inflammatory response syndrome, which is defined by varying combinations of fever or hypothermia, tachypnea, tachycardia, and polymorphonucleocystosis or leukopenia (94, 95). Associated with these manifestations, there is a variable local as well as systemic increase of many cytokines and other hormonal messenger molecules [e.g. TNF{alpha}, IL-1ß, IL-6, interferon-{gamma}, arachidonic acid derivatives, cortisol] (Table 2Go). Some of these substances, acting in a hemocrine and/or paracrine manner, are protective to the host; some may be harmful, and some may be either beneficial or damaging, depending on their concentrations, their timing, or the ambient humoral milieu. Alternatively, the increases of some of these substances may be epiphenomenal and exert no relevant bioeffects.

The clinical term, sepsis, has been used to indicate a systemic inflammatory response syndrome in which bacteria or microbial products have been shown or suspected to be the etiology. In some cases, infection cannot be documented because microbial culture does not reveal a pathogenic microbe. It is likely that in some of these culture-negative cases, the sepsis is indeed due to microbes, but the methods of detecting them are not completely reliable and, therefore, the pathogen may not be identified. Moreover, in other cases, toxic byproducts of the pathogen may be responsible for the syndrome. For example, the translocation through the gut wall of toxins [e.g. endotoxin (lipopolysaccharide, LPS)] from bacteria normally inhabiting the gastrointestinal tract may be the cause of the sepsis (96, 97).

In sepsis, the patient is not ill principally because of the initial injury or infection but because of a humoral and/or cellular overreaction of the host. The unrestrained or unbalanced cytokine and humoral response in this illness may progress sufficiently to cause multiple organ failure, characterized by varying degrees of severe occurrences, such as myocardial insufficiency, hypoperfusion, shock, coagulopathy, respiratory failure, hypoxemia, renal failure, and coma. Sepsis is the 11th leading cause of death in the United States. Approximately half of the fatalities in intensive care units result from this condition. The incidence of sepsis is increasing (currently approximately 750,000 cases per year), and the mortality remains unchanged (approximately 30%, with rates up to 75% in septic shock) (98).

CTpr as markers of inflammation, systemic infection, and sepsis. An initial publication in 1983 first called attention to increased serum levels of immunoreactive CT in patients with the staphylococcal toxic shock syndrome, a severe form of sepsis (99). The assay used an antiserum that was not selective for the amidated carboxyl terminal portion of CT(1–32) and hence would also bind to the immature CT within the ProCT and CT-CCP-I molecules. Gel filtration studies demonstrated that this immunoreactive CT was of large molecular weight, now known to correspond to ProCT and CT-CCP-I. This paper provided the inspiration for multiple subsequent studies of CTpr in inflammation, systemic infection, and sepsis.

The traditional clinical signs of severe infection (e.g. fever or hypothermia, tachycardia, tachypnea, or hypotension) and the routine laboratory tests (e.g. an abnormal leukocyte count, elevated serum C-reactive protein, or positive bacteriologic studies) may be nonspecific or may not occur at all. Furthermore, the classical associated proinflammatory cytokines of severe inflammation, systemic infections, and sepsis (i.e. TNF{alpha}, IL-1ß, or IL-6) commonly are increased in the serum only transiently or intermittently. In contrast, serum levels of CTpr are very frequently increased, sometimes attain levels several thousand-fold normal, and these high levels often persist for long periods of time. Moreover, the levels often correlate positively with the severity of the condition and mortality. Indeed, after the first systematic study of sepsis due to severe bacterial infection (100), many clinical studies have documented the considerable utility of serum CTpr to identify and follow the course of this illness and sepsis-like conditions (Fig. 2Go) (100, 101, 102).



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FIG. 2. Receiver operating-curve analysis of serum CTpr for the diagnosis of sepsis in an intensive care unit, as compared with values for circulating C-reactive protein, IL-6, lactate, and pentraxin-3 (PTX-3). The sensitivity of CTpr for the diagnosis of sepsis was 89%, specificity 94%, negative predictive value 90%, and positive predictive value 94% (as assessed by the commercially available LUMItest PCT). However, the other non-CTpr markers were considerably less sensitive and less specific and had relatively poor negative or positive predictive values (modified from Ref. 102 ).

 
Thus, serum CTpr have been shown to be extremely useful markers in sepsis, whether blood cultures are positive or negative, and also in sepsis-like conditions such as severe burns, pancreatitis, pneumonitis, inhalational injury, bacterial meningitis, heat stroke, and severe mechanical trauma after extensive surgery, and also in some infections of nonbacterial causation (e.g. severe malaria or systemic fungal infections) (100, 101, 102, 103, 104, 105, 106, 107, 108). This phenomenon also occurs in patients with a prior thyroidectomy (100, 109).

Molecular sizing by gel filtration and HPLC studies in sepsis and in the above septic-like conditions have demonstrated that serum components corresponding to ProCT, NProCT, CT-CCP-I, and the CCP-I peptide are all elevated to varying degrees (30) (Fig. 1Go). The individual components were quantified with antisera specific for CT(1–32), NProCT, CCP-I, and also a two-site assay that used antibodies specific to the CT and CCP-I regions of ProCT. Using capillary zone electrophoresis spectrometry and Edman sequence analysis, the serum ProCT in sepsis has been reported to lack the first two amino acids of the molecule (Ala-Pro), consequent to an aminoterminal truncation by the dipeptidyl peptidase IV enzyme (EC3.4.14.5) (110). In another study, immunoreactive ProCT and its cleavage products were extracted from pooled septic serum using octadecylsilyl columns and characterized by HPLC and Western blot analysis of electrophoresis-sizing gels. The peptides were identified using antisera specific for CT, NProCT, and CCP-I (111). These investigators found ProCT and multiple fragments of ProCT that appeared to differ in molecular size from those found in the serum of patients with MTC. Thus, whereas there is some uncertainty as to the absolute identities of some of the fragments of ProCT present in the sera of sepsis patients, there appears to be universal agreement that both ProCT and multiple fragments of ProCT are present. Interestingly, in all these sepsis and sepsis-like conditions, CT(1–32) remains undetectable or normal or slightly to moderately elevated (30, 100, 102) (see below).

There are four assays that have been created to measure serum CTpr. Currently none of these assays are commercially available in the United States. The authors developed a single-antibody RIA for NProCT, which quantitates ProCT and the free NProCT peptide (as well as ProCGRP and NProCGRP) (76, 101). This sensitive assay [10 pg/ml (1.6 fmol/ml)] detects CTpr in healthy nonsmoking persons [the upper limit of normal is 76 pg/ml (12 fmol/ml)]. There are two two-site rapid assays. In Europe, a commercially available assay (LUMItest PCT, B.R.A.H.M.S. Diagnostica GmbH, Henningsdorf/Berlin, Germany), shortly to be available in the United States, measures both ProCT and the conjoined CT-CCP-I by means of a luminometer (100). This assay, which has been used in many clinical studies, is inaccurate at levels less than 300 pg/ml (24 fmol/ml). This company manufactures another more sensitive double-antibody kit, which can reliably detect levels as low as 20 pg/ml (1.6 fmol/ml) (82). The sensitive assays, including a recently developed tracer technology Kryptor assay (B.R.A.H.M.S.), the latter of which is commercially available in Europe, are especially useful for the detection of early forms of infection and for follow-up determinations (112).

Ubiquity of expression of the calcitonin-I gene in response to sepsis. Normally, immunoreactive CT is found predominantly in some neuroendocrine cells such as the thyroid C cells and PNE cells, and CGRP is found predominantly in brain and neurologic tissues. However, immunoreactive CT is found in many tissues throughout the body (113). Furthermore, low levels of CT gene mRNAs have been reported to occur in liver (55) and also several other tissues (114). In septic hamsters, substantially increased levels of immunoreactive CT (apparently mostly ProCT as shown by gel filtration chromatography and HPLC) were found in liver, lung, kidney, pancreas, brain, heart, and small intestine (115). In this investigation, quantitative analysis of CT mRNA expression was determined by the CT/ß-actin ratio of the septic tissues in relation to the CT/ß-actin ratio of the respective control tissues (Taq-Man technology). Among the tissues studied, the relative increase of CT mRNA, compared with controls, was, in descending order, adrenal, spleen, spinal cord, brain, liver, pancreas, colon, lung, fat, testes, and stomach (115). This phenomenon of marked increase of CT mRNA in extrathyroidal tissues also occurs in the septic human (116). Of course, when evaluating the impact of each increase, consideration must be given to the total weight of each organ. Thus, for example, the demonstration of CT mRNA in fat of septic individuals assumes marked pathophysiological significance when one considers the great bulk of adipose tissue (116).

The extraordinary tissue-wide expression and secretion explains the enormous elevation of CTpr in the sera of septic patients. The substantial suppression of CALC-I gene expression by nonneuroendocrine cells that occurs in normal persons is altered in septic patients by a unique stimulus arising from the infectious and/or cytokine insult that then influences the transcriptional regulation of the gene. Indeed, in sepsis the mRNA is more uniformly up-regulated than are the mRNAs of the classical sepsis-related cytokines, TNF{alpha}, IL-1ß, and IL-6. In a broader sense, in sepsis the entire body becomes a CTpr-producing endocrine gland. It is because this novel form of secretion is intrinsic to the host response to sepsis, and is reminiscent of the expression of the classical cytokines in this condition, that CTpr are referred to as hormokines (115).

The lack of a significant increase of CT(1–32) in sepsis merits further investigation. In part, this is indicative of a shift away from the normal, regulated, neuroendocrine pathway (characterized by a progressive posttranslational processing, maturation, and secretion via secretory granules) to a constitutive pathway (characterized by a nonstoring, bulk-flow secretion from nonneuroendocrine cells); this latter pathway is deficient in the enzymatic processing required to produce CT(1–32) (31, 117). Furthermore, once secreted, ProCT and NProCT are extremely resistant to enzymatic degradation; in contrast, CT(1–32) is extremely labile. Thus, circulating CT(1–32) may not be increased in sepsis because of its rapid degradation. Yet another factor may be the influence of heat shock proteins. These stabilizers of cellular function are synthesized in response to heat, other stressful stimuli, and sepsis. In this latter illness, they may serve a protective role (118). Heat shock proteins also are found in the blood as well as intracellularly (119, 120). They bind to CT(1–32) (121), and perhaps this further augments the disposal of CT(1–32) or interferes with its immunologic detection.

Other members of the CT-gene family of peptides in sepsis: CGRP and adrenomedullin. There are five genes in the CT-gene family of peptides. The gene that was initially discovered gives rise to two alternative splice variants: CT mRNA, resulting in ProCT and its components, and CGRP mRNA, giving rise to CGRP-I (122, 123). In the healthy, noninfected state, there is a preferential synthesis of either CT(1–32) mRNA or CGRP-I mRNA according to the cellular phenotype. Additionally, there are other structurally related peptides originating from this superfamily of genes: one of the genes yields a very slightly different form of CGRP (CGRP-II), another gene gives rise to amylin, and another gives rise to adrenomedullin. Furthermore, there is a gene that gives rise to a CT receptor-stimulating peptide with some homology to CGRP (31, 124, 125).

Similar to the case for CT-mRNA, in septic hamster tissues but not in similar healthy control tissues, there also is a tissue-wide expression of CGRP mRNA (126). Here, as well, CGRP mRNAs are more specifically up-regulated than are the mRNAs of classical cytokines (e.g. IL-6 and TNF{alpha}). A similar phenomenon occurs for adrenomedullin. In contrast, amylin mRNA has not been found to be up-regulated in nonneuroendocrine septic tissues. Thus, in sepsis several members of the CALC gene superfamily escape from their normal tissue-selective expression pattern. In studies in septic humans, serum levels of CGRP and adrenomedullin are increased in sepsis, although to levels considerably below those found for CTpr.

ProCT as a toxic factor in severe inflammation, systemic infection, and sepsis

Development of animal models of sepsis and correlation of serum CTpr with sepsis and mortality. In humans, the concentration of serum CTpr often reflects the severity of sepsis and may be predictive of mortality. Accordingly, to study this phenomenon more extensively, a model for sepsis was developed in hamsters and pigs (127, 128, 129, 130, 131).

Sepsis in the hamster

Severe peritonitis was induced in hamsters by the ip implantation of pellets containing measured quantities of Escherichia coli, and serum CTpr was determined at intervals. According to the dosage of bacteria, the 72-h mortality increased proportionately from zero to approximately 20, 70, and 100%. Serum CTpr also demonstrated a dose-related increase; at the highest dose of bacteria, serum CTpr levels exceeded control values by nearly 200-fold. Thus, CTpr levels correlated both with the severity of bacterial insult and the mortality. Gel filtration and HPLC studies revealed that most of the CTpr in these septic animals was in the form of ProCT (127).

Relationship between serum CTpr and cytokines. Using this animal model of sepsis, the relationship of CTpr to the proximal proinflammatory mediators, IL-lß and TNF{alpha}, was studied (128). Whereas serum CTpr remained extremely high throughout the 24 h of this study, the increases of these cytokines in the serum were less than 2-fold greater than the baseline and, importantly, were transient in duration. In healthy hamsters, the iv administration of human ProCT caused no evident adverse effects and no changes in serum IL-1ß and TNF{alpha} levels. In septic animals, the ProCT injections, albeit markedly increasing mortality (see below), only modestly blunted IL-1ß levels and did not affect TNF{alpha} values. Interestingly, however, when TNF{alpha} was injected into healthy animals, there was a 25-fold increase of CTpr levels, compared with noninjected controls. Thus, as is the case in the human, the magnitude and duration of the CTpr elevation demonstrated its utility as a marker of sepsis in the hamster. It also revealed that ProCT does not secondarily enhance levels of IL-1ß or TNF{alpha} in the systemic blood. In contrast, it showed that in healthy animals, TNF{alpha} can induce a sepsis-like elevation of serum CTpr.

ProCT as a toxic factor. Based on these clinical and animal studies, it was hypothesized that ProCT per se may be a toxic factor in sepsis and may adversely influence survival. The iv administration of human ProCT, which appeared not to be overtly injurious to normal hamsters, doubled the mortality of septic animals (129). In contrast, administration of human CT(1–32) to septic hamsters was without effect. Furthermore, a goat antiserum raised to this hormone, and shown to be completely cross-reactive with ProCT, was found to increase survival whether administered prophylactically or therapeutically.

Sepsis in the pig

To investigate in detail the physiologic and metabolic consequences of sepsis and evaluate how ProCT immunoneutralization might affect these parameters, a larger animal model of a rapidly fatal porcine polymicrobial peritonitis was then developed (130, 131). The subsequent sepsis is similar pathophysiologically to that encountered in human disease but far more lethal.

Early immunoneutralization. After having determined the structure of porcine ProCT, and, in rabbits, having produced an antiserum that was specific to the NProCT portion of this peptide, sepsis was induced in Yorkshire pigs by the ip instillation of a suspension of cecal content (l g/kg) plus E. coli (2 x 1011 cfu). Simultaneous to the induction of the peritonitis, experimental pigs received an iv infusion of the ProCT-reactive purified rabbit IgG, whereas control animals received non-ProCT-reactive IgG. All animals had physiologic data (e.g. urine output, core temperature, arterial pressure, heart rate, cardiac index, and stroke index), and metabolic data (e.g. blood urea nitrogen, serum creatinine, arterial lactate, and pH) collected or recorded hourly until death or being killed (15 h after ip instillation) (130).

Similarly to what occurs in humans, in this large animal model of lethal peritonitis, serum CTpr levels were found to be significantly elevated. Most of the untreated animals died within 9 h, and none survived for the 15-h duration of the experiment. However, immune IgG administration resulted in a significant improvement or a beneficial trend in most of the measured physiologic and metabolic derangements induced by sepsis. Moreover, most of these treated animals survived until the time of being killed, in contrast to animals treated with the non-ProCT-reactive IgG (P = 0.007).

Immunoneutralization of moribund pigs. In this septic pig model, the physiologic and metabolic parameters of the untreated animals worsened rapidly, so that the animals were essentially moribund by 4 h after the induction of the peritonitis. This state is comparable with the syndrome of multiple organ failure that occurs in humans with preterminal sepsis. Accordingly, to determine whether these gravely ill animals might be rescued, an evaluation of iv therapeutic immunoneutralization was undertaken during the fourth hour of the experiment (131). In contrast to the parameters for control septic animals, all of which progressively worsened and ultimately died, most physiologic and biochemical parameters of the treated animals improved or stabilized after infusion of the ProCT-reactive IgG (Fig. 3Go). Although all untreated animals had died during the experiment, most of the treated pigs survived until being killed (P = 0.010). Thus, in this model, the findings indicate that the immunoneutralization of ProCT was useful in a clinically relevant situation in which sepsis was fully established and far advanced.



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FIG. 3. Top, Time course in septic pigs showing the changes in mean arterial pressure (MAP). Time point 0 h represents commencement of the fourth hour after induction of sepsis and is the time of infusion of purified nonreactive rabbit antibody to the control group and purified antiporcine ProCT rabbit IgG to the treated group. After time point 0, there is a sharp fall in MAP in the control group, but the MAP of the treated group remains near normal levels. *, Statistically significant data points (P < 0.017; mean ± SEM). Bottom, Time course showing the changes in serum creatinine. The graph shows very similar creatinine concentrations before antibody infusion, which is followed by a sharp rise in the control group but with concentrations that remain near normal for the treated group. Other physiologic or metabolic parameters also showed benefits. *, Statistically significant data points (mean ± SEM, P < 0.037), which were noted at 2, 4, and 6 h, after which all control animals died (from Ref. 131 ).

 
Mechanism of toxicity of ProCT in sepsis: hypotheses and unanswered questions

Currently the physiologic actions of ProCT are relatively unexplored, and it is not known how this polypeptide or its components might worsen the septic process. Although acute studies have not been done in the human, the experience in noninfected patients with MTC indicates that chronically high levels of ProCT do not appear to cause any obvious ill effects. Nonetheless, although administration of ProCT to healthy, noninfected hamsters had no apparent ill effects, its administration to hamsters that were septic significantly increased mortality. This indicates that ProCT is not an initial toxic factor but requires the prior presence of a proinflammatory stimulus followed by an altered cytokine milieu.

Initial studies of actions of CTpr. In addition to the osteoclast, it is well known that receptors for CT(1–32) are present in different cell types, and multiple experimental studies of this peptide have been performed. However, in the past, the effects of the various CTpr have very rarely been evaluated. Insofar as ProCT is concerned, one study revealed the presence of receptors to this prohormone in newborn rat calvarial cells (132). Also, the human NProCT peptide portion of ProCT has been reported to be mitogenic to human osteoblastic cells (U-2 OS osteosarcoma) and to induce an increase in intracellular cAMP (133), although a subsequent study was not confirmatory (134). In a preliminary and unconfirmed report, a large-molecular-weight species of immunoreactive CT, corresponding to an undetermined portion of the ProCT molecule, appeared to suppress prostaglandin-E2-stimulated osteoclastic bone resorption (135). It is because of the recent awareness of the phenomenon of greatly increased serum levels of CTpr in severe inflammation and sepsis that studies of the action of CTpr are now underway by several groups.

The monocyte. Although the cell that has been most associated with the action of CT is the osteoclast, it is well known that this cell is produced from a precursor monocyte/macrophage cell line that also gives rise to the mature monocyte (136, 137, 138, 139). Moreover, monocytes influence osteoclast activity and also directly induce bone resorption (139, 140, 141). Importantly, the monocyte plays an essential role in phagocytosis, T lymphocyte immune activity, and inflammation; thus, it is greatly involved in the initiation and course of sepsis. The awareness that monocytes have CT receptors (142, 143) has led to several experiments investigating the effects of CT(1–32) and CTpr on these cells.

CT(1–32) and the monocyte

Insofar as CT(1–32) is concerned, after exposure of the lymphocyte/monocyte/macrophage family to this peptide, cAMP increases (144). This cAMP response is inhibited after exposure of these cells to various mechanical or hormonal stimuli (145). Also, analogous to the finding of down-regulation of the osteoclast CT(1–32) receptor that occurs when hypercalcemic patients receive CT(1–32) therapy (146), a similar response to an excess of this hormone occurs in vitro when the monocyte CT(1–32) receptor is studied (143). The early cAMP response to relatively small amounts of CT(1–32) has been shown to be blocked by other hormones [e.g. PTH (147), epinephrine (148)].

A principal function of the monocyte is its migration to sites of inflammation, a phenomenon that has been demonstrated to be induced by the addition of CT(1–32); in contrast, when patients receive therapeutic doses, this motion is diminished, strongly suggesting down-regulation of CT(1–32) receptors (149).

The immunologic relevance of the monocyte response to CT(1–32) remains to be fully elucidated. In vivo immunologic activities of this hormone have been demonstrated in several studies (150, 151), and salmon CT(1–32) has been reported to diminish the local inflammation after various forms of in-jury in rats (152). In humans with rheumatoid arthritis, eel CT(1–32) decreased production of IgG immunoglobulin and inhibited IL-1ß (153). However, as mentioned above, CT(1–32) is not appreciably elevated in patients with severe inflammation or sepsis, and because of the marked increase of serum ProCT and its component CTpr in such patients in the serum, more interest is being focused on the effects of these latter peptides on the monocyte.

CTpr and the monocyte

In vitro studies of isolated human monocytes have demonstrated that not only CT(1–32) but also ProCT and the free CCP-I peptide act as chemoattractants, inducing migration, which is dosage dependent. This phenomenon is accompanied by an intracellular elevation of cAMP levels. Furthermore, paradoxically, these peptides may deactivate the migratory effects of other unrelated chemoattractants and/or modify monocyte surface signals (154, 155). Monocytes and neutrophils are stimulated by LPS and another proinflammatory product of bacteria, formyl methionyl leucyl phenylalanine peptide, which induces these cells to produce an important integrin, CD11b, a substance that is involved in chemotaxis. Both NProCT and CGRP decrease this cellular CD11b production (156). In an initial study (157), human ProCT was added to mononuclear cells of peripheral blood harvested from normal humans, and cytokine secretion was measured in the ambient culture media. When compared with background cytokine synthesis by unstimulated cells, IL-1ß secretion was augmented 4-fold, TNF{alpha} 2-fold, and that of IL-8 2-fold. These preliminary findings suggest that ProCT might stimulate cytokine secretion from monocytes in local circulatory pools. As mentioned, TNF{alpha} is a known stimulus to ProCT secretion, and in sepsis this cytokine might locally induce a yet further local production of this prohormone in a positive-feedback manner. The clinical impact of all of these actions and complex interactions on the monocyte awaits clarification.

CTpr and nitric oxide (NO). The production of the vasodilator, NO, is elevated in sepsis (158), and this agent has been proposed as a mediator of the shock that may occur during the course of this illness (159). However, others have reported that NO may have a beneficial role. When ProCT is added to cultures of vascular smooth muscle cells of normal rats that had previously been exposed to LPS, TNF{alpha}, and interferon-{gamma}, the prohormone amplified the expression of the inducible NO synthase gene as well as NO production (160). The potentially detrimental effects of such an occurrence in the septic process requires further evaluation.

CTpr and hypocalcemia. Hypocalcemia, involving particularly but not exclusively the ionized component, is a frequent concomitant of critical illness and sepsis (161, 162). Studies in the septic rat indicate that this hypocalcemia is accompanied by an increase of intracellular calcium (163), and a similar intracellular increase occurs in the septic human (164). Interestingly, it is known that calcium infusions may be harmful to septic humans (165, 166), and when septic hypocalcemic rats are administered calcium, the fatalities increase markedly (167). In a large study of critically ill patients, dose-related correlations were noted among the severity of illness, the degree of hypocalcemia, and the level of serum CTpr (161). Also, in this condition, serum ionized calcium is known to correlate inversely with levels of TNF{alpha} and IL-6 (162). The cause of the hypocalcemia is unknown. CT(1–32) has been demonstrated to augment the intracellular calcium concentration in several different cell lines (61, 168, 169, 170). However, CT(1–32) is rarely appreciably elevated in sepsis. A direct effect of other CTpr on intracellular calcium has not been demonstrated. In a study in hamsters, administration of human ProCT did not cause hypocalcemia; indeed, this prohormone was found to completely block the hypocalcemia normally induced by injection of CT(1–32) (171). Thus, further studies are required to define any relationships between the CTpr peptides and serum calcium.

Influence of other peptides of the CT gene family. Lastly, as discussed above, both CGRP and adrenomedullin are increased in sepsis. These hormones have been reported to exert antiinflammatory effects (172, 173, 174, 175), and these actions could potentially be beneficial in severe infections or sepsis (e.g. antibacterial, increased dilatation of coronary arteries, positive cardiac inotropic and chronotropic effects). Furthermore, the biologic effects of the members of the CT gene family of peptides are exerted via the same family of receptors. The physiological profiles of these receptors are modified by certain accessory proteins (receptor-activity-modifying proteins), thus perhaps modulating the action of the CT gene products according to ambient circumstances (176, 177, 178, 179, 180). Conceivably, the much higher serum levels of CTpr may interfere with receptors or with receptor-activity-modifying protein expression. Such an occurrence may block CGRP and/or andrenomedullin activity and hence impede their otherwise beneficial effects. Nevertheless, both CGRP and adrenomedullin have vasodilatory actions, and whether such effects may be beneficial (e.g. by increasing the blood supply to vital organs) or harmful (e.g. by inducing systemic hypotension) requires additional clarification.

Clearly, it is essential to further investigate the means by which ProCT exerts its toxicity in sepsis. ProCT and its components may exert actions that differ according to their target tissues as well as having actions that differ according to the ambient milieu of the host. Multiple in vitro and in vivo investigations will be required.

Potential advantages of ProCT as a target for immunoneutralization in the human

The administration of endotoxin results in a form of systemic inflammation that is associated with cellular activation and the release of inflammatory mediators (Table 2Go); albeit being different from sepsis, it is an experimental model of the mediators and the control mechanisms that are relevant to this devastating state. In humans, it was shown that endotoxin caused a rise in serum CTpr that persisted for the 24 h of the experiment, although the duration of this elevation was unknown (179). Therefore, an investigation was undertaken to evaluate the duration of the CTpr elevation (180). Serum CTpr were observed to increase by 3 h and attained peak values at 24 h. Subsequently values very slowly and progressively declined. Surprisingly, at 7 d, all volunteers still exhibited levels that were above normal (Fig. 4Go). In two of the subjects who were studied for a longer period, the levels did not normalize until 10–14 d. In contrast, when subjects were administered identical doses of endotoxin and cytokines were measured for 24 h, serum levels of the proinflammatory cytokine TNF{alpha} increased at 1 h, reached a peak at 1.5 h, and had normalized by 24 h. Similar patterns of secretion occurred for the IL-1 receptor antagonist (IL-1ra), IL-6, and granulocyte colony stimulating factor and also several other cytokines, some of which were even more transient (181, 182). Thus, these short-lived acute phase cytokine elevations contrast with the extremely prolonged elevation of serum CTpr after a systemic inflammatory episode in healthy humans; this suggests that CTpr not only have advantages as excellent markers of sepsis but also may offer a durable target for therapeutic immunoneutralization, even several days after the severe inflammatory illness has commenced.



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FIG. 4. Exposure of human volunteers to one injection of endotoxin illustrates the differences in the release and subsequent decrease of several humoral markers of critical illness: TNF{alpha}, IL-1 receptor antagonist (IL-1ra), IL-6, C-reactive protein (CRP), and CTpr (from Ref. 183 ).

 

    Conclusions
 Top
 Introduction
 Conclusions
 References
 
CT(1–32) was the initial peptide of the CT gene-related family to have been isolated from MTC and normal thyroid tissue, and experimental studies demonstrated its hypocalcemic activity, secretory responsivity, and marked effects on the osteoclast. This peptide rapidly became the classic marker for MTC. The relatively recent development of specific assays for the CT(1–32) molecule has considerably improved its use as an MTC marker as well as ensuring the accuracy and reproducibility of physiopathologic investigations. Many clinical studies demonstrated the utility of the this peptide as a therapeutic agent for osteoporosis and Paget disease. Nevertheless, in contrast to the continuing role of the assay of serum CT(1–32) in patients with MTC, the use of salmon CT(1–32) as a therapeutic agent for these conditions has markedly diminished.

Soon after the discovery of CT(1–32), this hormone was determined to be synthesized as part of the considerably larger prohormone, ProCT. ProCT and its components (CTpr) were found to be secreted by MTC and cultured C cells and were noted to be markedly elevated in the blood of patients with this tumor. Sensitive immunoassays for CTpr in the serum have been developed, and all of these peptides were found to circulate at low levels in the serum of normal persons. Clinically, serum CTpr measurement has shown promise of being of utility as markers for patients with MTC and perhaps for SCLC.

In severe inflammation, systemic infections, sepsis, and sepsis-like conditions, serum levels of CTpr are markedly elevated. The levels of serum CTpr correlate positively with the severity of the illness and mortality, and their clinical utility as markers for these conditions has been facilitated by the development of commercial assays for their measurement. In septic states, CTpr are produced throughout the body. Experimentally, ProCT is toxic to septic animals, as demonstrated by an increased mortality after its administration and also by an amelioration of the metabolic and physiologic parameters of illness as well as an increased survival after its immunoneutralization. The normal physiologic actions of ProCT and its component CTpr, if any, are unknown. Furthermore, the mechanism(s) by which this toxicity occurs also is unknown, and these are subjects that clearly merit further investigation.

The beneficial effects of immunoneutralization of ProCT in two different species, the efficacy of treatment at a time when the animals are nearly moribund, and the persistence of hyperprocalcitonemia for extremely long periods of time suggest that this prohormone may possibly be a useful target for therapeutic immunoneutralization in the human.


    Footnotes
 
Abbreviations: CCP-I, 21-Amino acid CT carboxyterminus peptide I; CGRP, CT gene-related peptide; CT, calcitonin; CTpr, CT precursors; LPS, lipopolysaccharide; MTC, medullary thyroid cancer; NO, nitric oxide; NProCT, 57-amino acid sequence at the amino terminus of ProCT; PNE, pulmonary neuroendocrine (cell); ProCT, procalcitonin; SCLC, small cell cancer of the lung.

Received September 16, 2002.

Accepted December 24, 2003.


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