Glucocorticoids and chronic inflammation.

Glucocorticoids are steroid hormones that once bound to their receptor interact with the DNA binding domain. Almost 1000-2000 genes are sensitive to their effects, including immune/inflammatory response genes. However, their role in pathophysiology and therapy is still debated. We performed a literature survey using the key words glucocorticoids, inflammation, autoimmune disease, rheumatology and adrenal glands in order to define important targets for this review on glucocorticoids. Considering endogenous/exogenous glucocorticoids in chronic inflammatory diseases brought up five major points for discussion: inadequately low production of endogenous cortisol relative to systemic inflammation (the disproportion principle); changes of the systemic and local cortisol-to-cortisone shuttle (reactivation and degradation of cortisol); inflammation-induced glucocorticoid resistance; highlights of present glucocorticoid therapy; and the role of circadian rhythms in action of cortisol. Much of this information becomes understandable in the context of neurohormonal energy regulation as recently summarized. The optimization of long-term low-dose glucocorticoid therapy in chronic inflammatory diseases arises from the understanding of the above mentioned aspects. Since glucocorticoid resistance is a consequence of inflammation, adequate anti-inflammatory therapy is mandatory.

Immunoreactive cortisone in droppings reflect stress levels, diet and growth rate of gull-billed tern chicks.

Blood levels of corticosterone have been traditionally analyzed to assess stress levels in birds; however, measuring steroid hormone metabolites in feces and droppings has gained much interest as a noninvasive technique successfully used for such purposed in vertebrates. Diet may affect these fecal metabolite levels (e.g., due to nutritional stress), however, this variable has not been taken into account in studies with chicks despite the great dietary flexibility of many avian species. In this study, we addressed for the first time this key issue and validated the technique in wild gull-billed tern chicks (Gelochelidon nilotica). Several enzyme immunoassays were used to determine the most appropriate test to measure the stress response. Subsequently, we performed an experiment in captivity to assess adrenocortical activity in gull-billed tern chicks fed with two diets: piscivorous vs. insectivorous. Finally, the relation between the chicks' growth rate and excreted immunoreactive glucocorticoid metabolites (EGMs) was also evaluated. We found the immunoreactive cortisone metabolites to be a good index of stress (as being an index of adrenocortical reactivity) in chicks of this species. Fish-fed chicks had higher levels of cortisone metabolites when comparing both concentration and total daily excreted metabolites. Within each treatment diet, cortisone metabolite levels and growth rates were negatively correlated. These findings suggest that the diet should be considered when using this technique for comparative purposes and highlight the trade-off between stress levels and chicks growth rates.

Comparison of salivary cortisol as measured by different immunoassays and tandem mass spectrometry.

Assessing the amount of bioavailable cortisol in saliva with immunoassays and thus sampling an endocrine marker of hypothalamus-pituitary-adrenal axis activity is of major interest in both research and clinical practice. However, absolute cortisol concentrations obtained with different immunoassays (IAs) are barely comparable precluding direct comparison between studies or individuals whenever cortisol analyses were not based on the same IA. The present technical report aims to solve this problem by evaluating the validity of, as well as agreement between the most commonly used immunoassays in psychoneuroendocrinological research (i.e., IBL, DRG, Salimetrics, DSL, and DELFIA) and a reference method (LC-MS/MS) in a sample of 195 saliva specimen covering the whole range of cortisol concentrations in adults. A structural equation modelling framework is applied to decompose systematic assay variance and estimate cortisol reference values, which are adjusted for measurement error and interference of salivary cortisone. Our findings reveal nonlinear relations between IAs and LC-MS/MS, which are discussed in terms of IA cross-reactivity with saliva matrix components. Finally guidelines for converting cortisol concentrations being obtained by these immunoassays into comparable reference values are proposed by providing conversion functions, a conversion table, and an online conversion tool.

Validation and application of a highly specific and sensitive ELISA for the estimation of cortisone in saliva, urine and in vitro cell-culture media by using a novel antibody.

It is generally acknowledged that local tissue concentrations of cortisol and cortisone are modulated by site-specific actions of 11ß-hydroxysteroid dehydrogenase (11ß-HSD) isoenzymes 1 and 2. Cortisone, the inactive metabolite of cortisol is produced by 11ßHSD type 2. To assess 11ß-HSD types 1 and 2 activities, the cortisol/cortisone ratio has to be accurately determined. Immunoassays to measure cortisone levels are not widely available and tend to lack specificity. The aim of this project was to develop a highly specific and sensitive ELISA method for the estimation of free cortisone levels in urine, saliva and in vitro media samples without chromatographic separation. Antibodies against cortisone were raised in rabbits using cortisone-3-CMO-KLH as immunogen. HRP-goat anti-rabbit IgG conjugate was used as enzyme tracer. Cross-reactivities of the untreated cortisone antiserum with major interfering steroids were minimal except for cortisol (3.15%). However, following an immune-affinity purification of the antibodies using CNBr-activated sepharose-cortisol-3-CMO-BSA, cross-reactivity of the purified cortisone antibody with cortisol was reduced to 0.27%. The minimum detection limit of cortisone ELISA was 28 pg/mL (77.7 pM). The validity of the cortisone ELISA was confirmed by the excellent correlation obtained before and after an HPLC fractionation step (Y=1.09X-0.21, R2=0.98). Intra-assay and inter-assay imprecision were 5.5-11.7% and 8.7-12.8% CV, respectively. Using this assay, salivary cortisone levels showed a circadian rhythm in men and women (11.2±7.3 nM at 08.00 h and 5.1±3.6 nM at 18.00 h), and the levels were reduced following liquorice ingestion. In media of adrenocortical H295 cell line incubations, basal cortisone levels were 4.24±0.22 nM that increased to 8.6±1.2 nM post forskolin treatment. Urinary free cortisone excretion levels in healthy subjects were 56.66±36.9 nmol/day. In human volunteers following ingestion of green coffee bean extract for 2 weeks, urinary free cortisol excretion reduced significantly from 66.67±22.3 to 42.66±17.5 nmol/day (p=0.02) and the cortisol/cortisone ratio from 2.04±1.33 to 1.49±1.13, p=0.05. In conclusion, a simple and highly specific and sensitive ELISA has been developed and applied to estimate cortisone levels in biological fluids and culture media.

Surface plasmon resonance immunosensor for cortisol and cortisone determination.

In this paper, we present a surface-plasmon-resonance-based immunosensor for the real-time detection of cortisol and cortisone levels in urine and saliva samples. The method proposed here is simple, rapid, economic, sensitive, robust, and reproducible thanks also to the special features of the polycarboxylate-hydrogel-based coatings used for the antibody immobilization. The sensor surface displays a high level of stability during repeated regeneration and affinity reaction cycles. The immunosensor shows high specificity for cortisol and cortisone; furthermore, no significant interferences from other steroids with a similar chemical structure have been observed. The suitability of the hydrogel coating for the prevention of nonspecific binding is also investigated. A good correlation is noticed between the results obtained by the proposed method and the reference liquid chromatography/tandem mass spectrometry method for the analysis of cortisol and cortisone in urine and saliva samples. Standard curves for the detection of cortisol and cortisone in saliva and urine are characterized by a detection limit less than 10 microg l(-1), sufficiently sensitive for both clinical and forensic use.

Alteration of the cortisol-cortisone shuttle in leprosy type 1 reactions in leprosy patients in Hyderabad, India.

Regulation of inflammation in leprosy may be influenced by local concentrations of active cortisol and inactive cortisone, whose concentrations are regulated by enzymes in the cortisol-cortisone shuttle. We investigated the cortisol-cortisone shuttle enzymes in the skin of leprosy patients with type 1 reactions (T1R), which are characterised by skin and nerve inflammation. Gene expression of the shuttle enzymes were quantified in skin biopsies from 15 leprosy patients with new T1R before and during prednisolone treatment and compared with levels in skin biopsies from 10 borderline leprosy patients without reactions. Gene expression of 11beta-hydroxysteroid dehydrogenase (11beta-HSD) type 2, which converts cortisol to cortisone, is down-regulated in skin from T1R lesions. However expression levels of 11beta-HSD type 1, which converts cortisone to cortisol, were similar in skin with and without reactions and did not change during anti-leprosy drug treatment. Prednisolone treatment of patients with reactions is associated with an upregulation of 11beta-HSD2 expression in skin. The down regulation of 11beta-HSD2 at the beginning of a reaction may be caused by pro-inflammatory cytokines in the leprosy reactional lesion and may be a local attempt to down-regulate inflammation. However in leprosy reactions this local response is insufficient and exogenous steroids are required to control inflammation.

Reduced capacity for the reactivation of glucocorticoids in rheumatoid arthritis synovial cells: possible role of the sympathetic nervous system?

OBJECTIVE: Cortisol, the biologically active glucocorticoid, is a major endogenous antiinflammatory factor in rheumatoid arthritis (RA). The aim of this study was to examine the local conversion of cortisol to biologically inactive cortisone and vice versa (the cortisol-cortisone shuttle) in RA and osteoarthritis (OA) patients. METHODS: Thin-layer chromatography and phosphorimaging were used to examine the cortisol-cortisone shuttle in mixed synovial cells. Double immunohistochemistry was used to assess the key enzymes 11beta-hydroxysteroid dehydrogenase 1 (11beta-HSD1) and 11beta-HSD2 and their possible cellular locations. RESULTS: Double immunohistochemistry demonstrated 11beta-HSD1/2+ macrophages in the sublining area. The ratio of 11beta-HSD2+ cells to 11beta-HSD1+ cells was significantly higher in RA than in OA patients. Cortisol was converted to inactive cortisone in mixed synovial cells from RA and OA patients, which was largely inhibited by carbenoxolone (11beta-HSD1 and 11beta-HSD2 inhibitor). Using metyrapone to inhibit the 11beta-HSD1 reducing reaction (cortisone --> cortisol), we demonstrated that the capacity for reactivation of cortisone to cortisol was significantly higher in OA than in RA patients. Although the capacity for the cortisone-cortisol shuttle was higher in synovial cells from less-inflamed OA tissue compared with inflamed RA tissue, it was obvious that synovial inflammation in RA, but not OA, was related positively to the reactivation of cortisone. This indicates that in RA, a cause other than typical inflammatory factors inhibits the reactivation of cortisone. Since isoproterenol and adenosine inhibited the cortisol-cortisone shuttle, the loss of sympathetic nerve fibers (loss of beta-adrenergic agonist and adenosine) may be the missing link that accounts for the increased cortisol-cortisone shuttle in RA. CONCLUSION: This study demonstrates a reduced capacity for local reactivation of cortisone in RA synovial cells. Since synthetic glucocorticoids also use this reactivation shuttle, the results also apply to therapeutic glucocorticoids. This defective reactivation of cortisone may be an important unrecognized pathophysiologic factor in RA.

Radioimmunoassay of cortisone in serum, urine, and saliva to assess the status of the cortisol-cortisone shuttle.

We have developed a new assay for cortisone (E) in serum, saliva, and urine involving Celite chromatography followed by RIA with 125I-labeled E and scintillation proximity assay. The chromatography step separates cortisol (F) from E, and in combination with their RIAs, permits assessment of the status of the F-E shuttle. We report the results of basal, postcorticotropin (ACTH), and postdexamethasone E and F concentrations and their circadian fluctuations in the serum, saliva, and urine of healthy volunteers. The serum and urine F/E ratios were increased in patients with ectopic ACTH secretion, whereas in adrenal adenoma and Cushing disease only the urinary ratio was increased. In chronic renal insufficiency this ratio was increased in serum (23.5 +/- 3.9) but diminished in saliva (0.38 +/- 0.11), and in apparent mineralocorticoid excess the ratios were high in serum (44.3 +/- 9.3) and urine (5.35 +/- 0.85) compared with those of healthy subjects (serum 9.8 +/- 3.5, urine 0.52 +/- 0.29, saliva 0.52 +/- 0.29).

Cortisol and immunity.

The relationship between adrenocortical function and immunity is a complex one. In addition to the well-known detrimental effects of large, pharmacologic dosages of glucocorticoids upon the immune process, there is impressive evidence that physiologic amounts of cortisol, the chief glucocorticoid normally produced by the human adrenal cortex, is necessary for the development and maintenance of normal immunity. This evidence is reviewed, and the importance of differentiating between physiologic and pharmacologic dosages and effects is discussed. The popular use of synthetic derivatives of cortisol, which differ greatly from the natural hormone in strength, and the dynamic nature of the normal adrenocortical response, which varies with the degree of stress being experienced, have contributed to the confusion. Further studies of the nature of the beneficial effect of cortisol, and possibly of other normal adrenocortical hormones, upon immunity in humans are needed, especially in view of recent evidence of a feedback relationship between the immune system and the hypothalamic-pituitary-adrenal axis, and with the increasing awareness not only that the immune process provides protection against infection, but also that its impairment seems to be involved in the development of autoimmune disorders, malignancies and the acquired immunodeficiency syndrome (AIDS).

Synthesis of 7 alpha- and beta-carboxymethyl derivatives of cortisol, corticosterone, deoxycorticosterone and cortisone. Immunogenic properties of cortisol, corticosterone and deoxycorticosterone derivatives.

7 alpha- and 7 beta-Carboxymethylderivatives of cortisol, corticosterone and deoxycorticosterone have been synthetized. After coupling to bovine serum albumin, they were used to elicit antibodies in rabbits. Highly specific antisera were obtained which may possibly be used for a direct radioimmunoassay of these steroids in human and rodent plasma. In the case of the derivatives of cortisol and corticosterone and stereoisomery of the coupling had an effect on the affinity and the specificity of the antisera. In all immunized rabbits the antisera obtained with the 7 alpha-derivative had a higher affinity and a narrower specificity than the antiserum obtained with the 7 beta-derivative.

Alveolar response to experimental Pneumocystis carinii pneumonia in the rat.

In order to characterize the alveolar response to Pneumocystis carinii pneumonia, light and electron miscropy were used to trace the development of experimental infections with P carinii in rats treated with cortisone acetate and a low-protein diet. The first changes were found by the eighth day of treatment and consisted of the selective attachment of Pneumocystis organisms, mostly trophozoites, to alveolar Type 1 pneumocytes; the host cells were undamaged, and no inflammatory response was seen. After approximately one month of treatment, the seemingly innocuous host-parasite interaction was succeeded by focal necrosis of the Type 1 pneumocytes adjacent to organisms; hyperplasia of nearby Type 2 pneumocytes also occurred, to replace the dead Type 1 pneumocytes. Even at this stage, inflammatory reaction was conspicuously absent except for occasional alveolar macrophages in the diseased alveoli; in addition, all cells of the alveolar-capillary membrane other than Type 1 pneumocytes appeared entirely normal. Not only does the present study clarify the nature of alveolar injury caused by Pneumocystis carinii, but it also provides an experimental animal model in which selective injury of the alveolar lining cells occurs.

Specificity of immunoassays. III. Use of two antisera of differing specificities to improve the specificity of steroid immunoassay.

The specificity of immunoassays can be improved by using a second antiserum to bind substances that cross-react. Both theory and practice show that the effectiveness of the procedure is dependent of the complex interplay of the concentrations of the two antibodies, the concentrations of the three antigens involved (the labelled tracer, the antigen whose concentration is to be measured and the substance that cross reacts) and the affinities of the antibodies for these antigens. Measured cross-reactions can frequently be reduced to zero in the most important concentration ranges thereby enabling one to perform assays upon unpurified materials which other wise would not be possible. Limitations of the method are discussed.

Compounds in the urinary pH 1 extract reacting with corticosteroid and tetrahydrocorticosteroid antisera. Separation of a nonpolar substance with affinity to a tetrahydroaldosterone antiserum.

In human urinary pH 1 extracts prepared for the aldosterone-18-glucuronide estimation, several other substances are present, crossreacting not only with aldosterone antisera, but also with various corticosteroid and tetrahydrocorticosteroid antisera. Aldosterone was measured before and after chromatographic purification. Further characterization of the non-aldosterone immunoreactive material was made by immunological analysis of paper chromatogram eluats. Pregnancy, and administration of ACTH, dexamethasone, and metopirone led to a change of excretion in the antigenic equivalents. A method for the separation of the antigenic material is described. For structural elucidation the gaschromatography-mass spectrometry (GC-MS) method was applied.

Experimental disseminated candidiasis. II. Administration of glucocorticosteroids, susceptibility to infection, and immunity.

A model of experimental disseminated candidiasis in inbred guinea pigs was used for study of the effects of both short-acting and long-acting glucocorticosteroid administration on the susceptibility to infection, the development of in vivo and in vitro parameters of cell-mediated immunity, and the expression of already established Candida-specific, cell-mediated immunity. Results revealed that long-acting glucocorticosteroids markedly potentiated infection, increasing mortality and suppressing already established cellular immune parameters. The development of cellular immunity to Candida was not impaired. Short-acting glucocorticosteroids, however, did not potentiate infection, and they affected neither the development nor the expression of immune parameters.