Enumeration of interleukin-1 alpha and beta producing cells by flow cytometry.

A technique for intracytoplasmic immunofluorescence staining to detect and quantify human interleukin-1 alpha (IL-1 alpha) and beta (IL-1 beta) in CD4, CD8, and CD14 positive lymphoid cells is described. Mononuclear cells stimulated in vitro with PHA to produce IL-1, were fixed and made permeable to antibodies by sequential exposure to paraformaldehyde and the detergent n-octyl-glucoside. Cytoplasmic and surface staining of both forms of IL-1 were demonstrated by indirect fluorescence using IL-1 beta and IL-1 alpha specific mouse monoclonal antibodies and quantified with flow cytometry.

The molecular and cellular basis of corticosteroid resistance.

Corticosteroids (CS) can modulate gene expression and are often used to treat a range of immunological and inflammatory diseases such as asthma, inflammatory bowel disease and rheumatoid arthritis. However, a proportion of patients fail to show an adequate response. On this basis patients have been subdivided into CS-sensitive (SS) and -resistant (SR) subgroups. The ability of CS to inhibit peripheral blood T cell proliferation in vitro has also been used similarly. In rheumatoid arthritis (RA), the in vitro-defined SS and SR subgroups correlate with the clinical responses to CS therapy. The mechanisms responsible for this observation are unknown but they appear to involve a number of known molecular events related to the described mechanisms of action of CS. These include alterations in the functional status of CS receptor-alpha, perturbations of the cytokine and hormonal milieu and intracellular signalling pathways. Peripheral blood mononuclear cells (MNCs) from SR significantly overexpress activated NF-kappaB. In vitro, CS fail to significantly inhibit concanavalin A (conA)-induced NF-kappaB activation in MNCs from SR RA patients. The alterations in the intracellular signalling pathways may explain in part our observations seen in SR RA subjects, CS fail to significantly inhibit conA-induced interleukin (IL)-2 and IL-4 secretion and lipopolysaccharide-induced IL-8 and IL-1beta secretion in vitro. CS therapy fails to reduce the circulating levels of IL-8 and IL-1beta in RA patients. In asthma, CS fail to induce L10 in SR asthma patients. Other molecular mechanisms such as enhanced AP-1 expression and alterations in the MAP kinase pathway are most likely to be involved too and we are currently investigating such possibilities. A full understanding of the molecular basis of SR will lead to the development of more rational therapeutic strategies.

Neuroendocrine, immunologic, and microvascular systems interactions in rheumatoid arthritis: physiopathogenetic and therapeutic perspectives.

OBJECTIVE: To review the "core" systems interactions in rheumatoid arthritis (RA): neuroendocrine, immunologic, and microvascular, and to interpret an integrated physiopathogenesis of the disease, beginning at a preclinical phase of risk factors to the later stages of manifest clinical inflammation. METHODS: Publications on stress reactions, serum hormonal levels, biological mediators of inflammation and vascular alterations in RA during its preclinical phase, course of active disease, including pregnancy, and hormonal therapy of active disease were retrieved. In addition, experimental reports on biological models of the disease were considered. Levels of adrenal and gonadal steroids (ie, glucocorticosteroids [GCS], dehydroepiandrosterone [DHEA], its sulfate [DHEAS], estradiol [E2], and testosterone [T]), as well as prolactin (PRL) and other hormones, biological mediators, vascular endothelial system (VES) interactions with hormones, and immunologic mediators of inflammation in RA, were reviewed and interpreted. RESULTS: Women with premenopausal onset of RA not previously treated with GCS had lower basal serum levels of adrenal androgens, that is, DHEA or DHEAS, both before and after onset of clinical disease, compared with controls. Risk factors, including hormonal, immunologic, and hereditary indicators, were found to be uniformly present many years before clinical onset in such younger women, as compared with a frequency of circa 15% in matched controls. Also, a history of heavy cigarette smoking significantly predicted the onset of RA in perimenopausal women, and in men, suggesting that vascular endothelial alterations predispose to the disease. In the same prospective study, 1 or more of 4 risk factors were present an average of 12 years before clinical onset of disease in 83% of male RA cases versus 26% in matched controls (ie, sensitivity of 83% and specificity of 74%). Early RA patients may have lower serum cortisol levels than normal controls, and less than expected for the degree of ongoing inflammation, as well as having upregulated PRL levels. CONCLUSION: Among persons genetically prone to RA, the "core" systems are hypothesized to become "remodeled" during a long preclinical phase as a result of chronic imbalances in their interactive homeostasis. This hypothesis needs to be critically assessed in further studies of such physiological precursors of disease as well as stressors in the development and course of RA. Optimal hormonal management of biological mediators of RA is also a priority challenge for disease control in the future. RELEVANCE: Evidence indicates that men and women who are susceptible to premenopausal onset of RA can each be identified long before their clinical onsets of disease, and that productive research in primary prevention is an achievable objective. Disease prevention objectives are central in the public health strategy of the National Arthritis Action Plan and of the US Public Health Service "Healthy People 2000" (and 2010 proposed). Success in such prevention goals can be expected to significantly reduce the enormous burden of this common disease, which affects all segments of the population.

Reciprocal interactions between the neuroendocrine and immune systems during inflammation.

The neuroendocrine and immune responses to inflammatory stress represent important integrated physiologic circuits for the regulation of inflammation whose basis has been reviewed. Proinflammatory cytokines such as IL-1 beta, TNF alpha, and IL-6 released from inflammatory foci initiate a local inflammatory response and travel by way of the blood-stream to the central nervous system, where they trigger a variety of neuroendocrine counterregulatory mechanisms. There is an important NEI loop. Stimulatory signals are received by the neural systems from inflammatory foci and are transduced by the hypothalamus, thereby initiating a complex hormonal and cytokine cascade of reactions aimed at modulating inflammation and returning the organism to normal physiologic homeostasis once the trigger has been neutralized. Conversely, a number of mechanisms that modulate the anti-inflammatory activity of the neuroendocrine responses to inflammation are also activated. Defects in the neuroendocrine-immune interactions can profoundly affect the susceptibility to developing chronic inflammatory disease and influencing survival after bacterial infections. The NEI loop has important pathophysiologic implications for disease processes.

The influence of the hormonal system on pediatric rheumatic diseases.

The role of NEI interactions in children with chronic inflammatory rheumatic disorders has not been systematically studied to the same extent as in adults. The data that are currently available suggest that NEI mechanisms are involved in the pathophysiology of some of the diseases. These include JRA, JSLE, and JAS. Prolactin has been most extensively investigated, showing interesting parallels with findings in adult rheumatologic diseases. Limited data on cortisol suggest a deficiency of production in JRA, a situation similar to that in adult RA. These findings suggest that there is a proinflammatory hormonal bias in children with JRA and JSLE. The data in children with chronic autoimmune inflammatory disorders seem to be identical to those seen in adults with RA and SLE, but a clear delineation of the role of the neuroendocrine-immune system in disease pathophysiology is still required. The neuroendocrine aspects of pediatric rheumatologic disease observed to date suggest a number of avenues for further research in the field of neuroendocrine immunology, which may open up novel therapeutic options.

Neuroendocrine immune features of pediatric inflammatory rheumatic diseases.

Juvenile rheumatoid arthritis (JRA) and juvenile systemic lupus erythematosus (JSLE) are the most common autoimmune rheumatic diseases in children associated with high levels of autoantibodies and immune reactivity. JRA and JSLE are more common in girls. Disease activity is worse in the morning, improves during the daytime and worsens at night suggesting that neuroendocrine immune mechanisms are involved in disease pathophysiology. Adult patients with RA and SLE have excessive levels of prolactin (PL) while cortisol (CS) production is down-regulated for the degree of ongoing inflammation. PL has potent proinflammatory properties. Normal to low levels of cortisol have been observed in children with active JRA despite the high serum levels of IL-6, IL-1 beta, and TNF-alpha, which activate the hypothalamic-pituitary-adrenal axis (HPA). The CS levels are in fact subnormal because inflammatory stress activates the HPA. Normal serum PL levels were seen in children with JRA, most of whom were not active with higher levels in those with active ANA +ve JRA complicated by uveitis. A trend toward high PL levels was seen in 33 children with JSLE. High serum PL levels are seen in patients with active juvenile ankylosing spondylitis (JAS) only. Growth retardation is a feature of JRA. Patients with JRA have low to normal levels of growth hormone (GH) and low levels of insulin-like growth factor 1 (IGF-1). IGF-1 mediates the effects of GH. The observation of low IGF-1 in JRA raises the therapeutic possibility with IGF-1. Overall, high levels of follicle stimulating hormone and luteinizing hormone are found in children with JSLE while the levels in JRA tend to be normal. Testosterone levels are low in patients with JRA. No significant differences in estrogen levels have been found between patients with JRA and those with JSLE and matched controls. There is evidence that the autonomic nervous function is defective in patients with JRA.

Prolactin and neuroimmunomodulation: in vitro and in vivo observations.

Prolactin (PL) is a mammotropic neuropeptide produced by the pituitary and extrapituitary cells existing as several isoforms and belongs to the growth and lactogenic hormone family, which includes growth hormone and placental lactogens. The secretion of pituitary PL is under hypothalamic control. The cytokines IL-1, IL-2, and IL-6 also stimulate production, while IFN-gamma and endothelin-3 are inhibitory. PL exerts its effects via PL receptors (PLr) which exist as three isoforms. PL regulates reproduction, osmoregulation, and behavior and has potent immunomodulatory effects. PL is structurally related to members of the cytokine/hematopoietic family such as erythropoietin, GM-CSF, growth hormone, and IL-2 to IL-7. The PLr is a member of the cytokine/hematopoietic receptor family. Interaction of PL with PLr activates the Jak kinases which phosphorylate latent STAT proteins resulting in the activation of transcription. PL counteracts the effects of corticosteroids by enhancing Th1 cellular responses. Perturbations of PL physiology have significant immunologic effects. Hypoprolactinemia impairs immune function while hyperprolactinemia enhances active systemic lupus erythematosus, Reiter's disease, juvenile and adult rheumatoid arthritis (RA), autoimmune thyroiditis, multiple sclerosis, and cardiac allograft rejection. PL gene polymorphisms have now been shown to be linked to RA. Thus, manipulation of PL may have significant clinical utility. Further study of the relationship of the PL/PLr complex, other hormones, and the immune system will provide further insights into the potential therapeutic utility of this complex in immune diseases.

Perturbations of arginine vasopressin secretion during inflammatory stress. Pathophysiologic implications.

Pro-inflammatory cytokines, such as interleukin-1 beta (IL-1 beta), interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNF alpha), released from inflammatory foci, can activate the hypothalamus to produce corticotrophin-releasing hormone (CRH) and arginine vasopressin (AVP). These hypothalamic peptides in synergy increase ACTH production by the pituitary gland and hence corticosteroid (CS) secretion by the adrenal cortices. CS dampens inflammation. The pituitary also produces prolactin (PRL), which is pro-inflammatory, and macrophage inhibitory factor (MIF), which by counteracting the anti-inflammatory and immunosuppressive effects of CS, is pro-inflammatory. Lewis rats develop a variety of induced-autoimmune inflammatory conditions, such as streptococcal cell wall arthritis, whereas the histocompatible F344 Fisher rats are resistant to this condition. Lewis rats have a defective hypothalamic-pituitary adrenal (HPA) response to a variety of hypothalamic stimuli, but have augmented systemic secretion of AVP. Patients with rheumatoid arthritis (RA) have deficient CS with exaggerated PRL responses to inflammatory stimuli. Within inflammatory foci, CRH is pro-inflammatory. AVP, which augments autologous mixed lymphocyte reactions, can replace the IL-2 requirement for gamma IFN production by T cells via V1a receptors, and potentiates primary antibody responses, is also pro-inflammatory. Lewis rats have significantly high plasma levels, hypothalamic content, and in vitro release of AVP in comparison to the inflammatory disease-resistant Fischer rats. Immunoneutralization of AVP attenuates inflammatory responses. In Sprague-Dawley rats, AVP potentiates PRL secretion. Preliminary studies in patients with RA have shown that the circulating levels of AVP are significantly increased, which might be a compensatory response to low CS levels or a result of elevated levels of IL-6 in these patients but could nevertheless contribute to rheumatoid inflammation. A similar observation has been made in patients with ankylosing spondylitis.

Targeting tumour necrosis factor in the treatment of rheumatoid arthritis.

Inflammation in rheumatoid arthritis (RA) is associated with an imbalance between pro- and anti-inflammatory factors, which leads to a persistent chronic inflammatory state in the joint. Molecular studies of the physiology of the inflammatory response have identified a hierarchy of cytokine activities. The identification of this hierarchy has provided new potential therapeutic targets for the treatment of RA. At present the majority of new therapeutic agents have been developed to neutralise the activity of tumour necrosis factor-alpha (TNF alpha), a cytokine at the top of the inflammatory cascade. These agents consist of recombinant proteins that bind and neutralise TNF alpha, and they are effective in the treatment of inflammation in RA. In this review we discuss the rationale behind targeting TNF alpha, the various recombinant proteins that have been used, their clinical effectiveness, the possible adverse effects of these agents and the development of new chemical inhibitors of TNF alpha synthesis.

Why do we need new treatments for rheumatoid arthritis?

Rheumatoid arthritis is a chronic systemic autoimmune inflammatory disease characterized by progressive joint damage. The classical treatments of the disease such as myocrisin and sulphasalazine, are not always effective at controlling the disease. This has necessitated the development of novel agents for treating rheumatoid arthritis. Most of these drugs are biological in nature and are targeted at specific sites of the inflammatory cascade of reactions. A number of clinical trials have been conducted. The clinical effects that have been observed are transient, necessitating repeated treatments and the risk of vaccination effects. Many of these agents have to be administered parenterally, production costs are very high. Consequently, chemical entities which can be taken orally need to be developed. Since the immune system is very complex with pleiotropic cytokines and redundancy in some of the regulatory networks, it may therefore be necessary to use multiple agents targeted at different specific sites of the inflammatory cascade or that different agents could be given at different stages of the disease, to induce disease remission and maintain the response to therapy. Cytokines such as tumour necrosis factor (TNF) and interleukin 1 (IL-1) play important physiological roles in the host's defence systems against infections and malignancy. The chronic inhibition of these cytokines by targeted therapies may therefore lead to the development of side effects. Thus, carefully controlled long-term studies will be required to assess the safety of selective targeting of processes involved in inflammation. A more recent novel approach is to target hypoxic tissues with bioreductive agents. Thus, some of the established rheumatoid arthritis treatments could be linked to bioreductive agents and released in hypoxic tissues where inflammation is occurring. This review summarizes the important developments in the therapy of rheumatoid arthritis. There is no doubt that despite these developments we need to develop new and advanced treatment modalities for rheumatoid arthritis.

Chlorpromazine and frusemide in the acute management of severe hypertension.

Fifty two patients with severe hypertension were treated with parentral chlorpromazine and frusemide. There was a gradual reduction of blood pressure without reflex tachycardia in 92.4% of the cases. No serious side effects were observed. This therapeutic manoeuvre is simple, effective and a safe alternative for the management of hypertensive emergencies, especially when continuous monitoring cannot be guaranteed. this drug regimen must not be administered concurrently with methyldopa as this exerts an additive effect leading to severe hypertension. These results compare very well with those obtained with the use of repeated small bolus injections or graded infusions of labetolol.

The differential expression of corticosteroid receptor isoforms in corticosteroid-resistant and -sensitive patients with rheumatoid arthritis.

OBJECTIVE: A proportion of patients with rheumatoid arthritis (RA) fail to respond adequately to corticosteroid (CS) therapy. Using an in vitro CS sensitivity bioassay, we have subdivided RA patients into steroid-sensitive (SS) and -resistant (SR) subgroups and this correlates with clinical responses to CS therapy. CSs exert their effects via the CS receptor (CR), which exists as two main isoforms, CRalpha and CRbeta. CRbeta can function as a negative inhibitor of CRalpha. We have hypothesized that steroid resistance in RA patients is due in part to a relative over-expression of the CRbeta. METHODS: Peripheral blood mononuclear cells (PBMCs) were isolated from SS and SR RA patients. CRalpha and CRbeta mRNA expression was determined by quantitative real time polymerase chain reaction (qRT-PCR). The ratio of CRbeta/CRalpha mRNA expression was determined. CRalpha and CRbeta protein expression by PBMCs was analysed by flow cytometry. RESULTS: qRT-PCR analysis showed a trend towards higher expression of both CRbeta and basal CRbeta/CRalpha ratio in SR RA patients. Stimulation of PBMCs in vitro with concanavalin-A induced a significantly higher CRbeta mRNA expression, and CRbeta/CRalpha ratio in SR RA patients compared with SS patients, which was not inhibited by hydrocortisone. Flow cytometry showed that the percentage of PBMCs staining for CRbeta protein was significantly lower in the SS RA group (SS 43.3 +/- 14.8% vs SR 88.6 +/- 8.6%; P < 0.0010). The mean intensity of fluorescence CRbeta staining was higher in the SR RA patients (P < 0.001). CONCLUSION: We show for the first time that CRbeta is over-expressed in SR RA patients and that hydrocortisone fails to inhibit concanavalin-A stimulated increase in CRbeta mRNA in SR RA patients. This mechanism may contribute in part to the CS hyporesponsiveness seen in some RA patients.

The neuroendocrine immunology of rheumatoid arthritis .

Rheumatoid arthritis patients have defective neuroendocrine-immune responses to the stress of inflammation, and currently available data shows that this contributes to the pathophysiology of the disease. The advances in neuroendocrine immunology have improved our understanding of the pathophysiological mechanisms involved in RA. These observations raise important therapeutic questions which are certainly worth further investigation as they may open up novel avenues for the management of the disease.

Neuroendocrine immune responses to inflammation: the concept of the neuroendocrine immune loop.

The neuroendocrine and immune responses to inflammatory stress represents an integrated circuit whose basis is reviewed in this chapter. Pro-inflammatory cytokines such as IL-1 beta, TNF-alpha and IL-6 released from inflammatory foci initiate local anti-inflammatory mechanisms and travel via the blood stream to the brain where they trigger a variety of neuroendocrine counter-regulatory mechanisms. There is therefore an important neuroendocrine-immune loop in which stimulatory signals are received by the neural systems from inflammatory foci. These signals are transduced by the hypothalamus which initiates a complex hormonal cascade reaction aimed at modulating inflammation and returning the organism to normal physiological homeostasis once the trigger has been neutralized. Abnormalities in this cross-talk can profoundly influence the susceptibility to developing chronic inflammatory disease. Thus, in conclusion, the neuroendocrine-immune loop has important pathophysiological implications for disease processes.