S.L. Lightman

Corticotrophin-releasing factor-like immunoreactivity and mRNA in human leukocytes

Unstimulated normal leukocytes were studied for their expression of corticotrophin-releasing factor (CRF)-like mRNA and peptide. In situ hybridization demonstrated CRF mRNA in T and B lymphocytes and in neutrophils. Northern blot analysis also detected the presence of a CRF mRNA species of approximately 1.7 Kb from lymphocytes and a peptide that reacted with anti-CRF antiserum was also detected by radioimmunoassay. This lymphocyte-derived peptide eluted earlier than standard CRF on high performance liquid chromatography. These findings suggest that regulatory mechanisms within the hypothalamus and the immune system may utilize closely related peptides.

Differential effects of β-endorphin fragments on human natural killing

The endogenous opiate peptide, β-endorphin, has two effects on human natural killing (NK). Preincubation of effector lymphocytes with between 10−7 and 10−11M β-endorphin increases NK. Preincubation with lower concentrations results in a reduction in NK. Endorphin peptides containing an unmodified N-terminal sequence, and which are known to bind only to opiate receptors, increase NK. Sequences reported to bind only to nonopiate receptors reduce NK.

Action of interleukin-2 and interleukin-4 on CRF mRNA in the hypothalamus and POMC mRNA in the anterior pituitary

We have used the technique of in situ hybridization histochemistry to determine corticotropin-releasing factor (CRF) mRNA in the hypothalamic paraventricular nucleus (PVN) and proopiomelanocortin (POMC) mRNA in the anterior pituitary of rats given a single ip injection of either interleukin (IL)-2 or IL-4. IL-2 increased POMC mRNA in the anterior pituitary in a dose-dependent manner. The effect was apparent both at 4 and 24 h after injection. No effect of IL-2 on CRF mRNA in the PVN was detected in these animals, suggesting that the increase in POMC mRNA was not driven by an increase in CRF. IL-4 was without effect at the hypothalamic level although this cytokine did result in a decrease in POMC mRNA in the anterior pituitary.

Substance P and substance K in the median eminence and paraventricular nucleus of the rat hypothalamus.

We have used specific radioimmunoassays coupled with reversed-phase high-performance liquid chromatography (HPLC) to measure and characterise substance P (SP) and substance K (SK) in subdivisions of the rat hypothalamus. SP and SK levels in the paraventricular nucleus (PVN) were 968 +/- 61 and 381 +/- 22 pg respectively; in the supraoptic nucleus (SON) were 210 +/- 21 and 79 +/- 8 pg; and in the median eminence/arcuate nucleus (ME) were 1044 +/- 66 and 451 +/- 20 pg. Reversed-phase HPLC revealed that immunoreactive (ir) SP was present solely in the non-oxidised form in all tissue extracts. The principal form of ir-SK in the PVN and SON coeluted with synthetic SK on HPLC, but some immunoreactivity eluted in a later position. This material represented less then 5% of the total ir-SK in extracts of the PVN and SON, but increased to 35-40% of the total in the ME. Gel chromatography and HPLC characterised this compound as being slightly smaller and more hydrophobic than SK. These results establish that ir-SK is present within the hypothalamus in varying amounts and molecular forms. The location of significant amounts of both SP and SK in the PVN and ME, the principal regions of CRF-41 synthesis and release, is compatible with a role for neurokinins in the modulation of CRF-41 and consequently ACTH release.

Interleukin-1β induces corticotropin-releasing factor-41 release from cultured hypothalamic cells through protein kinase C and cAMP-dependent protein kinase pathways

Abstract Interleukin-1β (IL-1β) induces a dose-dependent increase in the release of corticotropin-releasing factor-41 (CRF) from dispersed rat fetal hypothalamic cells in culture. This release of CRF could be inhibited by the protein kinase C inhibitor H-7, and by the protein kinase A inhibitor IP-20. This suggests that both protein kinase C and protein kinase A-dependent pathways are involved in the response of CRF to IL-1β. Dexamethasone also blocked the CRF response to IL-1β, indicating that activated glucocorticoid receptors can inhibit the response of CRF to IL-1β.

Effects of cyclosporine A on the hypothalamic-pituitary-adrenal axis and anterior pituitary interleukin-6 mRNA expression during chronic inflammatory stress in the rat.

In the rat, adjuvant arthritis (AA) is an inflammatory joint disease associated with chronic stimulation of the hypothalamic-pituitary-adrenal (HPA) axis. We have investigated the effects of the immunosuppressive agent cyclosporine A (CsA) on plasma levels of adrenocorticotropin (ACTH) and corticosterone (B), as well as on anterior pituitary proopiomelanocortin (POMC) and interleukin (IL)-6 mRNA accumulation in control and adjuvant-injected animals. In control animals, CsA reduced basal anterior pituitary POMC and IL-6 mRNA and decreased plasma levels of ACTH and B. Adjuvant-injected animals that were treated with CsA showed no clinical signs of AA. Moreover, CsA inhibited the arthritis-induced increases in pituitary POMC and IL-6 mRNA levels and in circulating ACTH and B. In vitro, CsA reduced the POMC mRNA content of cultured anterior pituitary cells and diminished the stimulatory effects of corticotropin-releasing hormone (CRH) on POMC mRNA expression and ACTH secretion from these cells. These data indicate that CsA has a direct action on the HPA axis and also reduces the activation of the HPA axis seen in chronic inflammatory arthritis.

Interleukin-6 and corticotrophin-releasing hormone mRNA are modulated during differentiation of human neuroblastoma cells

Two cell clones [BE(2)-C and BE(2)-M17] derived from the human neuroblastoma cell line SK-N-BE(2) express corticotrophin-releasing hormone as well as interleukin-6 mRNA. Both genes are overexpressed, although with a different time course, following exposure to 5 microM retinoic acid, in parallel to the induction of neuroblastic differentiation. On the contrary, we are unable to detect interleukin-1 beta mRNA in these cell lines. Both cytokines are known to increase hypothalamic CRH mRNA. The production of cytokines and neuropeptides by neuroblastoma cells indicate a complex dialogue between tumour cells and anti-tumour immunity.

Substance P and substance K in the rat hypothalamus following monosodium glutamate lesions of the arcuate nucleus.

Adult rats treated neonatally with monosodium glutamate (MSG) exhibit lesions in the arcuate nucleus of the hypothalamus. Following MSG lesioning, dopamine content in median eminence/arcuate nucleus (ME/AN) tissue extracts declined by 60-70%. Substance P (SP) content as determined by radioimmunoassay was significantly decreased in the paraventricular nucleus (PVN) (531 +/- 30 pg, mean +/- SEM) compared to controls (871 +/- 110 pg) but was unchanged in ME/AN extracts. Substance K (SK) content decreased to 257 +/- 20 pg in the PVN of lesioned animals compared to controls (367 +/- 31 pg) and the ME/AN content of SK was also significantly decreased (236 +/- 36 pg compared to control levels of 619 +/- 65 pg). The CRF-41 content of the PVN and ME/AN was unchanged by MSG lesioning, indicating that these areas are not affected by MSG. The partial depletion of SP and SK in the PVN following MSG treatment provides evidence that at least some of the neurokinin content of the PVN may originate in cell bodies of the arcuate nucleus. However, the lack of response of ME/AN SP to MSG treatment may suggest that the arcuate nucleus is not the major source of SP in the median eminence.

Corticotrophin-releasing factor and arginine vasopressin in the hypothalamo-hypophyseal portal blood of rats following high-dose glucocorticoid treatment and withdrawal

Hypothalamo-hypophyseal portal blood was obtained from rats treated with chronic, high-dose prednisolone in the drinking water and after subsequent withdrawal of the steroid for up to 7 days. Corticotrophin-releasing factor (CRF) immunoreactivity in portal blood was reduced by treatment with prednisolone but not completely abolished. There was a rapid recovery of CRF to control values within 3 days of withdrawal of prednisolone. There was no significant change in arginine vasopressin (AVP) in portal blood over the time-course of the experiment.

Neuropeptide Y immunoreactivity in the spleen and thymus of normal rats and following adjuvant-induced arthritis.

Immunoreactive neuropeptide Y (irNPY) was detected by radioimmunoassay within the rat thymus and spleen. Total spleen and thymus irNPY contents in control animals were 77 +/- 3 ng and 23 +/- 1 ng respectively (means +/- S.E.M., n = 10). Total tissue contents of irNPY 14 days following bilateral adrenalectomy or induction of inflammatory arthritis were not significantly altered compared to controls. Most spleen irNPY coeluted with synthetic NPY after reversed-phase high-performance liquid chromatography, but two peaks of irNPY were detected in thymic extracts. This suggests that NPY may be differentially expressed in tissues of the immune system.