Human recombinant interleukin-1 receptor antagonist prevents adrenocorticotropin, but not interleukin-6 responses to bacterial endotoxin in rats.

The present study was designed to analyze the role of endogenous interleukin-1 (IL-1) in the ACTH, corticosterone (CORT), and IL-6 responses of rats to bacterial endotoxin. Recombinant rat IL-1 beta (rIL-1 beta) when given ip resulted in dose-dependent increases in plasma ACTH, CORT, and IL-6 concentrations. Plasma ACTH and CORT responses could be induced by low rIL-1 beta doses that did not elevate plasma IL-6 levels. The half-maximally effective dose of rIL-1 beta was less than 0.6 microgram/kg for the ACTH and CORT responses and higher than 2.5 micrograms/kg for the IL-6 response. Time-course studies indicated that plasma ACTH and CORT concentrations were already elevated 30 min after the injection of rIL-1 beta (2.5 micrograms/kg, ip), with peak values after 1-2 h, followed by a subsequent decline. In contrast, plasma IL-6 concentrations became elevated 2 h after the injection of rIL-1 beta. In another set of experiments, the administration of endotoxin resulted in a dose-dependent elevation of the plasma ACTH, CORT, and IL-6 concentrations. The dose-response characteristics for ACTH, CORT, and IL-6 were different. The half-maximally effective dose for the ACTH and CORT, and IL-6 responses were approximately 2.5 micrograms/kg and more than 10 micrograms/kg, respectively. Time courses of plasma ACTH, CORT, and IL-6 responses to endotoxin (2.5 micrograms/kg, ip) were similar, with peak values measured after 2 h. When given alone, the human IL-1 receptor antagonist (IL-1RA; 1 or 2.5 mg/kg, ip) did not affect resting plasma ACTH and CORT concentrations and reduced plasma IL-6 concentrations in one experiment. At a dose of 1 mg/kg, IL-1RA inhibited ACTH and IL-6 responses to rIL-1 beta (2.5 micrograms/kg, ip) by 75% and 90%, respectively. Administration of IL-1RA (2.5 mg/kg, ip) 30 min after endotoxin (2.5 micrograms/kg, ip) completely prevented the ACTH response and partially inhibited the CORT response, but did not affect the IL-6 response measured 2.5 h after endotoxin administration. We conclude that 1) IL-1 receptors are involved in the ACTH and IL-6 responses to rat IL-1 beta; 2) the ACTH response, but not the IL-6 response, to a low dose dose of endotoxin in rats requires IL-1 receptor activation by endogenous produced IL-1; and 3) circulating IL-6 is not a prime mediator involved in ACTH and CORT responses to low doses of rIL-1 beta and endotoxin.

Domains of rat interleukin 1 beta involved in type I receptor binding.

In the present study the inhibitory effects of a panel of 21 monoclonal antibodies (moabs) to rat interleukin 1 beta (rIL-1 beta) on the binding of 125I-labeled rIL-1 beta to murine type I IL-1 receptors on EL4 cells were investigated. Furthermore, the epitopes of these moabs were determined by the use of the pepscan technique, and these epitopes were visualized on a three-dimensional model of rIL-1 beta. Some moabs (SILK 3, 4, 5, 6, and 22) inhibited receptor binding of radioiodinated rIL-1 beta at concentrations that are similar to the dissociation constant values of antibody-rIL-1 beta binding. Another group of moabs (SILK 7, 11, 20, 21, and 23) also inhibited receptor binding but only at concentrations that are 10-150 times higher than their dissociation constants. A large group of moabs did not affect receptor binding in the concentration range tested, and two moabs enhanced the binding of rIL-1 beta to type I receptors. The result of pepscan analysis shows that the moabs bound to one or more of the amino acid sequences 35-49, 66-85, 78-97, 106-124, and 123-143 of mature rIL-1 beta. Modeling of rIL-1 beta shows that the binding domains of SILK 4, 5, 6, and 22 (sequence 123-143) is located at the closed end of the molecule, indicating that this part of rIL-1 beta harbors domains that are crucial for type I receptor binding. The binding domain of SILK 3 (sequence 66-85) is also located at this end of the molecule. In contrast, the binding domains of SILK 7, 11, 20, 21, and 23 (sequence 78-97) are located at the open end of the molecule, which is at the same face as the amino- and carboxy-terminals. The binding domain of SILK 16 (sequence 106-124) is positioned at the center of the molecule. It is concluded that the closed end of rIL-1 beta contains sequences that are crucial for its binding to type I receptors on murine EL4 cells. Because of the high concentrations of antibodies to residues 78-97 of rIL-1 beta that are needed to interfere with receptor binding, the importance of these domains in binding to type I receptors remains uncertain.

Activation of the hypothalamus-pituitary-adrenal axis by bacterial endotoxins: routes and intermediate signals.

Peripheral administration of endotoxin induces brain-mediated responses, including activation of the hypothalamus-pituitary-adrenal (HPA) axis and changes in thermoregulation. This paper reviews the mechanisms by which endotoxin affects these responses. The effects on thermoregulation are complex and include macrophage-dependent hyperthermic and hypothermic responses. Low doses of endotoxin, given IP, activate peripheral macrophages to produce interleukin (IL)-1 beta, which enters the circulation and acts as a hormonal signal. IL-1 may pass fenestrated endothelium in the median eminence to stimulate corticotropin-releasing hormone (CRH) secretion from the CRH nerve-terminals. In addition, IL-1 may activate brain endothelial cells to produce IL-1, IL-6, prostaglandins, etc., and secrete these substances into the brain. By paracrine actions, these substances may affect neurons (e.g., CRH neurons) or act on microglial cells, which show IL-1-induced IL-1 production and therefore amplify and prolong the intracerebral IL-1 signal. In contrast, high doses of endotoxin given i.v. may directly stimulate endothelial cells to produce IL-1, IL-6, and prostaglandin-E2 (PGE2) and thereby activate the HPA axis in a macrophage-independent manner.

Effects of monoclonal antibodies to specific epitopes of rat interleukin-1 beta (IL-1 beta) on IL-1 beta-induced ACTH, corticosterone and IL-6 responses in rats.

Recently, we developed a panel of monoclonal antibodies (MoAbs) to rat IL-1 beta and found that MoAbs binding to the aminoacid sequences 66-85 and 123-143 of mature rIL-1 beta inhibited the binding of rIL-1 beta to murine EL4 cells. Here we study whether MoAbs to these and other domains of IL-1 interfere with the biological effects of rIL-1 beta in adult male rats in vivo. Administration of rIL-1 beta (1 or 5 micrograms/kg i.v.) enhanced the plasma concentrations of ACTH, corticosterone (CORT) and of IL-6 in a time- (0.5-4 h) and dose-dependent manner. Because 2 h after 5 micrograms/kg i.v., all three parameters were consistently elevated, this dose and time interval was used for further studies. Prior to injection, rIL-1 beta was incubated alone or in the presence of a MoAb (10 mg/kg) for 30 min at 37 degrees C or at 4 degrees C. Plasma ACTH, CORT and IL-6 responses to these mixtures are compared to those obtained after preincubation of rIL-1 beta with a non-IL-1 binding MoAb (PEN7). SILK 3, a MoAb that binds to the 66-85 domain of rIL-1 beta, reduced the ACTH and IL-6 responses by 48 and 45% respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of conditioned media from murine granulosa cell lines on the growth of isolated bovine preantral follicles.

Isolated morphologically normal bovine preantral follicles (40 to 70 microm) were cultured for 8 d in collagen gel in control medium or in 1 of 3 conditioned media from the murine granulosa cell lines GRMO1L, GRMO2 and GE2. The percentages of follicles at Day 1 that remained nomal at Day 8 were similar for follicles cultured in the conditioned and control media (84 to 90%). A significantly higher percentage of follicles cultured in each of 3 conditioned media started to grow (89%; P < 0.05) and their increase in diameter was greater than that of follicles cultured in control medium (72%; P < 0.05). The mitotic activity of the granulosa cells from follicles cultured in conditioned media was increased (P < 0.05) indicated by a higher percentage of nuclei that incorporated BrdU compared with that of follicles cultured in control medium. Follicular viability was measured by the presence of nonspecific esterase activity, active mitochondria and dead cells in cultured follicles using the fluorescent probes calcein-AM, rhodamine 123 and ethidium homodimer-1 in combination with confocal laser scanning microscopy. The percentages of follicles with esterase activity and active mitochondria present in their granulosa were similar for follicles in all groups. Culturing in GRMO2 or GE2 tended to lower the number of granulosa with dead cells. The percentage of follicles with oocytes without esterase activity and active mitochondria was lower (P < 0.05) in follicles cultured in GRMO2 or GE2 compared with those cultured in control medium. Moreover, the percentages of dead oocytes tended to be higher in follicles cultured in GRMO1L and GE2 compared with oocytes of follicles incubated in control medium. Taken together, the conditioned media stimulated follicular growth and granulosa viability as well as enhance mitotic activity of the granulosa cells. However, they negatively affected oocyte viability.

ACTH response to a low dose but not a high dose of bacterial endotoxin in rats is completely mediated by corticotropin-releasing hormone.

In experimental animals and humans, bacterial endotoxin activates the hypothalamus-pituitary-adrenal (HPA) axis. The pathways by which endotoxin stimulates adrenocorticotropic hormone (ACTH) and corticosterone secretion are uncertain. In the present study we compared the role of hypothalamic corticotropin-releasing hormone (CRH) in the activation of the HPA axis by a low (2.5 micrograms/kg) and a high (2.5 mg/kg) dose of bacterial endotoxin. Two experimental models were applied using chronically cannulated male Wistar rats. In the first model, rats were subjected to lesions of the hypothalamus that interrupted dorsal, lateral and frontal input to the median eminence (anterolateral deafferentation) or to sham operation and rats were used 7 days later. Before and at hourly intervals after endotoxin (2.5 micrograms/kg i.p.), blood samples were taken for the determination of plasma ACTH and corticosterone concentrations. Deafferentation of the hypothalamus strongly attenuated the elevations in plasma ACTH and corticosterone concentrations by a low dose of endotoxin compared to the responses in sham-operated animals. The second model involved passive immunization to CRH using a monoclonal antibody to rat/human CRH (PFU83). PFU83 (90 nmol/rat) abolished the elevation of plasma ACTH concentrations and attenuated corticosterone responses to a low dose of endotoxin (2.5 micrograms/kg i.p.) compared to that in control IgG-treated rats. Since the corticosterone responses to endotoxin were less effectively inhibited by the antibody than the ACTH responses, we postulate that non-ACTH-dependent mechanisms may contribute to the corticosterone response to endotoxin.(ABSTRACT TRUNCATED AT 250 WORDS)

Circulating tumor necrosis factor-alpha does not mediate endotoxin-induced hypothermia in rats.

The present study was designed to investigate the role of macrophages and circulating tumor necrosis factor-alpha (TNF-alpha) in the endotoxin-induced hypothermic responses in rats. Intravenous as well as intraperitoneal administration of endotoxin to male Wistar rats (0.5 mg/kg) led to increased plasma TNF-alpha concentrations and a transient hypothermia, which reached its nadir after 90 min. The hypothermia and plasma TNF-alpha responses to endotoxin were abolished after elimination of peripheral macrophages. Seven days after the first challenge, tolerance of the hypothermic response was found if the same dose was administered intraperitoneally but not if it was administered intravenously. Tolerance of the TNF-alpha response was induced irrespective of the route of endotoxin administration. We hypothesize that, after intravenous administration of endotoxin, macrophage-dependent and -independent mechanisms are activated, whereas the hypothermic response to intraperitoneal endotoxin involves primarily macrophage-dependent mechanisms. These mechanisms may relate to the prime targets reached by endotoxin, such as macrophages and endothelial cells. Because the development of tolerance of the hypothermic response is dependent on the route of endotoxin administration, whereas that of the plasma TNF-alpha response is not, we conclude that circulating TNF-alpha is not the macrophage-derived cryogenic signal that triggers the hypothermic response.

Ultrastructure and viability of isolated bovine preantral follicles.

Techniques for the isolation of ovarian follicles and maturation of oocytes in vitro have enormous reproductive potential. Preservation of normal tissue function is vital. This study emphasizes the ultrastructure and viability of mechanically isolated bovine small (diameter 40-100 microm) preantral and large (140-450 microm) preantral/early antral follicles. Viability studies were performed for small preantral follicles. The presence of esterase activity, active mitochondria and dead cells served as parameters of oocyte and granulosa cell viability. After 1 day of culture, all follicles had a viable granulosa, displaying active mitochondria and/or esterase activity in all their cells, although a few (generally <5) dead granulosa cells were present in 17% of the follicles. Of the oocytes, 35 and 80% had esterase activity and active mitochondria respectively, whereas 8% appeared dead. The percentages of oocytes showing esterase activity and active mitochondria decreased during culture, whereas the percentage of follicles with dead oocytes or dead granulosa cells strongly increased. More than 90% of the isolated small follicles showed a poor ultrastructure, especially of their oocyte, which points to a negative selective isolation of poor follicles in the present study and/or an isolation procedure-induced damage of follicles. With respect to large preantral follicles, 42% of those distributed in the cortex and 64% of those isolated and cultured for 1 day had a poor ultrastructure. In contrast with the small ones, the percentage of ultrastructurally poor large preantral follicles had decreased to 27% after 5 days of culture, possibly due to better isolation and culture conditions. It is recommended to use ultrastructural and/or viability cell markers for in-vitro grown follicles to evaluate their quality, and particularly that of their oocytes.

Gradual inhibition of inducible nitric oxide synthase but not of interleukin-1 beta production in rat microglial cells of endotoxin-treated mixed glial cell cultures.

In cultures of purified microglial cells and astrocytes from newborn rats, the immunocytochemical localization of interleukin-1 beta (IL-1 beta) and inducible nitric oxide synthase (iNOS) using recently developed antibodies, as well as the release of IL-1 beta and nitric oxide (NO), was studied following exposure of the cells to endotoxin [lipopolysaccharide (LPS)]. In the absence of LPS, IL-1 beta- and iNOS-immunoreactive microglial cells and IL-1 beta or NO release were not observed, whereas in the presence of the endotoxin, the production of NO and IL-1 beta by microglial cells dramatically exceeded their synthesis and release by astrocytes. Interestingly, microglial cells cultured for 4-8 days in the presence of astrocytes appeared to lose their ability to produce iNOS, whereas the release of IL-1 beta remained unaltered. Moreover, endotoxin-stimulated microglial cells appeared to regain their ability to synthesize iNOS following their separation from astrocytes. These data show that microglia are primarily responsible for NO and IL-1 beta production in mixed glial cell cultures upon endotoxin stimulation. Moreover, in the presence of astrocytes the induction of iNOS, but not that of IL-1 beta in microglial cells is gradually inhibited.

Nonionic block polymer surfactants modulate the humoral immune response against Streptococcus pneumoniae-derived hexasaccharide-protein conjugates.

Nonionic block polymer surfactants (NBPs) were tested for the capacity to stimulate the antibody response against hexasaccharide (HS), derived from Streptococcus pneumoniae type 3 capsular polysaccharide (S3PS), which was conjugated to proteins. The immune response was evaluated in the (CBA/N x BALB/c)F1 progeny, in which female mice are phenotypically normal whereas male mice carry an X-chromosome-linked immunodeficiency. NBPs L101, L121, 1101, and 1501 were able to increase anti-HS immunoglobulin M (IgM) and IgG levels in both normal and X-chromosome-linked immunodeficient mice (with up to 74-fold stimulation of antibody titers). Distribution of S3PS-specific antibodies over the various IgG isotypes was restricted after immunization with either HS-bovine serum albumin or HS-keyhole limpet hemocyanin (HS-KLH). Addition of NBPs (in particular 1501) resulted in a more diverse immune response with either antigen as judged by isotype distribution. Isoelectric focusing of individual sera and subsequent detection of S3PS-binding antibodies in these sera by immunochemical staining revealed a restricted number of different spectrotypes in the course of the immune response. Upon immunization of mice with HS-KLH, spectra of secreted antibodies were slightly more complex and more densely stained than after immunization with HS-bovine serum albumin. Furthermore, NBPs 1101 and 1501 appeared to be able to stimulate the secretion of antibodies, which were secreted only in small amounts without the use of NBPs. Different explanations for increased spectrotype diversity after immunization with KLH as the carrier and after administration of NBPs as the adjuvant are discussed.