Rat Interleukin Research Articles

Comparison of anti-rheumatic effects of local RNAi-based therapy in collagen induced arthritis rats using various cytokine genes as molecular targets

RNA interference (RNAi) provides a powerful means of sequence-specific gene silencing. Several studies show that RNAi may provide promising strategies to treat human diseases by suppressing disease responsible genes in vivo. In locomotor diseases, the progression of collagen-induced arthritis (CIA) is suppressed by tumor necrosis factor-α (TNF-α)-specific small interfering RNA (siRNA) delivered into the joint. The aim of this study, is to compare the effects of intraarticularly administered siRNAs targeting TNF-α, interleukin-1β (IL-1β), interleukin-6 (IL-6) and receptor activator of NF-κB ligand (RANKL) on CIA in rats. We confirmed that the silencing effects of siRNA duplexes specific for rat TNF-α, IL-1β, IL-6 and RANKL in vitro. Each siRNA was also delivered into the knee joint of CIA rats by the in vivo electroporation method 7, 10, 13 and 16 days after immunization with collagen. Local delivery of TNF-α or IL-1β-specific siRNA ameliorated CIA in rats effectively at the gross morphological, radiographical and histological evaluations. Our results suggested that TNF-α and IL-1β were the cytokines to be targeted in the joint for the treatment of rheumatoid arthritis. The in vivo siRNA transfection method may be useful for selection of target molecules to be silenced for treatment of joint diseases.

Interleukins and Ischemic Stroke: Effect of Neutralization of Rat IL-6 Bioactivity on the Degree of Ischemic Cerebral Damage Caused by Experimental Middle Cerebral Artery Occlusion in the Rat

The purpose was to investigate the effect of neutralization of rat interleukin-6 (IL-6) bioactivity on the activity levels of Na+-K+/Mg++ adenosine-5′-triphosphatase and superoxide dismutase, and the degree of infarct size in ischemic brain produced by experimental middle cerebral artery occlusion in the rat. Seventy adult male Wistar rats weighing 280 to 310 g were used in this study. The animals were randomly allocated to groups: group I (n=10) consisted of normal controls, group II (sham-operated, n=20) underwent surgical exposure of the middle cerebral artery but no cauterization, and the remaining 40 rats were subjected to middle cerebral artery occlusion. Immediately after occlusion, each of these 40 rats was randomly assigned to either the occlusion-only group (group III, n=20) or the occlusion plus IL-6 antibody treatment group (group IV, n=20). All evaluations were performed in blinded fashion. Our data provide evidence that neutralization of IL-6 bioactivity correlates with less secondary damage in long-term treatment after focal cerebral ischemia in the rat.

Comparison of anti-rheumatic effects of local RNAi-based therapy in collagen induced arthritis rats using various cytokine genes as molecular targets

RNA interference (RNAi) provides a powerful means of sequence-specific gene silencing. Several studies show that RNAi may provide promising strategies to treat human diseases by suppressing disease responsible genes in vivo. In locomotor diseases, the progression of collagen-induced arthritis (CIA) is suppressed by tumor necrosis factor-alpha (TNF-alpha)-specific small interfering RNA (siRNA) delivered into the joint. The aim of this study, is to compare the effects of intraarticularly administered siRNAs targeting TNF-alpha, interleukin-1beta (IL-1beta), interleukin-6 (IL-6) and receptor activator of NF-kappaB ligand (RANKL) on CIA in rats. We confirmed that the silencing effects of siRNA duplexes specific for rat TNF-alpha, IL-1beta, IL-6 and RANKL in vitro. Each siRNA was also delivered into the knee joint of CIA rats by the in vivo electroporation method 7, 10, 13 and 16 days after immunization with collagen. Local delivery of TNF-alpha or IL-1beta-specific siRNA ameliorated CIA in rats effectively at the gross morphological, radiographical and histological evaluations. Our results suggested that TNF-alpha and IL-1beta were the cytokines to be targeted in the joint for the treatment of rheumatoid arthritis. The in vivo siRNA transfection method may be useful for selection of target molecules to be silenced for treatment of joint diseases.

Interleukin-1 receptor antagonist reduces the magnitude of the pressor response to acute stress

The purpose of the present study was to establish the effect of chronic central interleukin-1 receptors blockade and central chronic infusion of interleukin-1 beta (IL-1β) on cardiovascular response to an acute stressor. The experiments were performed on 12–14-week-old, male WKY rats, divided into three experimental groups. Each group was subjected to chronic intracerebroventricular (ICV) infusion of one of the following compounds: saline (control, group C), recombinant rat IL-1 receptor antagonist (IL-ANT group) or interleukin-1 beta (IL-1B group). After 5 days of the ICV infusions mean arterial blood pressure (MABP) and heart rate (HR) were recorded continuously under baseline conditions and after the application of an air jet stressor. The stressor was applied three times with 10-min intervals. There were no significant differences in MABP and HR between groups under baseline conditions and immediately before the application of the three consecutive air jets. After the first stressor the IL-ANT group responded with a significantly lower increase in blood pressure than the control and IL-1B group. After the application of the two following air jets only the trend for an intergroup difference was present. The results of the present study provide further evidence that cytokines play an important role in the regulation of the circulatory system. The most important new finding is that the magnitude of the pressor response to the alarming stress is strongly influenced by IL-1 receptors in the brain.

Interleukin‐1 induced increases in blood pressure are mediated by cerebral‐vasculature‐associated perivascular cells

Interleukin‐1 (IL‐1) induces a sympathetically mediated increase in mean arterial pressure (MAP) and heart rate (HR) in awake rats. The response can be attenuated with peripheral injections of non‐specific prostaglandin (PG) synthesis inhibitors, but a role for central PGs has not been established. ED‐2 positive perivascular cells (pvcs) are the sole source of Cox‐2 within the brain in response to low doses of IL‐1 and their loss has been shown to attenuate IL‐1 induced HPA activation suggesting central PG synthesis by pvcs mediates the response. This study tested the hypothesis that the increase in MAP and HR induced by IL‐1 was dependent on brain production of PG's by pvcs. Male Sprague Dawley rats were chronically instrumented with an iv jugular catheter, and an abdominal transmitter (Data Systems International) for recording MAP and HR. One week later, they received intraventricular injections of clodronate‐containing (clod‐lip) or saline and DiI‐containing liposomes (sal‐lip) to selectively deplete the brain of pvcs, or label them respectively. Clod‐lip injections produced a > 90% depletion of brain pvcs. Baseline MAP and HR values were not significantly different between groups (sal‐lip, 105 ± 4 mmHg, 413 ± 13 bpm vs. clod‐lip, 107 ± 4 mmHg, 408 ± 10 bpm). Recombinant rat IL‐1 (2 μg/kg, iv) increased MAP and HR by 10 ± 4 mmHg and 38 ± 24 bpm respectively at 10 minutes after injection and remained elevated for approximately 20 minutes. Liposome‐depletion of ED2‐positive pvcs significantly reduced the MAP response to −3 ± 6 mmHg and HR to 5 ± 11 bpm. These results suggest that the cardiovascular changes induced by IL‐1 are mediated by pvcs and implicate a novel signaling mechanism at the blood brain barrier used by cytokines to regulate the sympathetic nervous system.

Intraperitoneal recombinant interleukin-2 activates leukocytes in rat endometriosis

The aim of this double-blinded study was to determine changes in leukocyte populations in blood, peritoneal lavage fluid, eutopic and ectopic endometrium after treatment with recombinant rat interleukin-2 (IL-2) using an in vivo experimental model of rat endometriosis. The in vivo model involved transplanting four square fragments of autologous endometrium onto the inner surface of the abdominal wall in 20 Wistar rats. The control group was constituted by 20 sham-operated rats. Both groups were randomly treated (1-month interval treatment) with 2 intraperitoneal doses of glucose solution (5%) that did or did not contain recombinant IL-2, and animals were sacrificed 4 weeks after the last dose of treatment. Blood and peritoneal lavage were obtained during the initial and final laparotomy, whereas eutopic and ectopic endometrium were collected at the end of the experiment. Endometriotic implants were measured in each laparotomy to determine any change in size. Leukocyte populations were analyzed by flow cytometry and immunofluorescence microscopy. Cytometric results were similar in blood and peritoneal lavage. CD25+ and natural killer (NK) cell levels in peripheral blood were lower in rats with endometriosis treated with IL-2, whereas NK cells increased in lavage compared to placebo group. The percentage of macrophages and dendritic cells in blood were higher in all rats treated with IL-2, as well as peritoneal dendritic cells. Implant size of these rats decreased significantly, showing a greater number of activated lymphocytes, macrophages, NK and dendritic cells inside them. In conclusion, recombinant IL-2 induced recruitment of activated leukocytes into endometriotic-like foci, and this was related to a reduction of the implant size, suggesting potential effectiveness of IL-2 as an immunomodulatory agent in this pathology.

An initial investigation of spinal mechanisms underlying pain enhancement induced by fractalkine, a neuronally released chemokine

Fractalkine is a chemokine that is tethered to the extracellular surface of neurons. Fractalkine can be released, forming a diffusible signal. Spinal fractalkine (CX3CL1) is expressed by sensory afferents and intrinsic neurons, whereas its receptor (CX3CR1) is predominantly expressed by microglia. Pain enhancement occurs in response both to intrathecally administered fractalkine and to spinal fractalkine endogenously released by peripheral neuropathy. The present experiments examine whether fractalkine-induced pain enhancement is altered by a microglial inhibitor (minocycline) and/or by antagonists/inhibitors of three putative glial products implicated in pain enhancement: interleukin-1 (IL1), interleukin-6 (IL6) and nitric oxide (NO). In addition, it extends a prior study that demonstrated that intrathecal fractalkine-induced mechanical allodynia is blocked by a neutralizing antibody to the rat fractalkine receptor, CX3CR1. Here, intrathecal anti-CX3CR1 also blocked fractalkine-induced thermal hyperalgesia. Furthermore, blockade of microglial activation with minocycline prevented both fractalkine-induced mechanical allodynia (von Frey test) and thermal hyperalgesia (Hargreaves test). Microglial activation appears to lead to the release of IL1, given that pretreatment with IL1 receptor antagonist blocked both fractalkine-induced mechanical allodynia and thermal hyperalgesia. IL1 is not the only proinflammatory cytokine implicated, as a neutralizing antibody to rat IL6 also blocked fractalkine-induced pain facilitation. Lastly, NO appears to be importantly involved, as l-NAME, a broad-spectrum NO synthase inhibitor, also blocked fractalkine-induced effects. Taken together, these data support that neuronally released fractalkine enhances pain via activation of spinal cord glia. Thus, fractalkine may be a neuron-to-glia signal triggering pain facilitation.

The Roles of Eosinophils in Allergic Inflammation

Eosinophils are one of the cells that play a critical role in the pathogenesis of allergic diseases. The increase in the number of eosinophils in such diseases is regulated by interleukin-5 (IL-5). The author have prepared recombinant rat IL-5 using a baculovirus expression system and examined its biological activities in rat eosinophils. It was demonstrated that recombinant rat IL-5 prolongs the survival of mature eosinophils and differentiates immature eosinophils into mature eosinophils, suggesting that rat IL-5 is a factor for eosinophilia in rats. Recombinant rat eosinophil-associated ribonuclease (Ear)-1 and Ear-2 were also prepared. Eosinophil granule proteins are thought to cause tissue damage due to their cytotoxic activity, but using recombinant rat Ear-1 and Ear-2, it was found that rat Ear-1 and Ear-2 have strong RNase A activity and bactericidal activity, suggesting that these proteins play critical roles in host defense. Finally, the important role of acetylation of histones was clarified in the differentiation of HL-60 clone 15 cells into eosinophils using the histone deacetylase inhibitors sodium n-butyrate, apicidin, and trichostatin A. These findings would be useful for further investigations of the role of eosinophils in allergic inflammation.

Interferon alpha induction of metallothionein in rat liver is not linked to interleukin-1, interleukin-6, or tumor necrosis factor alpha

Synthesis of metallothionein (MT) is induced by interferon-alpha (IFN-α) in vitro and in vivo. In addition, IFN-α promotes redistribution of zinc (Zn) from the plasma to the liver in mice. However, it is not clear if IFN-α induces hepatic MT synthesis directly or indirectly via liberation of other cytokines. In order to address this issue, we determined hepatic MT levels, Zn concentration in plasma, liver, and urine, and plasma levels interleukin-1 (IL-1), interleukin-6 (IL-6), and tumor necrosis factor-α (TNFα) in rats following intramuscular injection of human IFN-α (1.5 × 10 6 UI/m 2). Animals were housed in metabolic cages and sacrificed at various times after IFN-α administration. Zn concentrations in serum, urine, and hepatic tissue were determined by atomic absorption spectrophotometry. MT protein was measured using the MT silver saturation method and expression of MT-1 and MT-2 mRNA was measured by RT-PCR. Plasma levels of rat IL-1, IL-6, and TNFα were determined using an ELISA method. Hepatic MT levels began to increase at 2 h following IFN-α administration and reached maximum levels at 12 h post-treatment. Induction of MT gene expression was confirmed by increases in MT-1 and MT-2 mRNA levels 6, 12, and 18 h after IFN-α administration. IFN-α treatment also resulted in biphasic increases in hepatic Zn, with levels peaking at 2 h, the time-point when MT levels are first increased, and again at 18 h. Concurrently, there were decreases in serum Zn levels at these time points, suggesting IFN-α induced movement of Zn from the blood to hepatic tissue. The decrease in serum Zn was not due to increased excretion since urinary Zn levels were unaffected following IFN-α treatment. IFN-α administration had no effect on plasma IL-1, IL-6, and TNFα levels. These results show that IFN-α promotes the increase of hepatic MT levels and plasma/liver redistribution directly, without IL-1, IL-6, or TNFα participation.

Secretory production of biologically active rat interleukin-2 by Clostridium acetobutylicum DSM792 as a tool for anti-tumor treatment

The search for effective means of selectively delivering high therapeutic doses of anti-cancer agents to tumors has explored a variety of systems in the last decade. The ability of intravenously injected clostridial spores to infiltrate and thence selectively germinate in the hypoxic regions of solid tumors is exquisitely specific, making this system an interesting addition to the anti-cancer therapy arsenal. To increase the number of therapeutic proteins potentially useful for cancer treatment we have tested the possibility of Clostridium acetobutylicum to secrete rat interleukin-2 (rIL2). Therefore, rIL2 cDNA was placed under the control of the endo-β-1,4-glucanase promoter and signal sequence of C. saccharobutylicum. Recombinant C. acetobutylicum containing the relevant construct secreted up to 800 μg l −1 biologically active rIL2. The obtained yield should be sufficient to provoke in vivo effects.

Exogenous interleukin-6 increases cold allodynia in rats with a mononeuropathy

Interleukin-6 (IL-6) is a pleiotropic cytokine, signaling intracellularly via its unique membrane-bound receptor IL-6R and gp130. In peripheral nerve injury models, IL-6 and IL-6R are increased at the injured nerve and the respective dorsal root ganglion. IL-6 is increased at the ipsilateral dorsal and ventral horn of the spinal cord. IL-6 is known to affect neuronal survival, differentiation and regeneration. It is involved in synaptic plasticity and hyperexitability and induces the synthesis or release of other substances with known neuroprotective or neuromodulatory effects. In this study, intrathecal administration of recombinant rat IL-6 to rats with a chronic constriction injury of the sciatic nerve, induced a logarithmic dose-dependent increase in cold allodynia with a threshold of 10 pg IL-6 and a maximal effect at 100 ng IL-6. Intrathecal administration of saline or denaturated IL-6 was without effect. In rats with a chronic constriction injury, systemic administered IL-6 did not induce a hyperalgesic effect, illustrating that IL-6 acts at the level of the dorsal root ganglion or the spinal cord. Intraplantar injection of 100 ng IL-6 in the operated hind paw resulted in an increased cold allodynia. This study demonstrates that the sensitivity to exogenous intrathecal or peripheral IL-6 increases in rats with a mononeuropathy.

Oxidative Stress Mediates Sodium Arsenite-Induced Expression of Heme Oxygenase-1, Monocyte Chemoattractant Protein-1, and Interleukin-6 in Vascular Smooth Muscle Cells

Arsenic exposure is associated with an increased risk of vascular disorders, and results in increased oxidative stress in endothelial cells and vascular smooth muscle cells (VSMCs). Since oxidative stress is involved in regulating the expression of genes related to atherogenesis, we investigated its involvement in the enhanced expression of three atherosclerosis-related genes coding for heme oxygenase-1 (HO-1), monocyte chemoattractant protein-1 (MCP-1), and interleukin-6 (IL-6) in VSMCs treated with inorganic sodium arsenite (iAs). In human VSMCs (hVSMCs) and rat VSMCs (rVSMCs), HO-1, MCP-1, and IL-6 mRNA levels were significantly increased by iAs treatment. An increase in HO-1 protein levels in hVSMCs was confirmed by Western blotting technique, while increased MCP-1 and IL-6 secretion by hVSMCs was demonstrated by enzyme-linked immunosorbent assay. Although modulators of oxidative stress inhibited this iAs-induced increase in the expression of these three genes, different modulators had differential effects. In iAs-treated rVSMCs, catalase, dimethylsulfoxide, and L-omega-nitro-L-arginine significantly inhibited the increase in expression of all three genes, allopurinol inhibited the increase in MCP-1 and IL-6 expression, but had no effect on HO-1 expression, while superoxide dismutase had no significant effect on HO-1 expression, but had an inhibitory effect on IL-6 expression and a stimulatory effect on MCP-1 expression. Therefore, iAs may enhance the expression of HO-1, MCP-1, and IL-6 in VSMCs via different reactive oxygen molecules. Furthermore, using tin protoporphyrin IX (SnPP) and anti-MCP-1 antibody to abolish iAs-induced HO-1 and MCP-1 activity, respectively, shows that HO-1 has protective effect against iAs-induced injury in VSMCs and MCP-1 is chemoattractive to human monocytes, THP-1.

Insights into the role of interleukin-6 in the induction of hepatic injury after trauma-hemorrhagic shock.

Although systemic interleukin-6 (IL-6) level is elevated, hepatocellular function is impaired and liver injury occurs after trauma-hemorrhage (T-H), it remains unknown whether a causal relationship exists between elevated IL-6 levels and liver injury after T-H. We hypothesized that IL-6 is causative in the development of hepatic dysfunction and injury after T-H. To examine this, adult male Sprague-Dawley rats underwent a 5-cm midline laparotomy and were subjected to hemorrhagic shock (blood pressure = 35 mmHg for approximately 90 min), followed by resuscitation (Ringer lactate, 4 times the shed blood volume). At 2, 5, and 24 h thereafter, blood samples were collected and the liver isolated and perfused for 60 min. Portal inflow pressure was measured, and perfusate samples were collected to measure IL-6, alanine aminotransferase (ALT), and lactate dehydrogenase (LDH) levels. A significant positive correlation between plasma levels of IL-6 and ALT and perfusate levels of IL-6 and LDH levels was observed. In a second series of experiments, rats were treated with immunoglobulin G (IgG) or antibodies against rat IL-6 (anti-IL-6) at the onset of resuscitation. At 5 h after resuscitation, anti-IL-6 treatment attenuated the T-H induced increases in plasma ALT and thromboxane B(2) (a thromboxane A(2) metabolite) levels, and bile flow was normalized to sham levels. Perfusion of livers from normal rats with IL-6 did not alter portal pressure; however, perfusion of a stable thromboxane A(2) analog dose dependently increased portal pressure. Thus IL-6 plays a significant role in the induction of hepatic dysfunction and liver injury after T-H that appears to be in part mediated by increased thromboxane A(2) levels.

LONG TERM FOLLOW-UP (10 YEARS) OF A PROSPECTIVE TRIAL ASSAY COMPARING LO-TACT-1 ANTIBODY VERSUS ANTI-THYMOCYTE GLOBULIN INDUCTION THERAPY IN KIDNEY TRANSPLANTATION.

P760 Aims: To evaluate the long term graft survival, acute and chronic rejections, infectious diseases and malignancies following an induction therapy with a rat interleukin 2 receptor monoclonal antibody Lo-Tact-1 versus anti-thymocyte globulin (ATG). Patients and Methods: Forty recipients of primary renal transplantation were prospectively randomised between May 1990 and June 1991. Twenty recipients were treated with Lo-Tact-1 IV 10 mg daily (group I) and twenty with ATG 15 ml/day (group II) during the first 14 days of transplantation. All patients were treated with azathioprine, steroids, cyclosporine. Creatinemia, proteinuria and clinical events were collected during ten years. Results: Median age was 42.1 years in group I and 39.3 years in group II. There was no difference between the two group for sex ratio, rate of anti-HLA antibodies, HLA A, B, DR mismatches, cold ischemia time. Six recipients died within 10 years of follow-up (n=5 in group I, n=1 in group II). All of them had a functional graft. At 10 years the graft loss was 35 % in group I and 45 % in group II (p=ns). Nine acute rejections occurred in group I and 10 in group II (p=ns). All were cortico-sensitive. Chronic allograft rejection was significantly higher in group II (n=9) than in group I (n=3), p<0.05. Six patients had a nephropathy due to cyclosporine toxicity (n=4 in group I, n=2 in group II, p=ns). Viral infectious diseases including cytomegalovirus, herpes virus and zoster virus diseases were significantly higher in group II (n=8) than in group I (n=2), p < 0.05. Bacterial infectious including pneumonia, pyelonephritis, airways infections, septicaemia were significantly higher in group II (n = 16) than in group I (n=10), p<0.05. At ten years three patients had malignancies (cutaneous lymphoma n=1 in group II, epidermoidis carcinoma n=1 and cervical uterus carcinoma n=1 in group I). Conclusions: Although both Lo-tact-1 and ATG were effective and safety induction therapy in kidney transplantation, less bacterial and viral infections and chronic allograft rejection were noted in Lo-tact-1-treated patient after a long follow-up.

Development of a novel antigen capture-ELISA using IgY against porcine interleukin-6 and its application

Interleukin-6 (IL-6) is introduced as a marker of disease. At present, a variety of method may be used to quantify expression of this protein. Antigen capture-ELISA is a sensitive and accurate quantification method previously used with ovine, rat, and human IL-6 proteins. However, it has never been reported to quantify porcine IL-6 protein using capture ELISA. In this study, we generated and characterized a set of IgY and mono-specific polyclonal antibodies to recombinant porcine IL-6 (rpIL-6), and combining these with a sensitive and specific capture-ELISA for a diagnostic purpose. cDNA encoding the mature protein coding region of porcine IL-6 was cloned and expressed with pQE-30UA expression vector. rpIL-6 was then expressed and purified by using Ni-NTA resin. Protein mass of 24 kDa was found with SDS-PAGE and the identity of the protein was confirmed by Western-blot. Production of polyclonal antibodies against rpIL-6 was performed using the purified rpIL-6 in mice and hens. An antigen capture-ELISA was developed with the antibodies after their extraction. To compare the IL-6 level in the different sanitary state of farms, pig sera were randomly collected and concentration of IL-6 in the sera was measured with the antigen capture-ELISA. The capture-ELISA with the optimal concentration of antibodies, in this study, was able to detect about 10 ng/ml of rpIL-6. IL-6 levels determined with the capture-ELISA in pig sera showed positive correlation with the sanitary states of the farms. These results suggested that the developed antigen capture-ELISA could be a good tool for the screening of microbial infection in pig farms.

Reduced production of hyperalgesic substances by mononuclear cells from aged rats incubated with carrageenan: role of interleukin 2 and prostaglandins.

To compare the production of hyperalgesic substances by cells from aged (A; 24-month) and juvenile (J; 2-month) rats. 4 x 10(5) purified mononuclear cells from J and A were 2 h-stimulated (test) or not (control) by 250 microg lambda-carrageenan/well. Supernatants (0.1 ml) were intraplantarly (ipl) injected in rat paws and development of mechanical hyperalgesia, in grams, evaluated. Rat interleukin 2 (IL 2) and prostaglandin E2 (PGE2) were also assessed for hyperalgesia development. Test supernatants from A compared with J induced significantly less hyperalgesia (-56 +/- 8.1 and -88.4 +/- 4.6 g, respectively, p < 0.05, ANOVA t test). Local injection of a specific, but not a control, antiserum against IL 2 significantly blocked both pure IL 2- and stimulated supernatants-derived hyperalgesia. In contrast to PGE-like materials, IL 2 content in supernatants was compatible with hyperalgesia development. Hyperalgesia induced by test supernatants was significantly less intense when derived from aged animals. IL 2 may have accounted for such hyperalgesia.

Effect of neutralization of rat IL-6 bioactivity on collateral blood supply from retrograde flow via cortical anastomoses in the rat central nervous system

The purpose of this study was to determine whether neutralization of rat interleukin-6 (IL-6) bioactivity increases the collateral blood supply from retrograde flow via the major middle cerebral artery branches after experimental middle cerebral artery occlusion in the rat. Seventy rats were randomly allocated to four main groups: Group I (n = 10) consisted of normal controls; Group II (n = 20) underwent craniectomy only; Group III (n = 20) was subjected to middle cerebral artery occlusion; and Group IV (n = 20) underwent middle cerebral artery occlusion and treatment with anti-rat IL-6 antibody. Half of the rats from each of Groups II, III and IV were killed at 24 h and the other half at 72 h after craniectomy alone or occlusion. A single dose of antibody did not affect middle cerebral artery caliber, but administration of three doses resulted in a significant increase in the diameter of middle cerebral artery compared to the findings in the corresponding occlusion-only groups. The results suggest that neutralization of rat IL-6 bioactivity in longterm recovery increases the collateral blood supply from retrograde flow via cortical anastomoses after experimental arterial occlusion in the rat brain. [Neurol Res 2002; 24: 405-408]

Analysis of maturation states of rat bone marrow-derived dendritic cells using an improved culture technique.

In this study, we examined in more detail the development of rat bone marrow-derived dendritic cells (BMDC). A two-stage culture system was used to propagate BMDC from rat bone marrow precursors. BMDC developed within clusters of proliferating cells after repetitive addition of rat granulocyte/macrophage colony-stimulating factor and rat interleukin (IL)-4 at a concentration of 5 ng/ml to the cultures. Fluorescence-activated cell sorter analysis performed at an early stage of development (day 6) revealed an immature phenotype with intermediate levels of major histocompatibility complex (MHC) class II expression and low levels of the costimulator molecules CD80 and CD86. Upon further culture, a strong upregulation of MHC class II, costimulatory and adhesion molecules could be observed, whereas macrophage marker antigens were downregulated. Late-stage BMDC (day 10) showed a high expression of MHC class I and II, ICAM-1, Ox62 and CD11c, and revealed a split pattern of B7-1 and B7-2. The cell yield was about 40% of the initially plated bone marrow cells with 80% MHC class II-high and less than 20% MHC class II-low positive cells. Full maturation of rat BMDC (day 12) with an almost uniform expression of B7 was achieved by subsequent subculture and further stimulation with rat tumour necrosis factor alpha (TNF-alpha), lipopolysaccharide (LPS) or soluble CD40 ligand (CD40L). Analysis of the cell supernatant revealed a strong IL-12 production after LPS or CD40L, and to a lesser extent after TNF-alpha stimulation. Additionally, LPS-treated, but not CD40L-treated BMDC secreted TNF-alpha into the supernatant. Early-stage BMDC sufficiently triggered a T cell receptor (TCR) downregulation, but did not stimulate naive T cells in an allogeneic mixed leukocyte reaction (MLR) and revealed a low stimulatory capacity in an antigen-specific T cell assay. In contrast, late-stage BMDC and especially fully mature BMDC strongly induced TCR internalisation, elicited high T cell responses in the allogeneic MLR similar to those obtained by mature rat spleen dendritic cells and efficiently activated antigen-specific T cells. In conclusion, this protocol allows easy access to large numbers of rat BMDC at defined maturation stages and selective studies for the manipulation of immune responses in rat models.

Interleukin-1 is a potent growth factor for immature rat Sertoli cells

Testes from rats of different maturational ages were explored for presence of paracrine Sertoli cell growth factors. Pubertal and adult testes contained a 17 kDa protein, with potent stimulatory effect on immature Sertoli cell multiplication in vitro. The bioactivity of this protein was mimicked by rat interleukin-1 (IL-1) and neutralized by IL-1 receptor antagonist. A receptor-mediated action was further supported by the demonstration of IL-1 receptor type I mRNA and protein expression in the cultured Sertoli cells and in intact immature rat testes. IL-1α showed higher efficacy in stimulating proliferation than IL-1β and follicle-stimulating hormone (FSH), and displayed synergistic action in combination with FSH. As IL-1α is constitutively produced by the rat testis and IL-1β readily inducible by proinflammatory stimuli, our results suggest that IL-1 may serve as a growth factor for Sertoli cells under physiological and pathophysiological conditions.

Cloning of rat interleukin 11 and interleukin 11 receptor alpha chain and analysis of their expression in rat uterus in the peri-implantation period

Studies in mice have shown that interleukin 11 (IL-11) signalling is required for female fertility. In the absence of IL-11, decidualization is markedly retarded and implantation fails. IL-11 acts via a heterodimeric receptor composed of a ligand-specific receptor alpha chain (IL-11R alpha) and the signalling moiety gp130. This study reports the cloning of genes encoding rat IL-11 and IL-11R alpha. RNase protection was used to demonstrate that expression of IL-11 is upregulated in the rat uterus at the initiation of implantation at 5.5 days after mating. Expression of the genes encoding the two receptor components, IL11Ra and gp130, did not change throughout the peri-implantation period. In situ hybridization studies revealed that, as in mice, expression of IL-11 was high in the primary decidual zone at the time of the attachment reaction, whereas IL11Ra was expressed throughout primary and secondary decidua. Conservation of the temporal and spatial expression of IL-11 and IL-11R alpha in the uterus of the mouse and rat during the peri-implantation period will facilitate future studies investigating the role of IL-11 in fertility.