Detection of mRNA in megakaryocytes using in situ hybridization]

The usefulness of in situ hybridization for detecting mRNA encoding various kinds of proteins in megakaryocytic cell lines (K562, HEL and CMK) was examined. Using in situ hybridization, glycoprotein (GP) IIIa mRNA was found to be present in K562, HEL and CMK cells. Both GPIb and GPIIb mRNA were also present in HEL and CMK cells, while only HEL cells expressed platelet factor 4 mRNA. These findings were identical to those obtained with Northern blotting. It should be emphasized that in situ hybridization was useful to clearly define each cell expressing platelet specific protein mRNA. The expression of interleukin-6 (IL-6) and IL-6 receptor mRNA in mononuclear cells prepared from bone marrow aspirates was examined. In situ hybridization study demonstrated the presence of IL-6 receptor mRNA in the recognized megakaryocytes only, while IL-6 mRNA was found to be present in the megakaryocytes and a few mononuclear cells. These findings suggest that differentiation and proliferation of normal megakaryocytes might be controlled by an IL-6 autocrine loop, and detection of IL-6 receptor mRNA might be useful to identify megakaryocytes.     

Inflammatory cytokine expression in swine experimentally infected with Actinobacillus pleuropneumoniae.

An Actinobacillus pleuropneumoniae infection model in swine was established to study the expression of inflammatory cytokines during acute respiratory disease. Lavage fluid, lavage cells consisting primarily of alveolar macrophages, and lung tissue were analyzed for the presence of various cytokines at 2, 4, 8, and 24 h following endotracheal inoculation of A. pleuropneumoniae. Interleukin-1 beta (IL-1) and IL-8 mRNA levels were elevated within 2 h in lavage cells of animals inoculated with A. pleuropneumonia but not in cells from controls treated with saline-bovine serum albumin, based on Northern (RNA blot) analysis. Tumor necrosis factor (TNF) mRNA was present at low levels in all animals, and the level was not increased at any time point. In situ hybridization was more sensitive than Northern blotting and revealed elevations of all three cytokines in lavage cells within 2 to 4 h of A. pleuropneumoniae inoculation. IL-6 was detected in lavage cells by in situ hybridization but not by Northern blotting. In lung tissue obtained 18 to 24 h after A. pleuropneumoniae instillation, all cytokine mRNAs, including that of IL-6, were detected by Northern blot analysis. The levels of bioactive IL-1 and IL-6 in lavage fluids increased approximately 1,000-fold following A. pleuropneumoniae inoculation, but TNF bioactivity was not detected. Morphological localization of cytokine mRNAs by in situ hybridization indicated markedly increased levels of TNF, IL-1, and IL-8 mRNAs at the periphery of focal lung lesions. These findings indicate that inflammatory cytokines, particularly IL-1 and IL-8, are associated with the development of pleuropneumonia and may contribute to disease severity.     

信号转导子和转录激活子6及其mRNA在哮喘大鼠气道炎症中的作用

目的：探讨信号转导子和转录激活子6(STAT6)及其mRNA在哮喘大鼠气道炎症中的作用。 方法： 20只二级雄性SD大鼠随机分为2组, 对照组和哮喘组。以卵清白蛋白(OVA) 致敏激发法复制大鼠哮喘模型，每只大鼠左肺留取肺组织，右肺进行支气管肺泡灌洗并留取支气管肺泡灌洗液(BALF)。对BALF进行细胞总数、嗜酸性粒细胞(EOS)计数和分类计数；应用双抗体夹心酶联免疫吸附试验（Sandwich ELISA）法测定BALF和血清中IL-4浓度；采用免疫组化法和原位杂交法测定STAT6蛋白和STAT6 mRNA的表达情况。 结果： (1) BALF和血清中白细胞介素4(IL-4)的浓度哮喘组均显著高于对照组(均P<0.01)；(2)免疫组化和原位杂交显示，哮喘组支气管STAT6蛋白和STAT6 mRNA的表达均显著高于对照组(均P<0.01)，其主要表达细胞是上皮细胞；(3)支气管上皮细胞STAT6蛋白及其mRNA含量分别与BALF中的IL-4浓度、EOS绝对值呈非常显著正相关。 结论： 哮喘大鼠STAT6蛋白和mRNA高表达，上皮细胞是其主要表达细胞，并与IL-4浓度、EOS募集密切相关。     

Interleukin-5 mRNA in mucosal bronchial biopsies from asthmatic subjects

Using in situ hybridization, we have investigated the expression of interleukin-5 (IL-5) mRNA in bronchial biopsies from asthmatics (n = 10) and controls (n = 9). The number of IL-5-nRNA-positive cells were compared with the number of CD25+ and EG2+ cells and total eosinophil counts. Specific hybridization signals for IL-5 mRNA were demonstrated in 6 out of the 10 asthmatic subjects but in none of the controls. The 6 IL-5-mRNA-positive asthmatics tended to have more severe disease and showed a significant increase in the degree of infiltration of the bronchial mucosa by activated T lymphocytes and eosinophils.     

Perforin gene expression in stimulated human peripheral blood T cells studied by in situ hybridization and northern blotting analysis.

In situ hybridization was used here to monitor the mRNA level of the pore-forming protein perforin in mitogen-stimulated primary peripheral blood human T cells. In situ hybridization was performed using sense and antisense ribonucleotide probes specific for this granule mediator. After IL-2 treatment, an increase in perforin mRNA could be detected by 4 h; they peaked at 12 h, and decreased after 24 h. The perforin mRNA was also induced in T cells treated with a combination of phorbol ester PMA plus lectin or OKT3 mAb. This latter induction followed slower kinetics, peaking at 48 h. For all three mitogens used, even at peak induction times less than 10% of T cells were labeled with perforin probe. Similar patterns of mRNA expression were observed for both unprimed T cells and lectin-primed T blasts. The induction response of mRNA due to IL-2 stimulation is probably mediated by the IL-2 receptor p75 chain since its mRNA was upregulated by IL-2 with a kinetics comparable to that associated with an increase of perforin mRNA. The p55 IL-2 receptor chain increased much more slowly than p75.     

Expression of the interleukin 6 receptor in primary renal cell carcinoma.

AIMS: Interleukin 6 (IL-6) is expressed in the majority of renal cell carcinomas and has an important role in the proliferation of some renal cell carcinoma cell lines. This action is mediated by two membrane proteins, gp80 (the IL-6 receptor; IL-6R), which binds IL-6, and gp130, which transduces the signal. The soluble form of gp80 (sIL-6R) is able to activate gp130 when complexed to the IL-6 molecule. These considerations prompted an investigation of IL-6R expression in this malignancy. IL-6, C reactive protein (CRP), and sIL-6R were also measured in serum and correlated to clinical and pathological features. METHODS: Immunostaining was performed on cryostat sections from renal cell carcinoma tumours with M91, an anti-IL-6R monoclonal antibody, using the alkaline phosphatase antialkaline phosphatase technique. The proliferation index was measured using the KI-67 monoclonal antibody. CRP, IL-6, and sIL-6R were measured in serum before nephrectomy, using an immunoenzymatic or immunoradiometric assay. RESULTS: There were significant differences in survival in patients with tumours larger than 8 cm, metastasis at diagnosis, high nuclear grade tumours, detectable serum concentrations of IL-6 (correlated to CRP serum concentration), more than 4% proliferating cells, and the presence of the IL-6R in situ. Furthermore, the serum IL-6 concentration correlated with tumour size and stage. The mean serum sIL-6R concentration was not significantly different from that observed in 40 normal subjects. Tumour IL-6R expression was present in 10 samples. There was a significant association between the presence of the IL-6 receptor in tumours and tumour stage, nuclear grade, proliferation index, and serum IL-6. CONCLUSIONS: This study revealed the importance of IL-6/CRP and IL-6R expression in situ as potential new prognostic factors and opens the way to new therapeutic strategies in renal cell carcinoma.     

In situ production of interleukins in hyperplastic thymus from myasthenia gravis patients

We analyzed by in situ hybridization the expression of four interleukin genes (interleukin-beta [IL-1 beta], IL-6, IL-2, and interferon-gamma) in seven thymuses displaying a follicular hyperplasia. The seven thymuses were obtained from patients with myasthenia gravis. Interleukin-1 beta- and IL-6-producing cells were detected in similar amounts and with similar distributions: mainly in perifollicular areas and in the connective structures emerging from the septae at the site of cortex disruption. The comparison of in situ hybridization and immunohistochemical results suggested that thymic epithelial cells and/or perifollicular macrophages were responsible for this production. Interleukin-2-producing cells were detected in perifollicular areas and, to a lesser extent, inside follicles. They were clearly outnumbered by CD25-positive cells which were similarly distributed. Despite the expression of these molecular and immunohistochemical markers of T-cell activation, interferon-gamma-producing cells were extremely rare in myasthenic thymuses. The pattern of interleukin production (which was virtually absent in normal control thymuses) in myasthenic thymuses was different from that in benign hyperplastic lymph nodes. This interleukin production may play a role in the development of follicular hyperplasia in myasthenic thymuses, a phenomenon which is associated with the in situ production of autoantibodies.     

Heme oxygenase-1, heme oxygenase-2 and biliverdin reductase in peripheral ganglia from rat, expression and plasticity

The expression of inducible and constitutive heme oxygenase and biliverdin reductase was studied in normal and cultured peripheral ganglia from adult rats, using immunocytochemistry and in situ hybridization. Dramatic changes were induced by one to two days' culturing of dorsal root ganglia, nodose ganglia, otic ganglia, sphenopalatine ganglia and superior cervical ganglia. An up-regulation of inducible heme oxygenase was found in satellite cells of the cultured nodose ganglia, dorsal root ganglia, sphenopalatine ganglia and otic ganglia, whereas only a few satellite cells in the superior cervical ganglia responded with an increase in inducible heme oxygenase immunoreactivity. In the superior cervical ganglia inducible heme oxygenase also appeared in a subpopulation of macrophages. During culturing, expression of inducible heme oxygenase immunoreactivity also increased in axons and in nerve cell bodies. In situ hybridization corroborated the immunocytochemical findings, revealing a strong up-regulation of inducible heme oxygenase messenger RNA in satellite cells, and less pronounced up-regulation in nerve cell bodies. Constitutive heme oxygenase immunoreactivity was found in most neurons in all of the ganglia studied. No significant changes in constitutive heme oxygenase immunoreactivity could be observed in cultured ganglia. Biliverdin reductase immunoreactivity was barely detectable in any of the normal ganglia; however, after culturing it appeared in axons, single nerve cell bodies and nerve cell nuclei. The results show that inducible heme oxygenase is up-regulated in peripheral ganglia after axonal injury, and suggest a role for carbon monoxide in cellular signaling and a requirement for the antioxidant (bilirubin) during the regeneration process.     

布地奈德对哮喘大鼠信号转导子和转录激活子6的表达

目的研究布地奈德（BUD）对支气管哮喘大鼠支气管信号转导子和转录激活子6（STAT6）基因和蛋白表达的调控作用。方法30只清洁级幼年雄性SD大鼠随机分为对照组（A组）、哮喘组（B组）和BUD组。对支气管肺泡灌洗液（BALF）进行细胞总数、嗜酸性粒细胞（EOS）计数和分类计数；应用双抗体夹心酶联免疫吸附试验法测定BALF中IL-4、IL-12浓度；采用免疫组化法和原位杂交法分别检测STAT6蛋白和STAT6 mRNA表达的变化。结果（1）B组BALF中细胞总数、EOS绝对值和EOS占细胞总数的百分比（EOS％）均显著高于A组（P〈0．01），BUD组BALF中上述各项指标较B组均显著降低（P〈0．01）；（2）BALF中IL-4的浓度B组显著高于A组（P〈0．01），BUD组较B组显著降低（P〈0．01），而IL-12的浓度B组显著低于A组（P〈0．01），BUD组较B组显著升高（P〈0．01）；（3）B组支气管上皮细胞STAT6蛋白和STAT6 mRNA阳性表达较A组明显增强（均为P〈0．01）。BUD组较B组明显减弱（均为P〈0．01）；（4）支气管上皮细胞STAT6蛋白、STAT6 mRNA分别与BALF中的IL-4浓度呈显著正相关，与BALF中EOS绝对值呈显著正相关；而与IL-12浓度呈显著负相关。结论哮喘大鼠支气管STAT6及其mRNA较强表达，上皮细胞是其主要表达细胞；BUD有抑制气道炎症的作用，下调STAT6及其基因表达，使IL-4合成减少可能为其重要作用机制。     

In vivo production of interleukin-10 by malignant cells in AIDS lymphomas.

Expression of the interleukin (IL)-10/BCRF1 gene was studied by in situ hybridization in tissue samples from acquired immunodeficiency syndrome (AIDS) lymphomas using a BCRF1 probe which also recognizes the human IL-10 sequence. Hybridization was detected in 8 out of 15 lymphomas. In contrast, the IL-10/BCRF1 gene expression was detected in only 1 out of 11 lymphomas from human immunodeficiency virus (HIV)-seronegative patients (p = 0.05). In AIDS lymphomas, the number of cells labeled with a BCRF1-specific probe was dramatically lower than that of cells labeled with the IL-10/BCRF1 probe. Thus, the IL-10 rather than the BCRF1 gene was expressed. Production of IL-10 was associated with that of IL-10 mRNA, as shown by immunodetection of the protein in numerous cells. In contrast, BCRF1-producing cells were rarely detected. Both in situ hybridization and immunochemical experiments indicated that malignant cells were involved in this IL-10 synthesis. IL-10 production in AIDS lymphomas was associated with the presence of Epstein-Barr virus (EBV) in lymphomatous cells (p = 0.02). As IL-10 is a potent growth factor for human B lymphocytes, these results suggest that IL-10 may stimulate the proliferation of malignant cells in an autocrine pathway in a number of AIDS lymphomas, and that EBV and HIV may synergistically trigger its production.     

大鼠心内神经节雄激素受体和免疫因子白介素-6的共存

目的：研究雄激素受体(AR)和免疫因子白介素-6(IL-6)的表达，证实它们在心内神经节共存的可能性。方法：采用免疫细胞化学双重标记技术。结果：切片上观察到3种细胞：①AR单标细胞，胞核呈棕褐色；②IL-6单标细胞，胞浆呈红色；③AR／IL-6双标细胞，胞核棕褐色，胞浆为红色。双标细胞占全部阳性标记细胞的50--60％。结论：AR和IL-6可共存于同一大鼠心内神经节细胞，从而首次于细胞水平为神经免疫内分泌网络在心脏的存在提供了直接的形态学证据。     

In situ detection of interleukin-1 mRNA in human monocytes

The interleukin-1 (IL-1) family of soluble pleiotropic immunoregulatory and proinflammatory peptides has at least two distinct members, alpha IL-1 and beta IL-1. Since beta IL-1 is the predominant species in human monocytes, this study was undertaken to identify its mRNA in monocytes using in situ hybridization with a 35S-dCTP labelled beta IL-1 cDNA probe. Grain count analysis demonstrated that adherent lipopolysaccharide-stimulated monocytes were positive, while unstimulated monocytes, lymphocytes and neutrophils, and cells probed with vector only (35S-labelled pBR322) were all negative. We have also shown that in situ hybridization is approx. 13-fold more sensitive than conventional hybridization and in addition this technique allows visualization of mRNA coding for IL-1 in individual cells with morphology preserved. We conclude that in situ hybridization is a specific and sensitive technique for the detection of beta IL-1 mRNA in individual human peripheral blood monocytes.     

Macrophage subpopulations and macrophage-derived cytokines in sputum of atopic and nonatopic asthmatic subjects and atopic and normal control subjects

BACKGROUND: Previous studies have shown a prominent macrophage signal in the bronchial mucosa from nonatopic (intrinsic) compared with atopic (extrinsic) asthmatic subjects. This observation might have represented an expansion of a proinflammatory macrophage population or a homeostatic mechanism to decrease T(H)2-type inflammation. OBJECTIVE: The aim of the study was to investigate the numbers of macrophages and macrophage subpopulations and the expression of IL-10 and IL-12 in sputum from asthmatic and control subjects. METHODS: Eight atopic asthmatic (AA) subjects, 10 nonatopic asthmatic (NAA) subjects, 6 atopic control (AC) subjects, and 7 normal control (NC) subjects underwent sputum induction. Macrophages were enumerated by using Romanowsky stain and immunocytochemistry (CD68). RFD1 (interdigitating cell marker) and RFD7 (mature phagocyte marker) mAbs were used for immunocytochemical phenotyping, whereas IL-10 and IL-12 messenger (m)RNA was examined with in situ hybridization by using (35)S-labeled riboprobes. The phenotype of cells expressing IL-10 or IL-12 mRNA was examined by simultaneous in situ hybridization and immunostaining. RESULTS: No differences in the numbers of CD68(+) macrophages and RFD1(+), RFD7(+), and RFD1(+)/RFD7(+) subpopulations were found between AA, NAA, AC, and NC subjects. However, the numbers of IL-10 and IL-12 mRNA(+) cells were increased in AA subjects compared with NAA, AC, and NC subjects (P <.05). No other differences were found among the groups. Most of the IL-10 and IL-12 mRNA(+) cells in sputum from asthmatic subjects were macrophages (>80%), with less than 10% of mRNA colocalizing to epithelial cells. CONCLUSIONS: Sputum macrophage numbers, unlike tissue macrophages, as previously reported, were not elevated in NAA subjects. Increased IL-10 and IL-12 expression in atopic asthma may indicate the existence of a homeostatic mechanism to decrease lung inflammation. The lack of such cytokines in intrinsic asthma may predispose to bronchial inflammation in these subjects.     

Expression of pro-inflammatory cytokines in mouse blastocysts during implantation: modulation by steroid hormones

PROBLEM: Expression and hormonal regulation of pro-inflammatory cytokines and their role in blastocyst activation and implantation is poorly known. The present study is aimed at analysing the expression and hormonal modulation of two pro-inflammatory cytokines [interleukin-1alpha (IL-1alpha) and IL-6] in mouse blastocysts during implantation. METHOD OF STUDY: Blastocyst-uterine interactions are inhibited by progesterone during implantation and subsequent treatment with oestrogen triggers events that allow implantation to begin. Using this delayed implantation mouse model, dormant and activated blastocysts were recovered from mice treated with progesterone alone and progesterone plus oestrogen therapy, respectively. Expression of IL-1alpha and IL-6 messenger RNA (mRNA) was analysed in normal, dormant and activated blastocysts by in situ hybridization using specific labelled sense and antisense RNA probes, and the protein expression of the same was analysed by immunocytochemistry. RESULTS: In situ hybridization revealed IL-1alpha and IL-6 mRNA localization in normal, dormant and activated blastocysts and a differential expression was observed in relation to the exposure to progesterone and oestrogen. There was less expression in the dormant blastocysts as compared with the normal and activated ones, and the pattern was similar for both cytokines. Immunocytochemistry also revealed a similar pattern of protein expression to that of the mRNA expression for both the cytokines. CONCLUSIONS: Using a delayed implantation model, we show that mouse blastocysts express both IL-1alpha and IL-6 mRNA as well as their respective proteins. Both mRNA and the protein levels of IL-1alpha and IL-6 seem to be hormonally modulated in mouse blastocysts during implantation.     

Expression of cytokine and chemokine genes in Epstein-Barr virus-associated nasopharyngeal carcinoma: comparison with Hodgkin's disease

Nasopharyngeal carcinoma (NPC) and Hodgkin's disease (HD) are characterized by their association with Epstein-Barr virus (EBV) and the presence of an intense lymphoid stroma, consisting of T lymphocytes and other reactive cells. In both entities, the tumour cells express viral proteins known to provide target epitopes for cytotoxic T-cells (CTLs), yet in vivo, the tumour cells appear to escape CTL recognition. A comparative in situ hybridization study of cytokine and chemokine gene expression in NPC and HD has been undertaken, focusing on cytokines which are known to be inducible by EBV in vitro. Hodgkin and Reed-Sternberg (HRS) cells expressed interleukin (IL)-6, IL-8, and IL-10, and the thymus and activation regulated chemokine (TARC) in 15/22, 0/22, 5/22, and 16/21 cases, respectively. In NPC, the epithelial tumour cells showed expression of IL-6 in 3/43 cases and of IL-8 in 2/40 cases. There was no detectable expression of IL-10 and TARC in these cases. These data confirm that HRS cells frequently express cytokine and chemokine genes and suggest that this may enable HRS cells to modulate the immune response in their microenvironment and to escape CTL detection. In contrast, NPC tumour cells show only rare expression of IL-6 and IL-8 and no detectable expression of IL-10 and TARC. Thus, the results suggest that the mechanisms employed by the EBV-positive tumour cells to escape immune recognition and destruction differ between HD and NPC.     

In situ detection of intracerebral cytokine expression after human brain contusion

The study was undertaken to analyze intracerebral expression of pro- and anti-inflammatory cytokines after traumatic brain injury (TBI) in man in order to compare the findings with previous experimental data regarding the pathogenesis of secondary brain injury. Contused brain tissue biopsies were obtained from 12 consecutive patients undergoing surgery for brain contusions 3 h to 5 days after trauma. Cytokine expression was analyzed by in situ hybridization and immunohistochemistry. In patients undergoing surgery less than 24 h after trauma, strong expression of both the pro-inflammatory cytokines interleukin (IL)-1-beta, IL-6 and interferon (IFN)-gamma and the anti-inflammatory cytokine IL-4 was detected. In patients undergoing surgery between 3 and 5 days after trauma, IL-4 expression was significantly lower (P < 0.05) compared to the patients operated early. IL-1-beta and IFN-gamma expression remained strong in comparison to IL-6 and IL-4 expression (P < 0.05). Immunohistochemistry for IL-1-beta confirmed that the protein was produced with a temporal and regional pattern that corresponded to in situ hybridization results. The study provides in situ data on intracerebral cytokine expression after contusion in the clinical setting. Strong intracerebral cytokine expression occurs in the perilesional zone both in the early and the delayed phase after traumatic brain injury in humans. The temporal regulation of pro- and anti-inflammatory cytokines differs which reveals different therapeutic windows for pharmacological intervention.     

IL-21 and IL-21 receptor expression in lymphocytes and neurons in multiple sclerosis brain

IL-17-producing CD4(+) T cells (Th-17) contribute to the pathogenesis of experimental autoimmune encephalomyelitis and are associated with active disease in multiple sclerosis (MS). In addition to IL-17, Th-17 cells can also express IL-21, IL-22, and IL-6 under Th-17-polarizing conditions (IL-6 and transforming growth factor-β). In this study we investigated IL-21 and IL-21 receptor (IL-21R) expression in MS lesions by in situ hybridization and immunohistochemistry. We detected strongly IL-21(+) infiltrating cells predominantly in acute but also in chronic active white matter MS lesions in which IL-21 expression was restricted to CD4(+) cells. In contrast, IL-21R was much more broadly distributed on CD4(+), CD19(+), and CD8(+) lymphocytes but not major histocompatibility complex class-II(+) macrophages/microglia. Interestingly, in cortical areas we detected both IL-21 and IL-21R expression by neurons. These findings suggest role(s) for IL-21 in both the acute and chronic stages of MS via direct effects on T and B lymphocytes and, demonstrated for the first time, also on neurons.     

Epstein-Barr virus-immortalized B cells produce IL-6 as an autocrine growth factor.

The continuous proliferation of Epstein-Barr virus (EBV)-immortalized B cells is enhanced by autocrine as well as paracrine growth factors. In the present study, the possibility that EBV-immortalized B cells might produce interleukin-6 (IL-6) proteins in an autocrine manner was examined. It was found that culture supernatants from EBV-transformed B cells, but not from Burkitt's lymphoma lines, augmented the proliferation of an IL-6-dependent murine hybridoma clone, MH60.BSF2. This growth-promoting activity for hybridoma cells found in culture supernatants of EBV-transformed B cells was specifically neutralized by rabbit anti-recombinant (r) IL-6 antibody. The IL-6 activity in culture supernatants of EBV-transformed B cells, though much less than that of lipopolysaccharide (LPS)-stimulated monocytes, was increased by the addition of phorbol myristate acetate. Western blot experiments using rabbit anti-rIL-6 antiserum demonstrated that supernatants from cultured EBV-transformed B cells contained the distinct forms of IL-6, with a peak of 23,000 MW. When examined by in situ hybridization analysis, it was found that IL-6 mRNA were expressed on EBV-transformed B cells. It was noted that a fraction, but not all, of these cells expressed IL-6 mRNA strongly, implying their cell cycle-dependent expression. In addition, it was shown that rIL-6 promoted the growth of EBV-transformed B cells at low cell densities. The results suggest that IL-6 serves as an autocrine growth factor in EBV-transformed B cells.