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PubMed1987

Regulation of mRNA entry into polysomes. Parameters affecting polysome size and the fraction of mRNA in polysomes.

The kinetics of labeled histone mRNA entry into polysomes was studied in nuclease-treated reticulocyte lysates. Added mRNA rapidly bound 1 or 2 ribosomes. However, the formation of full size polysomes required at least 16 min. The amount of mRNA bound to ribosomes reached a maximum (73%) within 2 min after mRNA addition and then declined slowly for the remainder of the experiment. Two initiation inhibitors, aurintricarboxylic acid and 7-methylguanosine 5'-triphosphate, were found to affect polysome size and the fraction of mRNA in polysomes in an opposite manner. These results suggest that initiation and reinitiation events may be intrinsically different. The relatively long time period required for the formation of large polysomes can be explained by large polysomes having higher initiation and/or reinitiation rates or slower elongation rates. These possibilities are not mutually exclusive. The results suggest that there exist several levels of control which can regulate polysome size and the fraction of mRNA in polysomes.

Animals↗
PubMed1983

Molecular cloning of mRNA from 3T3 adipocytes. Regulation of mRNA content for glycerophosphate dehydrogenase and other differentiation-dependent proteins during adipocyte development.

We have constructed a recombinant bacterial library containing cDNA prepared from mRNA of adipose 3T3 cells. We have screened this library by several methods and isolated colonies containing sequences complementary to mRNAs for glycerophosphate dehydrogenase, two other major differentiation-dependent proteins of Mr = 28,000 and 13,000, and actin. These recombinants were identified by hybrid selection of total adipocyte mRNA, translation in vitro and subsequent immunoprecipitation, and two-dimensional electrophoresis of translated proteins. Three of the four cloned cDNAs hybridized to single adipocyte mRNA species of size close to that expected from the size of the polypeptide it specifies, but the mRNA for glycerophosphate dehydrogenase contained 3,550 bases, far larger than necessary to code for its polypeptide (Mr = 34,000). The increase in amount of mRNA during differentiation was 150-fold for the protein of Mr = 13,000 and considerably greater for glycerophosphate dehydrogenase and the protein of Mr = 28,000. The time course of mRNA accumulation was different for each of these mRNAs, indicating they do not respond synchronously during differentiation.

Adipose Tissue↗
PubMed1995

c-fos mRNA instability determinants present within both the coding and the 3' non coding region link the degradation of this mRNA to its translation.

The instability of oncogenic mRNA such as c-fos mRNA is controlled in cis by sequences present in both the coding and the 3' untranslated regions (3'UTR). The latter contains AU-rich elements (ARE) which, depending on the cellular context, mediate either their rapid degradation or inhibit their translation. These observations, along with the known increase of the life spans of many unstable mRNA promoted by inhibitors of protein synthesis, raise the possibility that both processes are linked. To investigate further the putative involvement of translation in both coding region and ARE-mediated rapid decay of c-fos mRNA, we designed an expression vector based on the use of the ferritin mRNA iron regulatory element (IRE). The latter structure links translation to intracellular iron concentration when inserted at the proper location within the 5'UTR. Rapid degradation of a beta-globin/c-fos 3'UTR construct was prevented by Desferrioxamine, an iron chelator, and facilitated by ferric ammonium citrate or hemin, while stability of other mRNAs not containing the IRE or the ARE were unchanged. The same conclusion was reached when the stability of a c-fos mRNA devoid of ARE was assessed in function of iron availability.

Adenine↗
PubMed1993

An 18-bp element in the 5' untranslated region of the Drosophila beta 2 tubulin mRNA regulates the mRNA level during postmeiotic stages of spermatogenesis.

During Drosophila spermatogenesis transcriptional activity is mainly restricted to premeiotic stages. Translation during sperm morphogenesis, however, proceeds for several days, requiring a high stability for mRNAs translated postmeiotically. We studied expression of the Drosophila beta 2 tubulin gene, which is expressed solely in the male germ line from the primary spermatocyte stage onwards. Cis-acting elements involved in the regulation of mRNA levels were investigated in transgenic fly strains. In adult testes, mRNA amounts from beta 2-lacZ fusion genes in the presence of an 18-bp AT-rich element, termed beta 2DE1, are elevated about threefold. The element is present at about the same position in the 5' untranslated regions of the beta 2 tubulin genes of the distantly related species Drosophila melanogaster and Drosophila hydei. Changing the position of the element on the mRNA reduces the stabilizing effect, while inversion of the beta 2DE1 abolishes its function. The element also acts in a combination with the beta 1 tubulin transcription start site, and the beta 2UE1, which is required to achieve tissue-specific expression. In all experiments done, the comparison of premeiotic with postmeiotic stages strongly implies that this element is involved in regulating mRNA stability. This mRNA stabilizing element acts in a position-independent manner and also on a heterologous mRNA, showing its autonomous functional activity.

Animals↗
PubMed1996

[Expression of somatostatin mRNA and coexistence of SOM mRNA and 5-HT in nucleus raphe dorsalis following noxious stimulation and electroacupuncture analgesia].

Using the methods of in situ hybridization (ISHH), ISHH combined with immunocytochemistry ABC and image processing, somatostatin (SOM) mRNA level and coexistence of SOM mRNA and 5-HT in the nucleus raphe dorsalis (RD) of rat were studied in the normal group, noxious stimulation group, simple electroacupuncture group and electroacupuncture analgesia group (EA). A number of oval and fusiform cells with a weak to moderate hybridization signals were mostly distributed in dorsomedial subnucleus of RD and concentrated on rostral part. An increase of SOM mRNA positive cells could be seen in noxious stimulation group and simple electroacupuncture group comparing with the normal group. In EA group the number of SOM mRNA positive cells increased more than any other groups. A few SOM mRNA and 5-HT doubled cells could be seen in RD in the normal group. Most of the doubled cells were distributed in ventromedial, lateral subnuclei of RD. Comparing with the normal group, doubled cells increased in noxious and simple electroacupuncture group and increased significantly in EA group. The results suggest: (1) SOM takes part in modulation of pain. (2) SOM plays a role in EA. (3) Coexistence of SOM mRNA and 5-HT-in RD suggests that SOM and 5 HT interact synergically in EA.

Acupuncture Analgesia↗
PubMed1998

Aging alters the rhythmic expression of vasoactive intestinal polypeptide mRNA but not arginine vasopressin mRNA in the suprachiasmatic nuclei of female rats.

Our laboratory has shown that the ability of the suprachiasmatic nuclei (SCN) to regulate a number of rhythmic processes may be compromised by the time females reach middle age. Therefore, we examined the effects of aging on the rhythmic expression of two neuropeptides synthesized in the SCN, vasoactive intestinal polypeptide (VIP) and arginine vasopressin (AVP), using in situ hybridization. Because both VIP and AVP are outputs of the SCN, we hypothesized that age-related changes in rhythmicity are associated with alterations in the patterns of expression of these peptides. We found that VIP mRNA levels exhibited a 24 hr rhythm in young females, but by the time animals were middle-aged, this rhythm was gone. The attenuation of rhythmicity was associated with a decline in the level of mRNA per cell and in the number of cells in the SCN producing detectable VIP mRNA. AVP mRNA also showed a robust 24 hr rhythm in young females. However, in contrast to VIP, the AVP rhythm was not altered in the aging animals. The amount of mRNA per cell and the number of cells expressing AVP mRNA also was not affected with age. Based on these results we conclude that (1) various components of the SCN are differentially affected by aging; and (2) age-related changes in various rhythms may be attributable to changes in the ability of the SCN to transmit timing information to target sites. This may explain why the deterioration of various rhythmic processes occurs at different rates and at different times during the aging process.

Aging↗
PubMed1998

Independence of estrogen-induced pituitary proliferation on local IGF-I mRNA and EGF mRNA expression. Modifying effects of tamoxifen and terguride.

Pituitary hyperplasia as well as proliferation of the endometrium are typical responses to estrogen administration in rodents. Both insulin-like growth factor-I (IGF-I) and epidermal growth factor (EGF) have been implicated as paracrine mediators and amplifiers of estrogen action in the rodent uterus. The auto/paracrine role of IGF-I, EGF, their receptors and IGF binding proteins in pituitary proliferation has not yet been solved. Here we have used a semi-quantitative reverse transcription polymerase chain reaction (RT PCR) assay to demonstrate the changes in IGF-I mRNA and EGF mRNA abundance in the proliferating male rat pituitary in response to estradiol benzoate (EB; 1 mg/kg b.w. twice weekly i.m. for 3 weeks) and modifying effect of drugs antagonizing the pituitary enlargement - antiestrogen tamoxifen (TAM, 5 mg/kg b.w. daily) and also the dopaminergic agonist terguride (TER, 0.66 mg/kg b.w. daily, routinely used for the treatment of prolactinomas). In three separate experiments, EB induced a 2.2-2.5 fold increase in pituitary weight. The abundance of IGF-I and EGF mRNAs in pituitaries of EB-treated animals did not differ from the controls in two experiments and in the third series with the most marked pituitary hyperplasia mRNAs of both growth factors were even significantly decreased. Antiestrogen TAM administered with EB partially blocked the EB-induced proliferation and significantly stimulated IGF-I mRNA (p=0.003) and EGF mRNA (p=0.023) expression, while EB or TAM alone did not stimulate mRNAs of the studied growth factors. Significant antiproliferative effect of dopaminergic agonist TER on EB-induced pituitary proliferation (p=0.006) was accompanied with decreased IGF-I mRNA (p=0.025), but not EGF mRNA abundance. Our results suggest that the estrogen-induced pituitary proliferation is independent of the local expression of IGF-I and EGF mRNAs.

Animals↗
PubMed1997

Molecular cloning, sequencing and expression of the mRNA encoding human Cdx1 and Cdx2 homeobox. Down-regulation of Cdx1 and Cdx2 mRNA expression during colorectal carcinogenesis.

Defining the molecular mechanisms involved in cancer formation and progression is still a major challenge in colorectal-cancer research. Our strategy was to characterize genes whose expression is altered during colorectal carcinogenesis. To this end, the phenotype of a colorectal tumour was previously established by partial sequencing of a large number of its transcripts and the genes of interest were selected by differential screening on high-density filters with mRNA of colorectal cancer and normal adjacent mucosa. Fifty-one clones were found over-expressed and 23 were underexpressed in the colorectal-cancer tissues of the 5 analyzed patients. Among the latter, clones 6G2 and 32D6 were found of particular interest, since they had significant homology with several homeodomain-containing genes. The highest degree of similarity was with the murine Cdx1 for 6G2, and with the murine Cdx2 and hamster Cdx3 for 32D6. Using a RT-PCR approach, complete sequence of both types of homeobox-containing cDNA was obtained. The amino-acid sequence of the human Cdx1 is 85% identical to the mouse protein, and human Cdx2 has 94% identity with the mouse Cdx2 and hamster Cdx3. Tissue-distribution analysis of Cdx1 and Cdx2 mRNA showed that both transcripts were specifically expressed in small intestine, in colon and rectum. Twelve tissue samples from colorectal adenocarcinomas and the corresponding normal mucosa were analyzed by Northern blot. Expression of the 2 types of mRNA was either reduced or absent in 10 of them. Several colon-cancer cell lines were also analyzed. Cdx2 mRNA was absent from LS174T cells and Cdx1 mRNA was absent in PF11, TC7 and SW480 cells; none was detected in HT29 cells. It was concluded that decrease in human Cdx1 and/or Cdx2 expression is associated with colorectal tumorigenesis.

Adenocarcinoma↗
PubMed2001

Effects of retinoids and thiazolidinediones on proliferation, insulin release, insulin mRNA, GLUT 2 transporter protein and mRNA of INS-1 cells.

Both 9-cis-retinoic acid (9cRA) and all-trans-retinoic acid (ATRA) are active metabolites of vitamin A (retinol). There exists an interaction between retinoid receptors and peroxisome proliferator-activated receptors (PPARgamma). To define their functions in an insulin secreting system the effects of ATRA, 9cRA and the PPARgamma agonist rosiglitazone on cell proliferation, insulin release and glucose transporter (GLUT) 2 of INS-1 cells were tested. Retinoic acid receptor (RAR-alpha and -gamma) and retinoid X receptor (RXR-alpha and -beta) proteins are present (immunoblots). Both 9cRA and ATRA inhibit INS-1 cell proliferation ([3H]-thymidine assay) in a concentration dependent manner. Both 9cRA and ATRA increased insulin release, but only ATRA ralsed the GLUT 2 mRNA in a bell-shaped concentration response curve after 48 h. The insulinotropic effect of one compound is not significantly superimposed by the other indicating that the same binding sites are used by 9cRA and ATRA. The acute and chronic effects of the PPARgamma agonist rosiglitazone on insulin release were additionally determined since glitazones act as transcription factors together with RXR agonists. At high concentrations (100 microM) rosiglitazone inhibited glucose (8.3 mM) stimulated insulin secretion (acute experiment over 60 min). Insulin secretion, however, was increased during a 24 h treatment at a concentration of 10 microM and again inhibited at 100 microM. Changes in preproinsulin mRNA expression were not observed. Rosiglitazone (100 microM) increased GLUT 2 mRNA paralleled by an increase of GLUT 2 protein, but only after 24 h of treatment. This data indicate that RAR and RXR mediate insulin release. The changes in GLUT 2 have no direct impact on insulin release; the inhibition seen at high concentrations of either compound is possibly the result of the observed inhibition of cell proliferation. Effects of rosiglitazone on preproinsulin mRNA and GLUT 2 (mRNA and protein) do not play a role in modulating insulin secretion. With the presence of an RXR receptor agonist the effect of rosiglitazone on insulin release becomes stimulatory. Thus the effects of RAR-, RXR agonists and rosiglitazone depend on their concentrations, the duration of their presence and are due to specific interactions.

Alitretinoin↗
PubMed1997

Enhanced expression of eotaxin and CCR3 mRNA and protein in atopic asthma. Association with airway hyperresponsiveness and predominant co-localization of eotaxin mRNA to bronchial epithelial and endothelial cells.

Eotaxin is a newly discovered C-C chemokine which preferentially attracts and activates eosinophil leukocytes by acting specifically on its receptor CCR3. The airway inflammation characteristic of asthma is believed to be, at least in part, the result of eosinophil-dependent tissue injury. This study was designed to determine whether there is increased expression of eotaxin and CCR3 in the bronchial mucosa of asthmatics and whether this is associated with disease severity. The major sources of eotaxin and CCR3 mRNA were determined by co-localization experiments. Bronchial mucosal biopsy samples were obtained from atopic asthmatics and normal non-atopic controls. Eotaxin and CCR3 mRNA were identified in tissue sections by in situ hybridization (ISH) using radiolabeled riboprobes and their protein product visualized by immunohistochemistry (IHC). Co-localization experiments were performed by double ISH/IHC. Eotaxin and CCR3 (mRNA and protein) were significantly elevated in atopic asthmatics compared with normal controls. In the asthmatics there was a highly significant inverse correlation between eotaxin mRNA+ cells and the histamine provocative concentration causing a 20% fall in FEV1 (PC20). Cytokeratin-positive epithelial cells and CD31+ endothelial cells were the major source of eotaxin mRNA whereas CCR3 co-localized predominantly to eosinophils. These data are consistent with the hypothesis that damage to the bronchial mucosa in asthma involves secretion of eotaxin by epithelial and endothelial cells resulting in eosinophil infiltration mediated via CCR3. Since selective (eotaxin) and non-selective C-C chemokines such as RANTES, MCP-3 and MCP-4 all stimulate eosinophils via CCR3, this receptor is potentially a prime therapeutic target in the spectrum of diseases involving eosinophil-mediated tissue damage.

Adult↗
PubMed2006

Maintenance of CCL5 mRNA stores by post-effector and memory CD8 T cells is dependent on transcription and is coupled to increased mRNA stability.

Immunological memory is associated with the display of improved effector functions by cells of the adaptive immune system. The storage of untranslated mRNA coding for the CCL5 chemokine by CD8 memory cells is a new process supporting the immediate display of an effector function. Here, we show that, after induction during the primary response, high CCL5 mRNA levels are specifically preserved in CD8 T cells. We have investigated the mechanisms involved in the long-term maintenance of CCL5 mRNA levels by memory CD8 T cells. We demonstrate that the CCL5 mRNA half-life is increased in memory CD8 T cells and that these cells constitutively transcribe ccl5 gene. By inhibiting ccl5 transcription using IL-4, we demonstrate the essential role of transcription in the maintenance of CCL5 mRNA stores. Finally, we show that these stores are spontaneously reconstituted when the inhibitory signal is removed, indicating that the transcription of ccl5 is a default feature of memory CD8 T cells imprinted in their genetic program.

3T3 Cells↗
PubMed1990

Cleavages in the 5' region of the ompA and bla mRNA control stability: studies with an E. coli mutant altering mRNA stability and a novel endoribonuclease.

We describe here the partial purification of a novel Escherichia coli endoribonuclease, RNase K. This protein catalyses site-specific cleavages in the 5' region of in vitro transcribed ompA and bla transcripts. Some of the resulting cleavage products are also found in cellular ompA mRNA, defining the in vivo activity of RNase K. The following evidence suggests that RNase K initiates mRNA degradation. First, RNase K cleavages are suppressed in the ams mutant, which has a generally prolonged mRNA half-life. Secondly, RNase K cleavage products seem to have very short half-lives in vivo, indicating that they are decay intermediates rather than processing products. Thirdly, the differences in in vivo half-life between the ompA and bla mRNAs are mimicked in in vitro decay reactions with purified RNase K. The relationship between RNase K and the ams locus might point to a more general role of RNase K in mRNA degradation. We discuss the influence of mRNA secondary structure on RNase K cleavage specificity.

Bacterial Outer Membrane Proteins↗
PubMed1992

Transforming growth factor-beta 1 (TGF-beta 1) up-regulation of collagen type II in primary cultures of rabbit articular chondrocytes (RAC) involves increased mRNA levels without affecting mRNA stability and procollagen processing.

The effect of transforming growth factor-beta 1 (TGF-beta 1) on collagen biosynthesis was investigated in confluent primary monolayer cultures of rabbit articular chondrocytes (RAC). Exposure to TGF-beta (0.1, 1, and 10 ng/ml) in serum-free medium caused a dose- and time-dependent stimulation of collagen biosynthesis associated with an increase of steady-state levels of procollagen type II mRNA. Elevation of the mRNA steady-state did not result from a stabilization of the transcript, as shown by measure of the mRNA half-life. Electrophoresis (SDS-PAGE) showed that TGF-beta stimulates the synthesis of most collagen isotypes, including type II, without qualitative change in their distribution. Moreover, pulse-chase experiments revealed that TGF-beta did not affect the processing rate of type II procollagen. TGF-beta slightly stimulated the production of prostaglandin E2 (PGE2), which could in turn exert an inhibition on collagen synthesis. However, addition of indomethacin to block prostaglandin synthesis did not further enhance the TGF-beta-induced stimulation of collagen production, suggesting that this mediator was not implicated in the effect. Moreover, TGF-beta increased steady-state levels of procollagen type II, I, and III mRNAs even in the presence of indomethacin. Despite these increased mRNA levels, only the production of type II collagen was significantly augmented, suggesting that type I procollagen mRNA was not fully translated. In addition, the TGF-beta-induced stimulation of collagen synthesis was observed whenever ascorbic acid is added or not in the culture medium. In conclusion, TGF-beta, which is present in great amount in bone and cartilage, can increase the collagen production of cultured RAC and might therefore play a role in the early events of cartilage repair, such as those observed in osteoarthritis.

Animals↗
PubMed1991

junD mRNA expression differs from c-jun and junB mRNA expression during male germinal cell differentiation.

The members of the jun family of protooncogenes (junB, c-jun, and junD) share a high degree of sequence homology and function as transcriptional regulators. Here we compare the pattern of junD mRNA expression during spermatogenesis to that of junB and c-jun (Alcivar et al.: J Biol Chem 265:20160-20165, 1990). junD transcripts are present at high levels in total RNA obtained from both prepuberal and adult intact testes, with the highest levels at stages containing predominantly premeiotic and postmeiotic germ cells. Analyses of cells isolated from testes of 8-day-old mice indicate that the level of the 1.8 kb junD mRNA is higher in type B spermatogonia than in type A spermatogonia. In testes of 17-day-old mice, the highest junD mRNA levels are detected in preleptotene spermatocytes compared to leptotene/zygotene and prepuberal pachytene spermatocytes. In cells from adult testes, the junD mRNA levels are higher in postmeiotic round spermatids and residual bodies/cytoplasts than in meiotic pachytene spermatocytes. An additional junD transcript of about 1.6 kb is detected in postmeiotic cells. Analyses of polysomal and nonpolysomal RNAs prepared from isolated testicular cells indicate that in early meiotic cell types the junD transcript is more efficiently loaded onto polysomes than in later cell types. In summary, the pattern of expression of junD differs from that of junB and c-jun during spermatogenesis most notably in that 1) junD mRNA levels do not increase following dissociation of testicular cells and 2) in contrast to the nearly undetectable levels of junB and c-jun mRNAs in adult postmeiotic testicular cells, high levels of junD mRNAs are seen.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1996

Comparison of four methods for isolating large mRNA: apolipoprotein B mRNA in bovine and rat livers.

The effectiveness of four methods for isolating large-size mRNA (14 kb) encoding for hepatic bovine apoprotein B (apo B) was compared. Total RNA of liver (positive controls) and lung (negative controls) samples taken in bovine and rat was extracted using the methods of Chirgwin et al., Chomczynski and Sacchi, Cathala et al., and RNAzol. The integrity of total RNA extracted by the four methods was demonstrated by electrophoresis on 1% agarose gel and staining with ethidium bromide. Yield of extraction was two- to four-fold higher for all samples with the methods of Chirgwin et al. and RNAzol than that with the other methods. By dot blot analysis, apo B mRNA in bovine and rat livers was revealed preferentially with Chirgwin et al. and RNAzol methods. By Northern blot analysis, a single band corresponding to apo B100 mRNA was shown in bovine and rat livers only with the method of Chirgwin et al., whereas a band corresponding to a medium-size mRNA (2.6 kb) encoding for rat phosphoenolpyruvate carboxykinase was revealed in rat liver samples with the four methods. These results showed the favorable role of guanidium isothiocyanate and guanidium hydrochloride used in the extraction and the purification of RNA, respectively, especially in the case of high-molecular-weight and/or low-represented RNA such as apo B mRNA in bovine liver.

Animals↗
PubMed1996

Nonisotopic quantitation of mRNA using a novel RNase protection assay: measurement of erbB-2 mRNA in tumor cell lines.

We have developed a nonisotopic RNase protection assay using RNA probes that are dual-labeled with biotin and fluorescein for detection. This system utilizes capture of the protected RNA probe hybrids to streptavidin-coated membranes attached to plastic dipsticks, complexing of anti-fluorescein-urease conjugate with the labeled RNA probe, and quantitative detection of the membrane-bound complex by a potentiometric silicon sensor. The dual-label RNase protection (RP) assay was capable of measuring beta-actin mRNA in cellular RNA samples at the 27- to 45-amol level (10-17 pg) with high precision (%CV < 7). We have used this method to quantitate the levels of erbB-2 mRNA in the human tumor cell lines SKBR-3, SKOV-3, and MCF-7. The levels of erbB-2 mRNA in these cells were 105, 190, and 0.9 amol per microgram of cellular RNA, respectively. The dual-label RP method should be useful for measuring the mRNA expression for other erbB-2 homologs such as erbB-3 and erbB-4 in tumor cells and tissues and can be a generally useful mRNA quantitative method for laboratories wishing to minimize radioisotope use.

Actins↗
PubMed1994

Overexpression of activin-beta A subunit mRNA is associated with decreased activin type II receptor mRNA levels in the testes of alpha-inhibin deficient mice.

Activins and inhibins are polypeptides of the transforming growth factor-beta family that participate in differentiation and growth of diverse cell types, and are involved in endocrine/paracrine regulation of the hypothalamo-pituitary-gonadal axis. Mice with alpha-inhibin subunit gene deletion develop large testicular tumors. In these animals, a 200-fold increase in testicular expression of activin beta A subunit mRNA was detected using S1-nuclease protection analysis. Northern blot analysis demonstrated that a predominant mRNA form of approximately 6.5 kb and a second minor form of 4.5 kb were overexpressed in the testes of the alpha-inhibin deficient animals. Testicular expression of the type II activin receptor was decreased 3-fold in these mice. In contrast, hypothalamic beta A and type II activin receptor mRNA levels remained unaltered. alpha-Inhibin may play a role to suppress the expression of beta A mRNA in the mammalian testes. These results demonstrate that increased expression of activin is accompanied by a tissue specific reduction in the expression of its own receptor mRNA in vivo.

Activin Receptors↗
PubMed2000

IgG-coated erythrocytes augment LPS-stimulated TNF-alpha secretion, TNF-alpha mRNA levels, and TNF-alpha mRNA stability in macrophages.

Previous studies have shown that IgG-coated erythrocytes (EIgG) augment the LPS-stimulated increase in serum TNF-alpha levels in animals and the LPS-stimulated secretion of TNF-alpha by isolated macrophages. The present study evaluated the mechanism for the effect of EIgG on LPS-stimulated TNF-alpha secretion in the murine macrophage cell line, RAW 264.7. Incubation of the macrophages with EIgG or IgG-coated glass beads caused a dose-dependent augmentation of LPS-stimulated TNF-alpha secretion. The addition of EIgG increased the rate of LPS-stimulated TNF-alpha protein secretion between 2 and 4 hr after LPS. Accordingly, EIgG increased the levels of TNF-alpha mRNA at 2 and 3 hr after LPS. The increase in the LPS-stimulated TNF-alpha mRNA levels caused by EIgG was associated with an increase in TNF-alpha mRNA stability. Thus, the augmentation of LPS-stimulated TNF-alpha secretion by EIgG was associated with an increase in TNF-alpha mRNA levels which at least partly resulted from an increase in the stability of TNF-alpha mRNA.

Animals↗