Biomedical subjects

B D Clark

Publications and source records attributed to B D Clark.

At least 55 records · Page 3Linked to original sources

PubMed1992

Interleukin-1 down-regulates gene and surface expression of interleukin-1 receptor type I by destabilizing its mRNA whereas interleukin-2 increases its expression.

The interleukin-1 receptor type I (IL-1RtI) plays an important role in the biological effects of IL-1, but regulation of its surface and gene expression remains unknown. We found that occupancy of 2-15% of the IL-1 surface receptor results in dramatic down-regulation of IL-1RtI both at the mRNA and cell surface level in murine D10S cells, a subline of T-helper type 2 cells. At these low occupancy levels (3 x 10(-12) to 3 x 10(-13) M), the reduction in IL-1RtI surface expression appears at 24 hr and continues to 48 and 72 hr. At the mRNA level, low occupancy of the IL-1R results in decreased IL-1RtI mRNA stability; steady state half-life of the IL-1RtI mRNA is reduced from 6 to 1 hr after exposure to 3 x 10(-12) M IL-1. This down-regulation of IL-1RtI by IL-1 is blocked by cycloheximide, suggesting de novo protein synthesis is necessary for decreased RNA stability. Low concentrations of human IL-1 beta, murine and rabbit IL-1 alpha or beta similarly down-regulated IL-1RtI, whereas low concentrations of human IL-1 alpha failed to reduce the receptor surface expression, despite inducing a full proliferative response. We also observed that the effect of IL-1 on this down-regulation was not through protein kinase C (PKC), since PMA rapidly increased IL-1RtI mRNA levels within 30 min and persisted for 24 hr. IL-2 up-regulated IL-1RtI in D10S cells at both mRNA and protein levels. These results demonstrate that low occupancy of IL-1 receptors induces down-regulation of IL-1RtI surface as well as mRNA expression. The regulation of IL-1RtI gene expression may be one of the mechanisms by which IL-1-mediated events are controlled.

Animals↗

PubMed1991

Enhancement of growth of virulent strains of Escherichia coli by interleukin-1.

Interleukin-1 (IL-1) is a polypeptide cytokine that mediates many physiological responses to infection and inflammation and is a growth factor for certain mammalian cells. Virulent and avirulent clinical isolates of Escherichia coli were grown in culture media in the presence of human IL-1. IL-1 beta, but not tumor necrosis factor or IL-4, enhanced the growth of virulent, but not avirulent, E. coli. This enhancement was blocked by the IL-1 receptor antagonist (IL-1ra). Radiolabeled IL-1 bound to virulent but not avirulent E. coli in a specific and saturable fashion; IL-1ra inhibited this binding. Thus, human IL-1 may recognize a functional IL-1-like receptor structure on virulent E. coli and may be a virulence factor for bacterial pathogenicity.

Culture Media↗

PubMed1991

Production of interleukin-1 receptor antagonist and interleukin-1 beta by peripheral blood mononuclear cells is differentially regulated.

We studied the relationship between the production of the 23-Kd interleukin-1 receptor antagonist (IL-1ra) and IL-1 beta in cultures of human peripheral blood mononuclear cells (PBMC) using a specific radioimmunoassay for IL-1ra that had a sensitivity of 166 +/- 11 pg/mL. PBMC cultured without human serum made little IL-1ra or IL-1 beta. In the presence of 1% AB serum, there was no increase in IL-1 beta (0.25 +/- 0.13 ng/mL) but IL-1ra production increased sevenfold to 3.4 +/- 0.5 ng/mL. IgG (2.5 to 100 micrograms/mL IgG) or granulocyte-macrophage colony-stimulating factor (GM-CSF) (1 to 100 ng/mL) had no significant effect on IL-1 beta production but increased IL-1ra production up to 18-fold (18.2 +/- 3.9 ng/mL). Using endotoxin as a stimulant, 82% +/- 2% of IL-1ra was secreted in comparison with 52% +/- 9% of IL-1 beta. Culture conditions of PBMC influenced the production of IL-1ra but not IL-1 beta. Rocking endotoxin-stimulated PBMC produced 75% less IL-1ra but the same amount of IL-1 beta when compared with PBMC cultured in stationary plastic tubes. Rocking IgG-or GM-CSF-stimulated PBMC also produced 75% to 80% less IL-1ra. GM-CSF or IL-1 beta at concentrations that elicited submaximal production of IL-1ra potentiated IgG-induced IL-1ra production. The production of IL-1ra and IL-1 beta are under differential regulation because serum, IgG, and GM-CSF were potent stimuli for the production of IL-1ra but not IL-1 beta, and the prevention of cell-cell contact of PBMC reduced IL-1ra but not IL-1 beta production.

Adult↗

PubMed1991

Interleukin-1 receptor antagonist competitively inhibits the binding of interleukin-1 to the type II interleukin-1 receptor.

The interleukin-1 receptor antagonist (IL-1ra) inhibits the binding of interleukin-1 (IL-1) to T-cell lines possessing the type I IL-1 receptor; evidence has been published (Carter, D. B., Deibel, M. R. J., Dunn, C. J., Tomich, C. S., Laborde, A. L., Slightom, J. L., Berger, A. E., Bienkowski, M. J., Sun, F. F., McEwan, R. N., Harris, P. K. W., Yem, A. W., Waszak, G. A., Chosay, J. G., Sieu, L. C., Hardee, M. M., Zurcher-Neely, H. A., Reardon, I. M., Heinrickson, R. L., Truesdell, S. E., Shelly, J. A., Eessalu, T. E., Taylor, B. M., and Tracey, D. E. (1990) Nature 344, 633-638; Hannum, C. H., Wilcox, C. J., Arend, W. P., Joslin, F. G., Dripps, D. J., Heimdal, P. L., Armes, L. G., Sommer, A., Eisenberg, S. P., and Thompson, R. C. (1990) Nature 343, 336-340) that IL-Ira does not bind to the type II IL-1 receptor (IL-1RtII). In this study we examined the ability of human recombinant IL-1ra to block the binding of IL-1 to the IL-1RtII on human polymorphonuclear leukocytes (PMN) and Raji human B-lymphoma cells. The binding of 125I-IL-1 beta to PMN was competively inhibited by IL-1ra. IL-1 beta was more potent in inhibiting the binding of 125I-IL-1 beta than IL-1ra. Incubating PMN with 125I-IL-1ra in the presence of increasing concentrations of IL-1 beta or IL-1ra showed that IL-1 beta was an approximately 40-fold more potent inhibitor of binding of 125I-IL-1ra than unlabeled IL-1ra. The IL-1ra was approximately 500-fold less potent in inhibiting the binding of 125I-IL-1 alpha than IL-1 alpha. IL-1ra was also able to competitively inhibit binding of 125I-IL-1 beta to Raji cells. PMN or Raji cells were also incubated with 125I-IL-1 in the absence or presence of IL-1 or IL-1ra. After cross-linking of IL-1 to cells followed by specific immunoprecipitation, sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed a band at 85 kDa corresponding to the 68-kDa IL-1RtII. However, in the presence of an excess of either unlabeled IL-1 or IL-1ra, the 85-kDa IL-1.IL-1RtII complex was not present. These findings demonstrate that the IL-1ra recognizes and blocks IL-1 binding to the IL-1RtII.

Binding, Competitive↗

PubMed1991

Detection of interleukin 1 alpha and 1 beta in rabbit tissues during endotoxemia using sensitive radioimmunoassays.

Interleukin 1 (IL-1) is a primary mediator of a wide variety of immunologic and inflammatory responses, including reactions to microbial infections. To study this cytokine in an animal model, we have developed specific and sensitive radioimmunoassays for the quantitation of rabbit IL-1 alpha and IL-1 beta. The sensitivity (limit of detection at 95% confidence level) of our assay for IL-1 alpha and 1 beta was 20-40 and 40-80 pg/ml, respectively. Recovery of IL-1 from tissues ranged from 75 to 107%, with a mean of 95% for IL-1 alpha and 89% (range 19-98) for IL-1 beta. We employed these assays in in vivo and in vitro studies. In an in vivo model, we measured the amount of rabbit IL-1 alpha and 1 beta protein present in brain, kidney, liver, lung, muscle, and spleen at various times after the injection of endotoxin. IL-1 was found in all tissues studied but largely in the spleen; IL-1 levels were transient, reaching peak levels by 4 h after injection of endotoxin and rapidly decreasing to low levels by 24 h. In similar in vitro studies, IL-1 alpha levels reached peak elevation 6 h after addition of endotoxin, whereas IL-1 beta was maximal at 24 h. IL-1 alpha was detected in all tissues; IL-1 beta was observed primarily in lung, kidney, and spleen. These studies establish the presence of IL-1 in various tissues during endotoxemia.

Animals↗

PubMed1991

Motor-unit recruitment in the decerebrate cat: several unit properties are equally good predictors of order.

1. Recruitment order was studied in pairs of motor units of the medial gastrocnemius (MG) muscle of decerebrate cats with the use of dual microelectrode recording from intact ventral root filaments. Excitation was provided by stretch of MG, stretch of synergists [lateral gastrocnemius (LG), plantaris (PL), and soleus (SOL) muscles] or electrical stimulation of the caudal cutaneous sural (CCS) nerve. Motor units were characterized by axonal conduction velocity (CV), tetanic tension (Pmax), twitch contraction time (CT), and fatigue index (FI). 2. Consistent with the recruitment pattern described by others, most often in relation to either CV or Pmax, the first unit of a pair to be recruited by MG stretch was typically the one with the lower CV and Pmax, and the higher FI and CT. The proportion of pairs that agreed in rank order of each property and recruitment order was as follows: for CT, 94%; for CV, 87%; for Pmax, 84%; and for FI, 75%. With a single marginal exception (CT vs. FI), no motor-unit property proved to be significantly better than the others at predicting recruitment (G test; P greater than 0.05). 3. In all 11 tested pairs containing one slow (type S) and one fast (type F) unit, the S was more easily recruited by stretch. Type F units divided into groups with high (type FR), low (type FF), and intermediate (type FInt) values for FI were recruited in order from FR to FInt to FF in 8/11 pairs. Thus our findings were similar to earlier demonstrations that recruitment proceeds in order by type. 4. Stretch of MG synergists usually recruited units in the same order as MG stretch. In two S-S pairs, recruitment order was switched with synergist stretch. 5. Stimulation of the CCS nerve was generally excitatory to the MG units sampled. Most unit pairs were recruited by CCS stimulation in the same order as by MG stretch, but, for 6 of 39 pairs, CCS stimulation switched the order produced by stretch. Thus, whereas sural afferent input can preferentially excite some units over others as suggested by Kanda et al., that effect is not widespread or selective for unit type under these conditions. 6. Assuming that all MG motor units cooperate as a single functional pool in homonymous stretch reflexes, we support others in concluding that a motoneuron's recruitment threshold is not strictly determined by its size. However, our data do not distinguish other schemes that predict recruitment order more accurately than the size principle.(ABSTRACT TRUNCATED AT 400 WORDS)

Action Potentials↗

PubMed1991

HLA class I epitopes accounted for by single residues.

We examined the serological reaction of 50,000 HLA antisera for correlations with amino acid substitutions at 16 variable residues on the A locus and 8 variable residues on the B locus. We identified antisera that produced mutually exclusive allelic reactions corresponding to amino acid variants at the various residues. We assumed that by this procedure, epitopes that were determined by a single amino acid substitution could be identified. Among 55 variable residues on the A locus, antibodies that reacted in an allelic fashion were found for 16, or 32%. These serologically determined epitopes were on the alpha helix for 13 of the 16 residues, and 3 were in the beta sheet. Among 54 variable residues for the B locus, antibodies were found to 8 which reacted in a mutually exclusive pattern. Thus, 13% of the residues were serologically defined. Sixteen "monospecific" HLA specificities were determined by a single residue. Interestingly, many "multispecific" sera could be accounted for by reactions to a single amino-acid defined epitope. We assume that most of the other monospecific and multispecific specificities are determined by conformational epitopes, as described in an accompanying article (Ch. 33).

Alleles↗

PubMed1991

Association of high sensitization to the structure of HLA class I alleles.

Some HLA Class I alleles or groups of alleles can be explained by exclusive residues which may explain their structural uniqueness. However, many sera are directed to alleles whose structural uniqueness is explained only by a combination of nonexclusive residues. A program implementing a combinatorial search algorithm was developed to analyze serological data by associating the primary structure of specific alleles to reaction patterns of broad multispecific sera. The method determines the residue or residues whose associated alleles best describe the serum specificity. The raw data reaction patterns direct the search, which utilizes known primary sequence information of HLA Class I alleles. Three-hundred highly positive sera from parous females were analyzed; 74 were highly correlated to the A locus and 125 to the B locus. Some of the remaining sera were associated with multiple loci. Reorganization methods were applied to the data. Clusters of residues associated with certain alleles focus attention on specific locations on the HLA Class I molecule. These regions may be included in, or in close proximity to, the serological determinant. Serological descriptions of certain broad specific sera have been termed "public epitopes." Specificities associated with distinct conserved regions of the Class I molecule offer a molecular basis for many public epitopes. Similar reaction patterns to 3 serologically allelic regions were found in pregnancy allosera, monoclonal antibodies, and transplant recipient sera. The correlated areas were: positions 166 and 167 of the alpha 2 domain alpha helix on the A locus, positions 79-83 of the alpha 1 domain alpha helix on the A locus, and positions 80-83 on the A and B loci.

Alleles↗

PubMed1991

Interleukin-1 gene expression in rabbit vascular tissue in vivo.

Cultured human vascular endothelial and smooth muscle cells express interleukin-1 (IL-1) genes when exposed to bacterial lipopolysaccharides (LPS) and a variety of inflammatory mediators. Local production of IL-1 may contribute to the pathogenesis of various vascular diseases. Therefore the ability of intact vascular tissue to accumulate IL-1 mRNA and synthesize de novo biologically active IL-1 protein was examined. Escherichia coli LPS (10 micrograms/kg) was administered intravenously to adult rabbits and total RNA was isolated from aortic tissue at various times after LPS injection. In saline-injected rabbits, RNA extracted from the thoracic aorta contained little or no IL-1 message detected by Northern analysis using IL-1 alpha and beta cDNA probes cloned from an LPS-stimulated rabbit splenic macrophage library. Lipopolysaccharide treatment promptly induced transient accumulation of mRNA for IL-1 alpha and IL-1 beta within the aorta (maximal 1-hour after injection). Short-term organoid cultures of rabbit aorta exposed to LPS in vitro synthesized immunoprecipitable IL-1 alpha protein. Extracts of aortic tissue excised 1.5 to 3.0 hours after intravenous LPS administration contained immunoreactive and biologically active IL-1 alpha. Anti-rabbit IL-1 alpha antibody neutralized the biologic activity (more than 90%). Microscopic and immunohistochemical studies did not disclose adherent or infiltrating macrophages in rabbit aorta at the time of maximal IL-1 mRNA accumulation after LPS administration (1.5 hours), indicating that intrinsic vascular wall cells rather than mononuclear phagocytes probably account for the IL-1 activity induced by LPS. In addition, aortic tissue from rabbits fed an atherogenic diet showed an enhanced ability to accumulate IL-1 alpha and beta mRNA and produce immunodetectable protein in response to LPS administration. These studies demonstrate inducible IL-1 gene expression in rabbit vascular tissue in vivo and support a local role for this cytokine in vascular pathophysiology.

Animals↗

PubMed1990

Dissociation between interleukin-1 beta mRNA and protein synthesis in human peripheral blood mononuclear cells.

Human peripheral blood mononuclear cells analyzed immediately after isolation did not express detectable mRNA for interleukin-1 beta (IL-1 beta). In the strict absence of lipopolysaccharide (LPS), incubation in glass or plastic resulted in expression of IL-1 beta mRNA without detectable IL-1 beta synthesis, even when cells were incubated for 20 h. The accumulation of IL-1 beta mRNA was most likely due to adherence since rotating the containers reduced the amount of mRNA. However, the cells were "primed" by 3 h adherence since subsequent stimulation with LPS resulted in more IL-1 beta (214%) 4 h after stimulation compared to freshly obtained, LPS-stimulated cells. Ratios of IL-1 beta mRNA induced by LPS versus adherence were 7.8, 36, and 20 at 4, 12, and 24 h, respectively; the corresponding ratios for IL-1 beta protein were 18, 160, and 180. Comparing Staphylococcus epidermidis versus LPS, the ratios of IL-1 beta mRNA were 3.2, 0.5, and 1.2 at 4, 8, and 24 h, respectively, however, the corresponding ratios for IL-1 beta protein were 65, 22, and 10. The differences in transcription versus translation in these studies are unlikely due to changes in stability of mRNA since the half-life of adherence-induced IL-1 beta mRNA was 2.5 versus 4 h for LPS-induced mRNA. There was also no evidence of superinduction of mRNA in peripheral blood mononuclear cells stimulated with LPS, whereas, tumor necrosis factor alpha mRNA was elevated in the presence of cycloheximide. Using different methods of cell surface stimulation, our results demonstrate that synthesis of IL-1 beta is regulated by at least two separate mechanisms, one at the level of transcriptional activation and the other one involving translational efficiency.

Blood Proteins↗

PubMed1990

Immunoreactive interleukin-1 beta localization in the rat forebrain.

To determine whether the cytokine, interleukin-1 beta, is present in the rat brain as has been reported in human brain, immunocytochemical studies were performed using an antiserum that recognizes recombinant, rat IL-1 beta. Immunoreaction product was present in the periventricular and medial hypothalamus, mossy fiber distribution in the hippocampus and olfactory tubercle. These studies demonstrate that IL-1 beta is part of a diffuse intrinsic neural system in the rat central nervous system, associated with regions involved with hypophysiotropic, autonomic, limbic and extrapyramidal functions.

Animals↗

PubMed1990

Modulation of interleukin 1 beta gene expression by the immediate early genes of human cytomegalovirus.

The immediate early (IE) genes of human cytomegalovirus (HCMV) can be expressed in monocytes/macrophages and are known to regulate other viral genes. The purpose of these studies was to determine if HCMV IE gene products also modulate expression of a monocyte/macrophage-derived gene, interleukin 1 (IL-1) beta. Steady-state cell-derived IL-1 beta mRNA was increased in lipopolysaccharide (LPS)-stimulated THP-1 cells when transfected with the HCMV IE1 + 2 genes, when compared to cells transfected with a control DNA. LPS-stimulated THP-1 cells also exhibited approximately 30-fold higher IL-1 CAT activity when cotransfected with IE1 + 2 than was observed for the same cells cotransfected with IL-1 CAT and a control plasmid containing the IE promoter alone. LPS increased IL-1 CAT activity in the absence of HCMV genes only twofold. IE1, by itself, increased IL-1 CAT activity in LPS-stimulated cells, whereas, IE2, by itself, caused no change in IL-1 CAT activity. These studies show that the IE1 gene of HCMV can regulate IL-1 beta gene expression. The observations further suggest that some of the inflammatory processes associated with HCMV infection may be due to an effect of HCMV IE genes on cell-derived genes, such as the IL-1 beta gene.

Antigens, Viral↗

PubMed1990

Interleukin 1 (IL-1) gene expression, synthesis, and effect of specific IL-1 receptor blockade in rabbit immune complex colitis.

Interleukin 1 (IL-1) may be a key mediator of inflammation and tissue damage in inflammatory bowel disease (IBD). In rabbits with immune complex-induced colitis, IL-1 alpha and beta mRNA levels were detectable at 4 h, peaked at 12 but were absent at 96 h after the induction of colitis. Colonic IL-1 tissue levels were measured by specific radioimmunoassays. IL-1 alpha was significantly elevated at 4 h (9.4 +/- 1.5 ng/g colon), progressively increased at 48 h (31 +/- 5.8 ng/g) and then decreased by 96 h (11.5 +/- 3.4 ng/g). IL-1 beta levels were 2.0 +/- 0.5 ng/g colon at 4 h, 5.0 +/- 1.6 ng/g at 48 h and undetectable by 96 h. By comparison, colonic levels of PGE2 and LTB4 were unchanged during the first 12 h and did not become elevated until 24 h. IL-1 alpha levels were highly correlated with inflammation (r = 0.885, P less than 0.0001), edema (r = 0.789, P less than 0.0001) and necrosis (r = 0.752, P less than 0.0005). Treatment with a specific IL-1 receptor antagonist (IL-1 ra) before and during the first 33 h after the administration of immune complexes markedly reduced inflammatory cell infiltration index (from 3.2 +/- 0.4 to 1.4 +/- 0.3, P less than 0.02), edema (from 2.2 +/- 0.4 to 0.6 +/- 0.3, P less than 0.01) and necrosis (from 43 +/- 10% to 6.6 +/- 3.2%, P less than 0.03) compared to vehicle-matched colitis animals. These studies demonstrate that (a) IL-1 gene expression and synthesis occur early in the course of immune complex-induced colitis; (b) are significantly elevated for 12 h before the appearance of PGE2 and LTB4; (c) tissue levels of IL-1 correlate with the degree of tissue inflammation and; (d) specific blockade of IL-1 receptors reduces the inflammatory responses associated with experimental colitis.

Antigen-Antibody Complex↗

PubMed1990

Intrinsic pituitary interleukin-1 beta is induced by bacterial lipopolysaccharide.

Using a specific antiserum recognizing recombinant rat interleukin-1 beta (IL-1 beta), immunoreactive material was localized to cytoplasmic granules in anterior pituitary endocrine cells and colocalized with TSH in thyrotropes. Authenticity was established by Northern blot hybridization using a specific rat IL-1 beta cRNA probe, revealing a 1.8-kilobase mRNA identical to that in the spleen. The marked increase in anterior pituitary IL-1 beta message after the administration of bacterial lipopolysaccharide, raises the possibility that IL-1 beta may be involved in paracrine or autocrine regulation of pituitary function during infectious challenge.

Animals↗

PubMed1990

HLA epitopes detected by serology.

There is evidence that many of the so-called multispecific cytotoxic antisera react to the amino acid-defined epitopes of the HLA-A and B loci molecules. This evidence is based on the finding that a group of sera produced allelic reactions which corresponded to amino acid substitutions. Antisera for 29 amino acid variants were identified at 67 variable residues on the A locus and for 17 of the 51 variable sites on the B locus. The results were validated by showing that the reactions produced by testing cells from 111 individuals on an epitope typing tray fit the Hardy Weinberg equilibrium. This means that the alleles defined by the antisera behaved as alleles in the random population. Tests of homozygous cells on the epitope typing trays produced completely mutually exclusive reaction patterns as expected on the basis of the amino acid substitutions. Two examples are cited in which immunization across a single HLA "antigen" difference existed between donor and recipient, resulting in a "multispecific" antiserum. The specificities contained in the serum exactly fit an amino acid difference between the donor and recipient.

Alleles↗

PubMed1990

HLA class II epitope detection by serology.

The amino acid sequence for the Class II specificities on 70 homozygous cell lines was compared with the cytotoxic reactions produced by 13,000 allogenic antisera against these lines. Many of the cytotoxic reaction patterns correlated completely with unique amino acids at a given residue. Therefore, we inferred that the antibodies were reacting directly with these amino acid-defined residues. Among 131 variable DRB1 residues, several well-correlated antisera were found to 64 (49%). Among 94 DQB1 residues, antibodies were found to 59 (63%). The location of these serologically defined epitopes on the molecule is shown in the table. We conclude that much of the serologic reactions of Class II can be explained by reactivity to the amino acid-defined epitopes. It can be shown that many of the sera that were previously considered multispecific are directed against these epitopes. These serologically defined epitopes are clearly immunogenic and are probably important in transplant matching.

Amino Acid Sequence↗

PubMed1989

Cytomegalovirus infection stimulates expression of monocyte-associated mediator genes.

Monocytes and tissue macrophages play important roles in host defense against virus infections and, in the case of human cytomegalovirus (HCMV) and HIV, may also be the reservoir for latent disease. Because these cells can also rapidly respond to most infections by secretion of inflammatory mediators, we were interested in determining if HCMV infection could have a direct activating effect on macrophage cytokine production. To do this, we primarily investigated the influence of HCMV infection on IL-1 beta-mRNA expression in peripheral blood monocytes and the promyelocytic cell line, ML-3 as well as the inflammatory response genes TNF-alpha, MAD-9, MAD-6, and MAD-2 in the promyelocytic ML-3 cell line. Exposure of ML-3 cells to the virus prior to induction of differentiation had little influence on mediator gene expression. However, induction of the macrophage phenotype by pretreatment of ML-3 cells with the phorbol ester, PMA, followed by HCMV challenge, resulted in a greatly extended period of expression of IL-1 beta, TNF-alpha, MAD-9, and CSF-1 but not MAD-6 and MAD-2. Constitutively expressed genes such as lysozyme and actin were not similarly modulated. Both RNA dot-blot and in situ hybridization studies demonstrated that infection of human peripheral blood monocytes with HCMV leads to sustained expression of IL-1 beta mRNA for up to 96 h, which contrasted markedly with mock-infected or LPS-stimulated monocytes. Flow cytometric analysis of the intracellular levels of IL-1 beta protein in ML-3 cells indicated that not only was there more protein produced in infected cells, but that the majority of the cells had responded. Enhanced levels of the intracellular form of IL-1 beta in monocytes was confirmed by Western blot analysis. Cotransfection experiments were performed using IL-1 beta-CAT chimeric plasmids together with plasmids encoding HCMV-immediate-early gene region products. Transactivation of the IL-1 beta gene by region 2 of the immediate-early gene was observed in ML-3 cells that had been induced to differentiate prior to transfection. No stimulation of IL-1 beta promoter activity was observed in ML-3 cells that were undifferentiated prior to transfection. In summary, HCMV infection, although not leading to productive infection, nonetheless may contribute to the pathology of the infection through enhancement of monocyte inflammatory mediator gene expression with subsequent stimulation of protein synthesis.

Cell Differentiation↗

PubMed1989

Rabbit IL-1. Cloning, expression, biologic properties, and transcription during endotoxemia.

The cloning, sequencing, expression, and biologic activities of rabbit IL-1 alpha and beta are described. A cDNA library was constructed in lambda gt10 by using polyadenylated RNA extracted from rabbit adherent splenic macrophages 4 h after stimulation with endotoxin. By using the cDNA for human IL-1 beta and IL-1 alpha as hybridization probes, cDNA for both forms of rabbit IL-1 were isolated. The cDNA for rabbit IL-1 beta encodes a precursor polypeptide of 268 amino acids with an overall homology to human IL-1 beta of 74% (81% in the mature region coding for a 17.5 kDa carboxyl-terminal protein). The similarity between the two rabbit IL-1 forms is 31% for the entire molecule and 34% for the mature protein. The mature polypeptides of both forms were expressed in Escherichia coli. The recombinant proteins were purified to homogeneity and tested in a variety of biologic assays. Both forms produced typical endogenous pyrogen fevers in rabbits and augmented murine thymocyte and Th cell proliferation. Rabbit IL-1 alpha and beta were more pyrogenic in rabbits than human rIL-1 beta, whereas human rIL-1 alpha and beta were slightly more potent lymphocyte-activating factors. The recombinant rabbit proteins induced PGE and IL-1 production from human PBMC in vitro. A RIA for human IL-1 alpha did not recognize rabbit IL-1 alpha or beta, but rabbit IL-1 beta cross-reacted (as much as 30%) in a RIA for human IL-1 beta. Rabbits were injected with endotoxin and mRNA for both forms of IL-1 were observed primarily in the spleen and liver. The mRNA reached maximal levels after 60 min, then declined rapidly over the next 3 h, but were still present after 24 h. Liver tissue removed 4 h after endotoxin infusion produced lymphocyte-activating factors which were neutralized by more than 90% with a combination of goat anti-rabbit IL-1 alpha and anti-IL-1 beta.

Amino Acid Sequence↗