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S S Macintyre

Publications and source records attributed to S S Macintyre.

12 recordsLinked to original sources
PubMed1995

Kinetic modeling and mathematical analysis indicate that acute phase gene expression in Hep 3B cells is regulated by both transcriptional and posttranscriptional mechanisms.

To evaluate the possible role of posttranscriptional mechanisms in the acute phase response, we determined the kinetics of transcription (by nuclear run-on assay) and mRNA accumulation of five human acute phase genes in Hep 3B cells incubated with conditioned medium from LPS-stimulated monocytes. Increase in mRNA accumulation was comparable to increase in transcription rate for fibrinogen-alpha and alpha-1 protease inhibitor, suggesting largely transcriptional regulation. In contrast, mRNA accumulation was about 10-20-fold greater than transcriptional increase for serum amyloid A, C3, and factor B, suggesting participation of posttranscriptional mechanisms. Since finding a disparity between the magnitudes of increase in mRNA and transcription does not definitively establish involvement of posttranscriptional mechanisms, we subjected our data to modeling studies and dynamic mathematical analysis to evaluate this possibility more rigorously. In modeling studies, accumulation curves resembling those observed for these three mRNAs could be generated from the nuclear run-on results only if posttranscriptional regulation was assumed. Dynamic mathematical analysis of relative transcription rates and relative mRNA abundance also strongly supported participation of posttranscriptional mechanisms. These observations suggest that posttranscriptional regulation plays a substantial role in induction of some, but not all acute phase proteins.

Acute-Phase Reaction↗
PubMed1992

Regulated export of a secretory protein from the ER of the hepatocyte: a specific binding site retaining C-reactive protein within the ER is downregulated during the acute phase response.

The half-time for secretion of the plasma protein C-reactive protein (CRP) by the hepatocyte decreases markedly in association with its increased synthesis during the acute phase response to tissue injury (Macintyre, S., D. Samols, and I. Kushner. 1985. J. Biol. Chem. 260:4169-4173). In studies in which subcellular fractions were prepared from cells incubated under pulse-chase conditions, CRP was found to be preferentially retained within the ER of normal hepatocytes, but secreted relatively efficiently in cells prepared from rabbits undergoing the acute phase response. On the basis of the detergent-dependency of specific binding of radiolabeled CRP, as well as EM visualization of biotinylated CRP identified with peroxidase-conjugated streptavidin, CRP was found to bind to the lumenal surface of permeabilized rough microsomes, while no binding was detected in Golgi fractions. As judged by both kinetic and equilibrium binding studies, rough microsomes from control rabbits were found to have two classes of specific binding sites for CRP; a high affinity site (Kd = 1 nM, Bmax = 1 pmol CRP/mg microsomal protein) as well as a much lower affinity (Kd = 140 nM) site. In contrast, only the lower affinity class was detected in microsomes isolated from rabbits undergoing the acute phase response. On nitrocellulose blots probed with radiolabeled CRP a 60-kD protein, distinct from BiP, was detected in extracts of rough microsomes isolated from control rabbits, but not in Golgi fractions or rough microsomes from stimulated animals. These findings correlate with previous observations of changes in secretion kinetics of CRP and are consistent with the hypothesis that the intracellular sorting of CRP could be rerouted by downregulation of a specific ER binding site during the acute phase response.

Acute-Phase Proteins↗
PubMed1990

Absence of a binding reactivity of human C-reactive protein for immunoglobulin or immune complexes.

Because C-reactive protein (CRP) has been identified as a component of circulating immune complexes from patients with inflammatory diseases, we sought to evaluate a potentially clinically important interaction of this acute-phase protein with immunoglobulin or experimentally-prepared immune complexes in vitro. Highly purified human CRP was incubated with a variety of immunoglobulin substrates, including monomeric immunoglobulin G1 (IgG1), a polyclonal IgG, heat-aggregated IgG, and human serum albumin/anti-serum albumin complexes. We were unable to detect a significant binding interaction of radioiodinated CRP with any of these materials, using either polyethylene glycol (PEG) precipitation or sucrose density gradient ultracentrifugation. In contrast, binding of radioiodinated human C1q to both aggregated immunoglobulin and immune complexes was readily detected by these techniques. Incubation of radiolabeled CRP with serum samples from 22 patients with active inflammatory diseases and high levels of circulating immune complexes disclosed no difference in the amount of PEG-precipitable CRP when compared with serum samples from healthy individuals. However, a radiolabeled commercial preparation of CRP did result in some PEG-precipitable radioactivity after incubation with aggregated IgG. These findings provide no support for a biologically important binding interaction of CRP with immunoglobulin or immune complexes, and they suggest that highly purified preparations of CRP should be used in functional studies of this acute-phase protein.

Acute-Phase Reaction↗
PubMed1989

Specific binding of human C-reactive protein to human monocytes in vitro.

The precise biologic function of C-reactive protein (CRP), a major acute phase protein in man, is unknown. The abilities of CRP to bind biologic substrates and to activate the C pathway, and its localization at sites of inflammation argue for an opsonic role for this protein. Such a role has been supported by recent reports of specific binding of CRP to neutrophils. Using highly purified radioiodinated human CRP, we have observed specific binding of this protein to human monocytes in vitro. The binding was reversible and rapid, with a t1/2 for the dissociation reaction of approximately 3 min. Binding was saturable at a CRP concentration of approximately 0.2 microM, with an estimated K from Scatchard analysis of 1.1 x 10(-7) M. Specific binding was calcium-dependent, with optimal binding occurring at calcium concentrations of more than 1.0 mM. No specific binding could be demonstrated to a non-adherent population of mononuclear cells (more than 80% lymphocytes). In other experiments, a 100-fold excess of human IgG failed to inhibit binding, although rabbit CRP produced competitive inhibition of binding which was quantitatively similar to human CRP. The binding was maximal at pH 7.4 and was sensitive to prior trypsin treatment of cells. These studies provide direct evidence for specific binding of soluble human CRP to human monocytes in vitro and thus provide further support for an important functional interaction of this acute phase protein with phagocytic cells in man.

Antigens, Differentiation↗
PubMed1988

Heterogeneous nature of the acute phase response. Differential regulation of human serum amyloid A, C-reactive protein, and other acute phase proteins by cytokines in Hep 3B cells.

Because a number of different cytokines have been reported to regulate the synthesis of human, murine, and rat acute phase proteins (APP), we studied the effect of cytokines on production of several major human APP in a single system, the human hepatoma cell line Hep 3B. Conditioned medium (CM) prepared from human blood monocytes activated with LPS in the presence of dexamethasone led to substantial induction of serum amyloid A (SAA) and C-reactive protein (CRP) synthesis whereas the defined cytokines IL-1 beta, TNF alpha, and medium from a human keratinocyte cell line (COLO-16), containing hepatocyte-stimulating factor activity, failed to induce these two major APP. Induction of SAA and CRP was accompanied by an increase in concentration of their specific mRNA. Size fractionation of CM from activated monocytes by fast protein liquid chromatography indicated that SAA- and CRP-inducing activity eluted as a single peak with a Mr of approximately 18 kDa. alpha 1-Antitrypsin, which also failed to respond to IL-1 beta or TNF alpha, was induced by both CM and medium from COLO-16 cells. The induction of AT by CM was accompanied by an increase in specific mRNA. Induction of ceruloplasmin and alpha 1-antichymotrypsin and decrease in the synthesis of albumin was achieved by both CM and IL-1 beta. Ceruloplasmin and albumin responded in a comparable fashion to both TNF alpha and medium from COLO-16 cells; the response of ACT to these cytokines was not evaluated. These results indicate that human SAA and CRP are induced in Hep 3B cells by products of activated monocytes but not by IL-1 beta, TNF-alpha, or some hepatocyte-stimulating factor preparations and that a group of heterogeneous mechanisms are involved in the induction of the various human APP.

Acute-Phase Proteins↗
PubMed1988

Secretion of rabbit C-reactive protein by transfected human cell lines is more rapid than by cultured rabbit hepatocytes.

C-reactive protein (CRP) is a major acute phase protein in humans and rabbits. Its synthesis by the liver varies over a 1000-fold range depending on the presence and severity of inflammatory stimuli. In previous studies of synthesis and secretion of rabbit CRP, we showed that secretion becomes more efficient over the course of the acute phase response as CRP synthesis rates increase (Macintyre, S.S., Kushner, I., and Samols, D. (1985) J. Biol. Chem. 260, 4169-4173). The current studies were undertaken to help distinguish between two alternative explanations for this finding: 1) that secretion efficiency may simply be a property of the rate of synthesis and intracellular concentration of CRP or 2) that secretion may be regulated by separate intracellular mechanisms. A fusion gene containing the mouse metallothionein I promoter linked to the protein coding region of the rabbit CRP gene was introduced into the human hepatoma cell line, NPLC, and the nonliver cell line, HeLa. In this system a graded response of the mouse metallothionein I promoter following exposure to increasing zinc concentrations results in increasing CRP synthesis. Unlike hepatocytes from rabbits undergoing the acute phase response, we found that rabbit CRP was secreted by these transfected cell lines with a very high degree of efficiency which was independent of the rate of CRP synthesis. This finding implies that normal rabbit hepatocytes retard the secretion of CRP and that this inhibition is diminished as the acute phase response progresses. It further indicates that the relationship between changes in synthetic rate and efficiency of secretion of rabbit CRP is not a causal one and that synthesis and secretion of CRP by rabbit hepatocytes are regulated by independent intracellular mechanisms during the acute phase response.

Animals↗
PubMed1988

C-reactive protein.

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Amino Acid Sequence↗
PubMed1985

Secretion of C-reactive protein becomes more efficient during the course of the acute phase response.

We studied the kinetics of synthesis and secretion of the acute phase plasma protein, C-reactive protein, in primary hepatocyte cultures prepared from rabbits manifesting differing degrees of the acute phase response to inflammatory stimulus. In cultures prepared from progressively more responsive animals, rate of C-reactive protein secretion increased to a much greater degree than did intracellular C-reactive protein content, resulting in a progressive decrease in the ratio of intracellular content to rate of secretion. This ratio, which represents the time required to secrete the amount of C-reactive protein contained within the intracellular pool, decreased from 18 h in cultures from unstimulated rabbits to 2.5 h in cells from highly responsive animals. In contrast, these ratios for albumin were short and fell within a narrow range (0.8-2.1 h). In pulse-chase labeling experiments, the time required for secretion of 50% of pulse-labeled C-reactive protein varied markedly, ranging from well over 6 h in cells from a minimally responsive animal to about 75 min in cells from a highly responsive rabbit. In contrast, the half-time for secretion of albumin was consistently about 45 min in the same cultures. Taken together, these findings indicate that the process by which C-reactive protein is secreted becomes more efficient during the course of the acute phase response. Recent studies have indicated that secretory proteins pass from the rough endoplasmic reticulum to Golgi at different and characteristic rates, possibly by a receptor-mediated process in which rate of transfer is determined by receptor affinity. We postulate that C-reactive protein secretion is regulated, during the course of the acute phase response, either by alterations in availability of specific receptors or by competition between different secretory proteins for a common receptor.

Animals↗
PubMed1983

In vivo studies of serum C-reactive protein turnover in rabbits.

We determined the plasma half-life of the acute phase protein C-reactive protein (CRP) both in normal rabbits and in rabbits that had received inflammatory stimuli. Rabbit CRP was purified from acute phase serum by Cx-polysaccharide affinity chromatography, radiolabeled, and rendered pyrogen-free. Six unstimulated rabbits were injected intravenously with (125)1-CRP prepared by the lactoperoxidase method and four were injected with CRP labeled by methylation using [(14)C]formaldehyde. Blood samples were obtained at 0.25 h and at intervals thereafter. Plasma half-life of CRP was calculated from the data generated during the first 12 h, by which time an average of 86% of labeled protein had disappeared from the blood stream. The mean half-life for CRP was 4.45+/-0.2 h, with no significant difference (0.40 < P < 0.45) between (125)1- and (14)C-labeled CRP. In six animals stimulated with either endotoxin or turpentine 24 h before injection of labeled CRP, a mean half-life of 5.8+/-0.6 h was found, not significantly different (0.30 < P < 0.35) from unstimulated rabbits. We equated fractional catabolic rate to fractional disappearance rate, since the rate constant for passage of CRP from vascular to extravascular compartment can be assumed to be relatively small compared to the observed fractional disappearance rate. Fractional catabolic rate was independent of serum CRP concentration; average fractional catabolic rate in all 16 animals was 14+/-0.8% h(-1) of the plasma pool. We were able to estimate rate of CRP synthesis, based on steady-state assumptions of pool sizes in those rabbits whose serum CRP levels did not change substantially during the period of study. Values as low as 6.7 mug/kg per h in the unstimulated animals and as high as 560 mug/kg per h in the stimulated animals were found.

Animals↗
PubMedDate not supplied

Familial occurrence of systemic sclerosis, rheumatoid arthritis and other immunological disorders: report of two kindreds with study of HLA antigens and review of the literature.

We report two Caucasian families with systemic sclerosis and other connective tissue and immunological disorders, including rheumatoid arthritis, discoid lupus erythematosus, psoriasis, psoriatic arthritis, ankylosing spondylitis, ulcerative colitis, asthma, Sjögren's syndrome, Raynaud's phenomenon and thyroid disease. In one of these families, two sisters are affected with systemic sclerosis. Clinical, serological, and HLA haplotype results are reported, along with a review of the medical literature on familial occurrence of systemic sclerosis.

Aged↗