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Educational Measurement↗
Biomedical subjects
Samuel C Silverstein
Publications and source records attributed to Samuel C Silverstein.
PAF-receptor antagonists, lovastatin, and the PTK inhibitor genistein inhibit H2O2 secretion by macrophages cultured on oxidized-LDL matrices.
Adhesion of mononuclear phagocytes (Macs) to extracellular matrices containing oxidized low-density lipoproteins (oxLDL) stimulates these cells to secrete reactive oxygen species (e.g., O2-, H2O2) that are believed to promote atherogenesis. Current in vitro systems designed to measure Mac H2O2 secretion in response to oxLDL show that these cells secrete H2O2 for only a few hours after plating. The slow onset and chronicity of atherogenesis, however, suggested to us that Mac ROS secretion might be sustained for much longer periods when Macs are maintained in an environment resembling that in the intima of arteries undergoing atherogenesis. The findings reported here confirm this suggestion. They show that Macs maintained on collagen IV matrices containing oxLDL in medium containing human plasma-derived serum secrete H2O2 continuously and in large amounts for at least 11 days. Using this system we tested the effects of compounds known to attenuate atherogenesis in vivo. Platelet-activating factor (PAF) receptor antagonists, lovastatin, and the isoflavone protein tyrosine kinase (PTK) inhibitor genistein each reduced H2O2 secretion by Macs maintained on oxLDL-containing matrices by approximately 60%. Lovastatin's inhibitory effect was blocked completely by addition of geranylgeranyl pyrophosphate to the medium. We conclude that matrix-bound and oxidized lipoproteins stimulate Macs to produce H2O2 continuously and in large quantities via a pathway that involves PAF receptors and PTK and is reversibly blocked by inhibitors of protein prenylation.
Atherosclerosis↗
Determination of the critical concentration of neutrophils required to block bacterial growth in tissues.
We showed previously that the competition between bacterial killing by neutrophils and bacterial growth in stirred serum-containing suspensions could be modeled as the competition between a first-order reaction (bacterial growth) and a second-order reaction (bacterial killing by neutrophils). The model provided a useful parameter, the critical neutrophil concentration (CNC), below which bacterial concentration increased and above which it decreased, independent of the initial bacterial concentration. We report here that this model applies to neutrophil killing of bacteria in three-dimensional fibrin matrices and in rabbit dermis. We measured killing of 10(3)-10(8) colony forming units/ml Staphylococcus epidermidis by 10(5)-10(8) human neutrophils/ml in fibrin gels. The CNC was approximately 4 x 10(6) neutrophils/ml gel in the presence of normal serum and approximately 1.6 x 10(7) neutrophils/ml gel in the presence of C5-deficient serum. Application of our model to published data of others on killing of approximately 5 x 10(7) to 2 x 10(8) E. coli/ml rabbit dermis yielded CNCs from approximately 4 x 10(6) to approximately 8 x 10(6) neutrophils/ml dermis. Thus, in disparate tissues and tissuelike environments, our model fits the kinetics of bacterial killing and gives similar lower limits (CNCs) to the neutrophil concentration required to control bacterial growth.
Animals↗
Adult mouse astrocytes degrade amyloid-beta in vitro and in situ.
Alzheimer disease (AD) is a progressive neurodegenerative disorder characterized by excessive deposition of amyloid-beta (Abeta) peptides in the brain. One of the earliest neuropathological changes in AD is the accumulation of astrocytes at sites of Abeta deposition, but the cause or significance of this cellular response is unclear. Here we show that cultured adult mouse astrocytes migrate in response to monocyte chemoattractant protein-1 (MCP-1), a chemokine present in AD lesions, and cease migration upon interaction with immobilized Abeta(1-42). We also show that astrocytes bind and degrade Abeta(1-42). Astrocytes plated on Abeta-laden brain sections from a mouse model of AD associate with the Abeta deposits and reduce overall Abeta levels in these sections. Our results suggest a novel mechanism for the accumulation of astrocytes around Abeta deposits, indicate a direct role for astrocytes in degradation of Abeta and implicate deficits in astroglial clearance of Abeta in the pathogenesis of AD. Treatments that increase removal of Abeta by astrocytes may therefore be a critical mechanism to reduce the neurodegeneration associated with AD.
Alzheimer Disease↗
A critical concentration of neutrophils is required for effective bacterial killing in suspension.
We have examined the effect of neutrophil concentration on killing of a clinical isolate of Staphylococcus epidermidis. Human neutrophils at concentrations varying from 10(5) to 10(7) per ml were mixed in suspension with S. epidermidis at concentrations varying from 10(3) to 10(8) colony-forming units/ml, and the concentration of viable bacteria was assayed after various times at 37 degrees C. The rate of bacterial killing depended on the concentration of neutrophils and not on the ratio of neutrophils to bacteria. Below a critical concentration of neutrophils, bacteria growth was greater than neutrophil killing of bacteria even when the ratio of neutrophils to bacteria was 100:1. We fitted the time course of bacterial concentration and its dependence on neutrophil concentration with an exponential function, the exponent of which is (-kp + g)t, where k is the second-order rate constant for bacterial killing, p is the neutrophil concentration, g is the first-order rate constant for bacterial growth, and t is time. We found that k approximately 2 x 10(-8) ml per neutrophil per min, and g approximately 8 x 10(-3)/min. Only when p is greater than g/k, which we call the critical neutrophil concentration, does the bacterial concentration fall. Under optimal assay conditions, the critical neutrophil concentration was 3-4 x 10(5) per ml, a value very close to that (< or =5 x 10(5) per ml) known to predispose humans to bacterial and fungal infections.
Blood Bactericidal Activity↗
The bacterial peptide N-formyl-Met-Leu-Phe inhibits killing of Staphylococcus epidermidis by human neutrophils in fibrin gels.
To study human neutrophil (polymorphonuclear leukocyte (PMN)) migration and killing of bacteria in an environment similar to that found in inflamed tissues in vivo, we have used fibrin gels. Fibrin gels (1500 microm thick) containing Staphylococcus epidermidis were formed in Boyden-type chemotaxis chambers. PMN migrated < 300 microm into these gels in 6 h and did not kill S. epidermidis when the gels contained heat-inactivated serum, C5-deficient serum, a streptococcal peptidase specific for a fragment of cleaved C5 (C5a), or anti-C5aR IgG. In contrast, in gels containing normal human serum, PMN migrated approximately 1000 microm into the gels in 4 h and into the full thickness of the gels in 6 h, and killed 90% of S. epidermidis in 6 h. fMLP reduced PMN migration into fibrin gels and allowed S. epidermidis to increase by approximately 300% in 4 h, whereas leukotriene B(4) stimulated PMN to migrate the full thickness of the gels and to kill 80% of S. epidermidis in 4 h. We conclude that both complement opsonization and C5a-stimulated chemotaxis are required for PMN bacterial killing in fibrin gels, and that fMLP inhibits PMN bactericidal activity in fibrin gels. The latter finding is surprising and suggests that in the presence of fibrin fMLP promotes bacterial virulence.
Adhesins, Bacterial↗
Scavenger receptors in neurobiology and neuropathology: their role on microglia and other cells of the nervous system.
Scavenger receptor class A (SR-A, CD204), scavenger receptor-BI (SR-BI), and CD36 are cell surface proteins that mediate cell adhesion to, and endocytosis of, various native and pathologically modified substances, and participate in intracellular signaling, lipid metabolism, and host defense against bacterial pathogens. Microglia, Mato cells, astrocytes, cerebral microvascular endothelial cells, cerebral arterial smooth muscle cells, and retinal pigment epithelial cells express one or more of these SR. Expression of SR-A and SR-BI by microglia is developmentally regulated. Neonatal microglia express SR-A and SR-BI, while microglia in normal mouse and human adult brain express neither. Astrocytes in adult brain express SR-BI. In Alzheimer's disease, microglial expression of SR-A is increased. Such findings, and evidence that SR-A and SR-BI mediate adhesion and endocytosis of fibrillar beta-amyloid by microglia and astrocytes, respectively, and that SR-A, SR-BI, and CD36 participate in secretion of reactive oxygen species by microglia, suggest roles for these receptors in homeostasis and neuropathology.
Animals↗
CD36, a class B scavenger receptor, is expressed on microglia in Alzheimer's disease brains and can mediate production of reactive oxygen species in response to beta-amyloid fibrils.
A pathological hallmark of Alzheimer's disease is the senile plaque, composed of beta-amyloid fibrils, microglia, astrocytes, and dystrophic neurites. We reported previously that class A scavenger receptors mediate adhesion of microglia and macrophages to beta-amyloid fibrils and oxidized low-density lipoprotein (oxLDL)-coated surfaces. We also showed that CD36, a class B scavenger receptor and an oxLDL receptor, promotes H(2)O(2) secretion by macrophages adherent to oxLDL-coated surfaces. Whether CD36 is expressed on microglia, and whether it plays a role in secretion of H(2)O(2) by microglia interacting with fibrillar beta-amyloid is not known. Using fluorescence-activated cell sorting analysis and immunohistochemistry, we found that CD36 is expressed on human fetal microglia, and N9-immortalized mouse microglia. We also found that CD36 is expressed on microglia and on vascular endothelial cells in the brains of Alzheimer's disease patients. Bowes human melanoma cells, which normally do not express CD36, gained the ability to specifically bind to surfaces coated with fibrillar beta-amyloid when transfected with a cDNA encoding human CD36, suggesting that CD36 is a receptor for fibrillar beta-amyloid. Furthermore, two different monoclonal antibodies to CD36 inhibited H(2)O(2) production by N9 microglia and human macrophages adherent to fibrillar beta-amyloid by approximately 50%. Our data identify a role for CD36 in fibrillar beta-amyloid-induced H(2)O(2) production by microglia, and imply that CD36 can mediate binding to fibrillar beta-amyloid. We propose that similar to their role in the interaction of macrophages with oxLDL, class A scavenger receptors and CD36 play complimentary roles in the interactions of microglia with fibrillar beta-amyloid.
Alzheimer Disease↗
Different G(i)-coupled chemoattractant receptors signal qualitatively different functions in human neutrophils.
fMLP- or TNF-alpha-stimulated neutrophils produced H(2)O(2) when they adhered to fibrinogen-coated surfaces but not when they adhered to collagen I-, collagen IV-, or Matrigel-coated surfaces. In contrast, LTB4- or IL-8-stimulated neutrophils did not produce H(2)O(2) when they adhered to any of these surfaces. fMLP and TNF-alpha were much more potent than LTB4 and IL-8 in stimulating neutrophils to up-regulate and to activate their alpha(M)beta(2) integrins, as measured by the binding of specific monoclonal antibodies. Pretreatment of neutrophils with pertussis toxin completely blocked their production of H(2)O(2) on fibrinogen-coated surfaces in response to fMLP and their migration through Matrigel in response to fMLP, LTB4, and IL-8. These data show that although the fMLP, LTB4, and IL-8 receptors are coupled to pertussis toxin-sensitive Galpha proteins, they signal neutrophils to initiate qualitatively different effector functions. We propose that the qualitative differences in effector functions signaled by different chemoattractants reflect qualitative differences in using G-protein beta and/or gamma subunits or other factors by their cognate receptors.
CD18 Antigens↗