Endothelial Cells and Bacterial Infection

to be able to kill the yeasts. Todetermine whether the initial growth of C.n in the IFN-treated cultures was occurring extracellularly or intracellularly phagocytosis of C.n was followed over the same time course (Fig IB). We found that the rate of phagocytosis in the IFN-treated wells was inhibited by IFN, which resulted in extracellular proliferation of C.n over the 6rst 24 h. At 60 h all organisms were intracellular. Phagocytosis of C.n is dependent on complement activation." Natural antibodies in the opsonizing serum have little effect on inducing C.n ingestion," although cooperation between Fc and complement receptors is important in defense against this organism." Upon reaction with fresh serum, a polysaccharide capsule such as that of C.n favors the deposition ofC3b with little conversion into C3bi. 9 The possibility that the expression of complement receptors, in particular CR1, was affected by IFN was therefore investigated. Monoclonal antibodies to CR1, CR3, CR4, and to a control antigen, HLA-DR, were used to determine the relative expression of these antigens on untreated and IFNtreated (48 h) HAM by indirect immunofluorescence. CRI was found to be downregulated by IFN with 51%, while expression of CR3 and CR4 did not change (98 and 89% of untreated HAM, respectively). HLA-DR expression was enhanced by IFN as previously reported." Antibodies to these surface structures were used to inhibit phagocytosis of C.n. Preincubation of the HAMs with antibodies to the various complement receptors at a concentration of 50 ILW ml inhibited the uptake of C.n, while anti-HLA-DR had little effect (Fig 2). This suggests that there may be an interaction among the three receptors in binding and phagocytosis of the yeast. Previous studies have shown that in addition to binding C3bi, CR3 and CR4 function as important adherence molecules in various cell-to-cell interactions," The effect of IFN on the expression of CRI could result in interference with the interaction between HAM and organism and therefore might be responsible for the reduced rate of phagocytosis. More definitive experiments are now in progress to investigate this point. These experiments show that IFN affects phagocytosis of C. n, possibly via its effect on CRI expression. A larger number of organisms may overwhelm the HAM defenses. When fewer yeasts are present, phagocytosis proceeds, and the IFN-induced intracellular killing of the organism may prevent further spread of the infection. REFERENCES 1 ~inberg PB, Becker S, Koren HS. Growth inhibition of C'1IPtococcus neoformans by human alveolar macrophages. Am Rev Respir Dis 1987; 136:1242-47 2 Fluorescence assays described in Becton Dickinson monoclonal antibody catalog. 3 Murray H~ Rubio BY, Rothermel CD. Killing of intracellular UiahmanitJ donovani by lymphokine stimulated human mononuclear phagocytes: evidence that interferon-gamma is the activating lymphokine. J Clio Invest 1983; 72:1506-10 4 Rothermel CD, Rubio BY, Jaffe EA, Murray HW Oxygen independent inhibition of intracellular Chlamydia psittaci growth by human monocytes and interferon-gamma activated macrophages. J Immunoll986; 137:689-92 5 Brummer E, Morrison CJ, Stevens DA. Recombinant and natural gamma interferon activation of macrophages in vitro:

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different dose requirements for induction of killing activity against phagocytizable and nonphagocytizable fungi. Infect Immunoll985; 49:724-30 Bbarwaj N, Nash ~ Horwitz MA. Interferon-gamma activated human monocytes inhibit the intracellular multiplication of ugionda pneumophilG. J Immunoll986; 137:2662-69 Davies SF, Clifford D~ Hoida! JR, Repine JF. Opsonic requirements for the uptake of Cryptococcus neoformans by human polymorphonuclear leukocytes and monocytes. J Infect Dis 1982; 145:870-74 GrifBn FM Jr. Roles of macrophage Fe and C3b receptors in phagocytosis of immunologically coated Cryptococcw neoforman&. Proc Natl Acad Sci USA 1981; 78:3853-57 Newman SL, Mikus LIC. Deposition of C3b and iC3b onto particulate activators of the human complement system. J Exp Med 1985; 161:1414-31 Becker S. Interferons as modulators of human monocytemacrophage differentiation: I. Interferon-gamma increases HLA-DR expression and inhibits phagocytosis of zymosan. J Immunoll984; 132:1249-54 Andenon DC, Miller LI, Schmalstieg FC, Rothlein R, Springer TA. Contributions of the Mae-I glycoprotein family to adherence dependent granulcoute functions: Structure-function assessments employing subunit-specific monoclonal antibodies. J Immunoll986; 137:15-27

Endothelial cells and Bacterial Infectlon* Una s. BrPn, Ph.D.;]. M. \finn, Ph.D.; and K. B. Nolop, M.D.

cells are recognized as primary targets of E ndothelial vascular damage in septicemia both as a result of direct

action of bacterial toxins or from indirect actions mediated by other cell types and their products (including cytokines, enzymes, and oxygen radicals). We have now demonstrated that endothelial cells phagocytize bacterial and are capable of bactericidal activity involving generation of oxygen free radicals. J We have examined mechanisms involved in recognition of bacterial strains, the mechanisms involved in signal transduction leading to the respiratory burst, and the molecular mechanisms controlling the release of reactive oxygen species." Endothelial cells distinguish between the mutant (Re), which is taken up in large numbers, and the wild (8) form, which is not phagocytized, of Salmonella minnesota. The Be mutant binds Clq (subunit of the first component of complement), while the 8 type does not. When bacteria or endothelial cells were incubated with Clq-enriched or Clq-depleted sera, data show that Clq mediates binding to the Be mutant by the globular head region of the molecule and to receptors on the endothelial surface via the collagen-like tail region. Further, the release of superoxide anion in response to phagocytosis appears to involve Clq as a stimulus,"Thus, Clq acts as an opsonin. We have also investigated the interactions of Staphylococcus oorew with bovine endothelial cells. Cultures were incubated with 100 S OOnmB per endothelial cell for 3.5 h, followed by elimination of extracellular S aureu« with lysostaphin and enumeration of intracellular S oureus: Followingphagocytosis of S OOreu8 by endothelial cells, the *From the University of Miami School of Medicine, Miami.

AspenLung Confer8nce: Infectiona and the Lung

intracellular S ooreus do not multiply but damage to endothelial cell monolayers can result. 4 Phagocytized S aureu« strains which produce a-hemolysin are cytotoxic to endothelial cells," Results indicated that an a-hemolysin nonproducing S oureus strain was killed after phagocytosis by endothelial cells. Bactericidal activity is reduced when bacteria are phagocytized in the presence of SOD or catalase, indicating a role for oxygen radicals. Addition of 10 JoLM adenosine (ADO), inosine (INO), or hypoxanthine (HX) simultaneously with S ooreus resulted in increased phagocytosis and decreased bactericidal activity Thus, ADO, INO, and HX may modulate endothelial cell resistance to pathogenic bacteria." Subsequent experiments using the nucleoside uptake blocker dipyridamole have shown that uptake of ADO, INO, or HX is required for these effects. Following their uptake, ADO, INO, and HX may act on the intracellular "Bsite" of adenyl cyclase to decrease cellular cAMP levels. The phosphodiesterase inhibitor isobutyl methylxanthine and the adenyl cyclase activator forskolin improve endothelial cell bactericidal activity but do not change the extent of phagocytic uptake from control levels. These results suggest that bactericidal activity is coupled to cellular cAMP levels. However, conditions that increase cAMP did not change phagocytic uptake, which suggests that regulation of phagocytic activity may not be tightly coupled to cAMP levels. Finally, studies of endothelial cell-monocyte interactions imply a monocyte factor that augments the effects of endothelial cell damage by bacterial endotoxin. Bacterial infection, whether localized in the vascular wall or systemic, also initiates an inflammatory response involving circulating leukocytes. Inadvertent endothelial cell injury (as observed in the adult respiratory distress syndrome) is an unfortunate byproduct of this host response to infection. Neutrophils are

the initial leukocytes to respond to an inflammatory stimulus and can produce endothelial cell injury when stimulated. Monocytes also can cause endothelial cell injury when coincubated in the presence of endotoxin. Although part of this injury is due to the release of soluble mediators, the full expression of the injury requires simultaneous coincubation of both cell types," Thus, endothelial cells play a complex role both in the initiation and progression of vascular changes associated with sepsis.a REFERENCES 1 Ryan US. Phagocytic properties of endothelial cells. In: Ryan US, ed. Endothelial cells (vol 3). Boca Raton, FIa: CRC Press, 1988:33-49 2 Ryan US, Vann JM. Endothelial cells: a source and target of oxidant damage. In: Simic MG, 1llylor KA, Ward AF, eds. Oxygen radicals in biology and medicine. New York: Plenum Publishing; 1987;963-68 3 Ryan US, Schultz DR, Goodwin JD, VannJM, Selvaraj M~ Hart MA. Role ofClq in phagocytosis of S. minnesota by pulmonary endothelial cells. (in press) 4 Vann JM, Proctor RA. Ingestion of Staphylococcus aureua by bovine endothelial cells results in time and inoculum-dependent damage to endothelial cell monolayers. Infect Immunol 1987; 55:2155-63

5 VannJM, Proctor RA. Cytotoxic effects ofingested Staphylococcus aumu on bovine endothelial cells: role of S. auIVUB alphahemolysin. Microbiol Pathogen 1988; 4:443-53 6 Ryan US, Vann JM. Cultured endothelial cells as probes for in vivo biology Altern Methods Toxicoll988; 6:49-59 7 Nolop KB, Brigham KL, Meyrick BO. Potentiation of endotoxininduced endothelial cell injury by monocytes and monocyteconditioned media. S Ryan US. The macrophagelilce properties of endothelial cells. NIPS 1988; 3:93-96

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