Phagolysosome formation in normal and colchicine-treated macrophages.

Intracellular lysosomal fusion has been evaluated in cultivated mouse peritoneal macrophages by measurement of transfer of acid phosphatase to polyvinyltoluene (PVT)-containing phagolysosomes. Enzyme transfer was found to be directly and significantly related to the uptake of PVT and to be independent of time allowed for phagolysosome formation over time periods of 15 min to 18 h. In addition, the extent of transfer of lysosomal enzyme to phagolysosomes was unaffected by treatment of the cells with 10(-6) M colchicine, a dose which eradicates morphologically identifiable microtubules in this cell type within 2 h. The data indicate that intracellular fusion of lysosomes with phagosomes in the macrophage does not require formed microtubules and suggest that fusion occurs promptly after interiorization of inert particles.

Colchicine effects on lysosomal enzyme induction and intracellular degradation in the cultivated macrophage.

The effects of colchicine on lysosomal fusion and lysosomal enzyme induction in the cultivated mouse peritoneal macrophage have been examined. Colchicine (10- minus 6 M), but not lumicolchicine, inhibited lysosomal enzyme induction by both phagocytic and pinocytic stimuli. In addition, the drug significantly retarded pinocytic uptake of [3-H] sucrose and transport of the amino acids [3-H] alpha aminoisobutyric acid and L-[3-H] leucine. In contrast, lumicolchicine had no effect on pinocytosis or amino acid transport. Thus, a role for intact microtubules in lysosomal enzyme induction, pinocytosis, and amino acid uptake in these cells is suggested. That colchicine inhibited lysosomal enzyme induction by phagocytic stimuli under conditions in which pinocytosis contributed little to the enzyme rise indicated that inhibition of pinocytosis was unlikely to account for colchicine effects on lysosomal enzyme induction. Effects of colchicine on degradation of phagocytized and pinocytized substrates were examined to determine if intact microtubules are required for fusion among lysosomes, pinosomes, and phagosomes. Colchicine did not alter the rate of intracellular digestion of radiolabeled bacteria by the cultivated macrophage. Similarly, it had no effect on enzymatic hydrolysis of intracellular [3-H] sucrose resulting from uptake of exogenous invertase. The finding that colchicine had no effect on the functional consequences of fusion of lysosomes with endosomes suggests that intact microtubules are not required for fusion among these constituents of the vacuolar apparatus.

Murine cytomegalovirus-induced macrophage dysfunction.

Macrophages infected in vitro with murine cytomegalovirus (MCMV) manifest depressed phagocytic uptake of a variety of particles within hours after the initiation of infection. Analysis of kinetics of uptake of radiolabeled Staphylococcus aureus by MCMV-infected macrophages indicates that the diminished uptake results from a depression in the calculated maximum velocity of uptake (Vmax) with the apparent Michaelis constant (KM) remaining unaltered. This pattern of altered uptake is typical of that seen after manipulations that affect the surface interactions of macrophages with ingestible particles. Coincubation of macrophages and radiolabeled Staphylococcus with opsonizing antibody resulted in normalization of the phagocytic rates. The surface localization of the defective phagocytosis was further confirmed by light and scanning electron microscopy of the macrophages incubated with Staphylococcus or latex spherules. These data indicate that defective macrophage surface that interferes with the initial macrophage-particle interactions that initiate nonimmune phagocytosis.

Infective endocarditis with negative blood cultures. An analysis of 52 cases.

The clinical records of 52 patients who were diagnosed clinically as having had infective endocarditis despite negative blood cultures have been reviewed. They differed at presentation from patients with positive blood cultures in more frequent receipt of antibiotics prior to culture and more frequent signs of major systemic emboli and congestive heart failure. Response of culture-negative patients with fever to empiric antibiotic therapy was correlated with survival, in that 92 per cent of the patients who became afebrile within the first week of therapy liver, whereas only 50 per cent of those who did not become afebrile lived. Deaths resulted primarily from major systemic emboli and from uncontrollable congestive heart failure due to valvular insufficiency. In 25 cases, valvular tissue was examined histologically. In 15 cases, vegetations were seen and organisms identified; in six cases, only vegetations were seen; and in four cases (16 per cent), the clinical diagnosis of infective endocarditis was not substantiated in the pathologic report.

Immunologic defects and vaccination in patients with chronic renal failure.

Patients with chronic renal failure suffer from defective host defenses which are directly the result of the renal impairment, in addition to those dependent on the primary illness leading to the renal failure. The mechanisms underlying the defective responses in phagocytic cells, lymphocytes and antigen processing are likely due to either failure to adequately eliminate suppressive compounds by the defective kidneys or to improper metabolic processing of the factors by the damaged renal parynchema. That some of the defects are reversed by transplantation and not dialysis suggests that renal parenchymal metabolic activities may be involved, although it is also possible that functioning glomerular cells are capable of filtering substances that membranes are not currently capable of eliminating. The current strategy for dealing with the immunodeficiency appears to be totally based on developing means to circumvent the defective function. The other approach, correction of the impaired function, cannot be even considered until the mechanisms underlying the defective function of the cells involved in defenses are better delineated. It seems possible that one or a few compounds are pivotal in altering the function of all the affected cell lines, since, with only a small amount of effort, it is possible to relate the dysfunction to abnormal cell membrane functions in phagocytic cells, dendritic cells and lymphocytes. Until the biochemical basis of the dysfunction of all the cell types affected are better defined, such exercises cannot be translated into better management of patients with chronic renal failure. Proper function of host defenses requires that appropriate cells can properly respond to threats to host viability. For the cells of the immune system (phagocytes and lymphocytes) this means that their response to regulatory molecules be appropriate, that their mobility be normal, that their adherence to substrates be preserved, and that they can generate the appropriate response to the challenge. For neutrophils, for example, it is necessary that they recognize and mobilize appropriately to chemotactic stimuli, that they be able to adhere to and migrate through endothelial lining, that their phagocytic activity be sufficient, and that they can kill and degrade endocytosed particles and generate appropriate secretions. Similar lists of requirements for good function can be generated for any cell type in the immune defense system. Uremia, as well as currently available treatments for uremia, directly or indirectly alters the function of all phases of appropriate immune cell function. Defective host responses in uremia have been recognized for decades and there has been considerable effort in the past decade to better define the extent and mechanisms of impaired defenses. Despite the multitude of major defects in humoral, cellular, and inflammatory processes, uremic patients who are cared for today, although they remain at higher risk of serious infectious complications, can and do maintain a good quality of life, with most remaining free of major infections for years and decades.

Interaction of cytokines and alveolar cells with Pneumocystis carinii in vitro.

Although deficient cellular immune function is a major predisposing factor in the development of Pneumocystis carinii pneumonia, the mechanisms involved in cellular immune surveillance against P. carinii have not been defined. When P. carinii were separated from rat cells by a semipermeable membrane, alveolar macrophages secreted substances lethal to P. carinii only when the macrophages were activated by interferon-gamma; normal macrophages were ineffective. Type II alveolar epithelial cells caused death of P. carinii whether or not interferon-gamma was present. The effects of soluble mediators also were tested; recombinant human tumor necrosis factor-alpha (TNF) but not recombinant rat interferon-gamma or endotoxin was directly lethal to P. carinii. These lethal effects were prevented when antiserum to TNF or antioxidants (catalase and superoxide dismutase) were included. These data suggest that TNF may be a major mediator involved in the killing activity of activated macrophages against P. carinii and that TNF's activity against P. carinii is related to induction of oxidative stresses.

Protection of staphylococci ingested by macrophages from the bactericidal action of rifampin.

The ability of rifampin to kill Staphylococcus aureus which have been ingested by normal mouse peritoreal macrophages in vitro has been investigated. In contrast to data which have been reported from experiments with other cell types, in this system rifampin was no more active than was penicillin against the two strains tested.

Kinetics of phagocytosis of Staphylococcus aureus by alveolar and peritoneal macrophages.

The rate of uptake of radiolabeled Staphylococcus aureus by macrophages in vitro was studied by use of Lineweaver-Burk analysis. It was found that competition for ingestion by excess unlabeled particles, either staphylococci or unrelated particles, resulted in diminished uptake of the labeled particles and that opsonization of particles with specific antiserum enhanced that uptake solely by altering the maximum velocity of uptake (Vmax). Uptake of radiolabeled staphylococci opsonized with specific antiserum was not inhibited by excess numbers of unopsonized organisms; the ingestion was inhibited by excess numbers of opsonized unlabeled organisms, and that inhibition was characterized by depression of Vmax. Inhibition of phagocytosis by indoacetate and cytochalasin B resulted from depression in both Vmax and Michaelis constate (Km). In addition, the phagocytic function of macrophages improved during in vitro culture, a phenomenon which was particularly striking for alveolar macrophages. That enhancement of activity resulted from improvements in both Vmax and Km. Addition of opsonizing antibody at any stage of in vitro maturation resulted in further increases in phagocytic uptake, increases which affected only Vmax. The in vitro maturation of phagocytic function by alveolar macrophages could be inhibited by both 2-deoxy-D-glucose and cycloheximide, but not by culture in hypoxia. The data indicate that the terms of Lineweaver-Burk analysis cna be correlated with functional aspects of phagocytosis and that Vmax represents the avidity of the macrophage surface for the particle, whereas Km is an index of the capacity of the cell for ingestion.

In vitro effects of antiprotozoan drugs and immune serum on Pneumocystis carinii.

The in vitro survival of Pneumocystis carinii isolated from the lungs of rats with glucocorticoid-induced pneumocystosis has been evaluated by quantitation of uptake of vital dyes by the organisms after exposure to a variety of drugs, immune serum, and medium enriched with macrophage lysosomal enzyme. On the basis of these tests, it appears that pentamidine and chloroquine are directly lethal to the organisms, but that suramin, trimethoprim, and sulfamethoxazole are not. Neither immune serum supplemented with complement and lysozyme nor the supernatant of phagocytosing macrophages appeared to affect the viability of the organisms. Inhibitors of glucose uptake and metabolism appeared to kill Pneumocystis, but neither cycloheximide nor iododeoxyuridine was effective.

Suramin effects on macrophage phagolysosome formation and antimicrobial activity.

The effects of suramin on phagolysosome formation and antimicrobial activity of mouse peritoneal macrophages cultivated in vitro have been studied. Prolonged in vitro pretreatment of macrophages with high concentrations of suramin caused macrophages to form large fragile phagolysosomes in which the concentrations of the various lysosomal enzymes were inferred to be diminished. In addition, suramin-treated macrophages demonstrated enhanced exocytosis of acid phosphatase during phagocytosis of polyvinyl toluene spherules. However, suramin was found not to inhibit formation of phagolysosomes in macrophages that had ingested Listeria monocytogenes when those cells were examined by the electron microscope. Suramin pretreatment did not alter the ingestion or intracellular killing of Staphylococcus aureus or of a strain of L. monocytogenes that was essentially avirulent for mice, but did protect macrophages from destruction by virulent L. monocytogenes ingested in vitro, an effect that appeared to have been mediated through enhancement of the bacteriostatic potential of the macrophages. However, at a single dosage level, the drug did not alter the mortality of mice challenged with virulent L. monocytogenes.

Effects of bacterial pneumonitis on development of pneumocystosis in rats.

Disease caused by the protozoan parasite Pneumocystis carinii complicates management of patients with a variety of defects in immune function and is most commonly observed in patients who receive long-term therapy with glucocorticoids. In the rat, disease is readily induced by chronic administration of glucocorticoids. However, rats that have had polymorphonuclear leukocytic pneumonitis induced by Pseudomonas are protected from development of pneumocystosis, whereas rats that have received an intratracheal injection of Staphylococcus, which does not induce a polymorphonuclear leukocytic alveolar exudate, are not protected. It is possibly that accidental contact of polymorphonuclear leukocytes with dormant Pneumocystis is an important element of control of the organism in healthy animals, and suppression of polymorphonuclear leukocytic inflammatory response underlies glucocorticoid-induced and spontaneous activation of the disease.

Phospholipid profile of Pneumocystis carinii and its interaction with alveolar type II epithelial cells.

Pneumocystis carinii is an obligate parasite of mammalian lungs, attaching to but not invading the alveolar epithelium. The alveolar air spaces are rich in phospholipids, which are secreted by steroid-responsive alveolar type II epithelial cells. P. carinii isolated from rat lungs was found to contain the expected structural phospholipids as well as a large amount of firmly attached disaturated phosphatidylcholine, the characteristic phospholipid of alveolar surfactant. In vitro, P. carinii cells synthesized phospholipids from simple radiolabeled precursors; disaturated phosphatidylcholine was not formed. However, washed P. carinii cells avidly adsorbed radiolabeled rat surfactant, a process that appeared to be saturable, not dependent on viability of the organisms, and abolished by incubation at 4 degrees C. The surfactant was neither harmful nor beneficial to in vitro survival of the organisms. With the exception of high concentrations of arachidonic acid, fatty acids found in rat alveolar lining material were also not toxic. In addition, cultures consisting primarily of rat type II alveolar epithelial cells were toxic to P. carinii when the organisms were added to monolayers of type II cells at less than or equal to 10:1 multiplicity. At higher multiplicities, the parasite survived (but did not increase in numbers), and the type II cells deteriorated. The mechanism for this effect has not been determined.

Pneumocystis carinii: oxygen uptake, antioxidant enzymes, and susceptibility to oxygen-mediated damage.

The ability of Pneumocystis carinii obtained by alveolar lavage of rats with glucocorticoid-induced pneumocystosis to utilize molecular oxygen, the concentrations of selected antioxidant enzymes, and the susceptibility of P. carinii to in vitro killing by oxygen radical-generating systems have been evaluated. As expected of an organism which has been found to convert radiolabeled glucose to CO2, the parasite utilizes molecular oxygen. No evidence for pathways of oxygen utilization other than the cytochrome pathway was found; cyanide virtually abolished oxygen consumption. Although readily detectable levels of the antioxidant enzymes superoxide dismutase, catalase, and glutathione peroxidase were present in the P. carinii preparations, only superoxide dismutase was present at levels that suggested that the activity was indeed a property of the parasite. Almost certainly, P. carinii does not possess effective concentrations of catalase. In addition, it was found that P. carinii is susceptible to the lethal actions of hydrogen peroxide and superoxide, but the parasite seems to be resistant to the effects of a hydroxyl radical-generating system.

Chronic glucocorticosteroid therapy impairs staphylococcal clearance from murine lungs.

Since the clearance of Staphylococcus aureus from murine lungs after aerosol exposure depends only on regional defense processes and does not require the recruitment of neutrophils or other systemic factors, we used this model for pulmonary clearance to evaluate the effect of chronic glucocorticosteroid therapy on intrinsic pulmonary defense responses. Mice treated with oral prednisolone for 2 or more weeks had delayed clearance of S. aureus at both 6 and 22 h after aerosol exposure. Mice treated with prednisolone for 1 week had delayed clearance at 22h, and mice treated for 2 days had normal clearance. In mice that had been treated for 2 weeks, clearance returned to normal after 2 weeks off therapy, but not after 1 week. Prednisolone did not appear to alter the number of phagocytes in bronchoalveolar spaces or their ingestion capacity. These results suggest that chronic steroid therapy can alter pulmonary clearance functions independent of any effect on immune or inflammatory responses.

Staphylococcal clearance and pulmonary macrophage function during influenza infection.

Direct infection of pulmonary macrophages with influenza virus in vitro does not alter macrophage functions necessary for staphylococcal clearance. To determine whether these functions are altered during viral pneumonitis, we evaluated macrophages recovered from influenza-infected mice which had undergone aerosol challenge with Staphylococcus aureus. Sublethal infection with influenza A/PR8 produced patchy hemorrhagic pneumonia in CF1 mice and significantly reduced the intrapulmonary killing of staphylococci inhaled during aerosol challenge. However, only a small fraction of macrophage monolayers established from animals with influenza expressed viral hemagglutinin on their plasma membrane, and alveolar macrophages from infected mice ingested staphylococci and yeast cells in vitro at the same rate as control macrophages. The in vitro intracellular bactericidal activity against staphylococci ingested in vivo was comparable in monolayers from control and PR8-infected mice. In experiments with more severe influenza infections (mortality greater than 50% by day 7), a larger fraction of the staphylococci recovered by bronchoalveolar lavage appeared to be ingested in vivo during the aerosol exposure in the PR8-infected mice than in the control mice, but intracellular killing by macrophages during in vitro incubation was similar in control and PR8 monolayers. Hence, the severity of viral infection did not influence intracellular bactericidal activity. In virus-infected mice, a significantly larger fraction of viable staphylococci in the lower respiratory tract at the end of aerosol exposure was adherent to the trachea and major bronchi. In summary, PR8 infection established by intranasal inoculation impaired staphylococcal killing in the lung even though these infections did not alter in vivo ingestion rates or in vitro intracellular killing rates of macrophage populations in bronchoalveolar spaces.

Effect of influenza infection on the phagocytic and bactericidal activities of pulmonary macrophages.

The effect of mouse-adapted influenza A/PR/8/34 virus on pulmonary macrophage function was evaluated by using an in vitro system which allowed direct virus interaction with macrophages and then separate analysis of the steps required for bacterial clearance by macrophages. Infection of macrophages with this virus resulted in the appearance of a hemagglutinating activity on the macrophage surface; expression of this activity was inhibited by amantadine, 2-deoxyglucose, and cycloheximide and by pretreatment of the virus inoculum with ultraviolet light and specific antiserum. Since there was no release of extracellular virus, this growth cycle appeared to be incomplete (abortive). After influenza infection, net ingestion of viable Staphylococcus aureus by macrophage monolayers was unaltered and there was no change in the fraction of the monolayer which ingested cocci over a wide range of bacterial inputs. Influenza-infected macrophages also inactivated intracellular S. aureus at a rate indistinguishable from controls. Therefore, these in vitro studies do not support the hypothesis that the defect in pulmonary antibacterial mechanisms associated with influenza infections results from a direct effect of virus infection on either the phagocytic or bactericidal activity of resident pulmonary macrophages.

Replication of murine cytomegalovirus in lung macrophages: effect of phagocytosis of bacteria.

Murine cytomegalovirus was found to replicate in lung and peritoneal macrophages of both CF-1 and BALB/c mice in vitro. Cytopathic changes typical of cytomegalovirus infection, including intranuclear inclusions, developed within the infected cells and eventually resulted in death of infected macrophages. Viral antigens were demonstrable by indirect immunofluorescence microscopy, and morphologically typical herpesvirus particles were observed in both nuclei and cytoplasm of murine cytomegalovirus-infected macrophages. Within 24 h after infection, at which time there was expression of viral antigens but no marcophage death, murine cytomegalovirus-infected macrophages demonstrated marked inhibition of phagocytosis of Staphylococcus aureus. Direct inhibition of macrophage function by cytomegalovirus infection in vivo could impair pulmonary defenses and may account in part for the frequent association of cytomegalovirus infection with other infectious agents.