William Lippert

Pulmonary Alveolar Macrophage DEFENDER AGAINST BACTERIAL INFECTION OF THE LUNG

The rate of ingestion of inhaled bacteria by pulmonary alveolar macrophages is an important determinant of host defense. This parameter was investigated by infecting rats with finely dispersed aerosols bearing Staphylococcus aureus in high concentrations (about 10(s) bacteria/ft(3)/min). These aerosols deposited more than 10(6) bacteria/murine lung. At 0, 2(1/2), and 5 h after infection, bacterial clearance rates were measured in the left lung, and the intracellular or extracellular location of 100 bacteria was determined histologically in the right lung (perfused in situ). The clearance rates at 2(1/2) and 5 h were 44.5% and 76.9%, respectively. The percentages of intracellular bacteria were: 0 h, 54.8%; 2(1/2) h, 87.1%: 5 h, 91.9%. When rats were exposed for 4 h to 2.5 ppm of ozone (O(3)), bacterial clearance did not occur - 15.3%, although 78.7% of the bacteria were intracellular. Clumps of more than 10 bacteria-usually intracellular-were also present. These experiments demonstrate that phagocytic ingestion is an exceedingly rapid process, that in this experimental model the inactivation of inhaled staphylococci results almost entirely from phagocytosis, and that ozone-induced reductions in bacterial clearance are due to severe impairment of intrapulmonary killing mechanisms and minor impairment of bacterial ingestion.

Ozone and nitrogen dioxide exposure: murine pulmonary defense mechanisms.

Since ozone and nitrogen dioxide impair pulmonary resistance to infection by inhibiting the function of the alveolar macrophage, we investigated the effect of combinations of these gases to determine if they interacted biologically in a synergetic, indifferent, or antagonistic manner. Mice were exposed to atmospheres of ozone and nitrogen dioxide for 17 hours prior to, or four hours after, infection with aerosols of Staphylococcus aureus labeled with radioactive phosphorus (32P). Animals infected and then exposed to various oxidant combinations manifested bactericidal dysfunction only when the level of one of the pollutants approximated its individual threshold value. Similar results were obtained when mice were exposed to pollutants for 17 hours before infection. Hence, the pulmonary consequence of exposure to ozone and nitrogen dioxide is equivalent to the injury that would be expected from each individual oxidant.

Polymorphonuclear leucocyte motility in patients with severe burns

Chemotaxis, chemokinesis and cellular orientation were measured for unstimulated and 10(-7) n-formyl methionyl leucyl phenylalanine (F-met-leu-phe) stimulated polymorphonuclear leucocytes (PMNS) of nine patients with recent 10-80 per cent burns using a computer-assisted image analysis technique. The technique records PMN movement, as viewed with a phase-contrast microscope on videotapes, and then uses computer programs to calculate the speed and direction of up to 50 PMNS over a 5-min period. Orientation was determined visually. Cellular adherence was also measured by attachment methods. PMNS from burn patients were slower (av. speed 16.8 microns/min), responded less well to F-met-leu-phe (av. speed 20.9 microns/min, av. McCutcheon index 0.32), were less often oriented towards the chemoattractant (av. 39 per cent) and were more adherent (av. 50 per cent) than control cells (av. speed 21.8 microns/min; av. speed F-met-leu-phe 32.2 microns/min; McCutcheon index 0.61; oriented 59 per cent adherent; 16 per cent). Thus PMNS from burn patients orient less well, are significantly slower and have less directionality in response to a chemoattractant, and are more adherent suggesting activation.

Group B streptococcal lung infection in neonatal rabbits.

Summary: The interaction of alveolar macrophages (AMs) and group B streptococci (GBS) was investigated in 1 and 2-day-old rabbits following infection in an exposure chamber containing 107 streptococci per cubic foot of air. The % of streptococci within AMs was similar at 0 and 4 h (36 and 65%) after infection for the two groups of rabbits. Twenty-four h after infection, the 2-day-old rabbits had a significantly higher % of ingested GBS (86 versus 68%). Sixty % of inspired GBS were inactivated by the older rabbits within 4 h after infection. This clearance persisted throughout the 72 h experimental period. Inspired GBS proliferated in the younger rabbits until 48 h (mean negative clearance of –17, –276, and –79% at 4, 24, and 48 h) before their numbers were reduced by inflammation. Sixty of 78 1-day-old rabbits had inflammatory responses between 24 and 72 h versus only 5 of 50 older rabbits. At 24 h after infection, AMs of 1-day-old rabbits contained significantly increased numbers of intracellular GBS microcolonies (17/ 20) than did AMs from 2-day-old rabbits (5/19). These observations suggest that the enhanced susceptibility to GBS infection in the immediate postnatal period is caused at least in part by ineffective intracellular killing by AMs.Speculation: If the ability of macrophages to inactivate intrapulmonary group B streptococci in the immediate postpartum period is due to an underdeveloped oxygen-dependent bactericidal system, then the bactericidal abnormality may be even more profound in the prenatal period when macrophages are exposed to lower in utero alveolar oxygen tensions. As a consequence of this reduced activity, group B streptococci aspirated prenatally are likely to proliferate uncontrollably, and either prenatal stimulation of the alveolar macrophage or early detection of intrauterine streptococcal infection followed by delivery of the infected fetus may be life saving.

Effect of 0.64 ppm ozone on alveolar macrophage lysozyme levels in rats with chronic pulmonary bacterial infection

A rat model of chronic pulmonary infection (CPI) initiated by Pseudomonas aeruginosa embedded in agar beads was used to test the effect of ozone on lysosomal enzyme levels in alveolar macrophages (AM). CPI was induced by intratracheal instillation of a 0.1-ml suspension of infected beads into the left lung. Ten days after infection half the rats were exposed to atmospheres of air and half to 0.64 ppm ozone for 4 weeks. Enzyme levels were measured using a scanning cytospectrophotometer linked to PDP/11 computer. Measurement of lysozyme in individual rat AM in situ showed a significant decrease in cell size and enzyme content in ozone-exposed uninfected animals. Cell size and enzyme content of ozone-exposed animals with CPI were further reduced, suggesting a synergistic effect between ozone exposure and chronic infection.

Cellular acid phosphatase activity: Correlation of cytochemical and biochemical measurements

SummaryMethods for comparing results of cellular acid phosphatase activities obtained by quantitative cytospectrophotometry with those obtained by biochemical analysis are needed to express the cytospectrophotometric data in biochemical units. Since naturally occurring cells have differing amounts of acid phosphatase, enzyme activity was measured cytochemically and biochemically in polymorphonuclear leukocytes and peritoneal and alveolar macrophages from male rats to determine if these measurements permitted construction of a line correlating the two parameters. Cellular acid phosphatase activity, as measured cytospectrophotometrically and biochemically, increased proportionately with polymorphonuclear leukocytes having the lowest activities and alveolar macrophages the highest. These values when subjected to linear regression analysis fixed a line with a correlation coefficient of 0.95 demonstrating that cytochemical and biochemical activities of acid phosphatase activity can be correlated using naturally occurring cells.

INCREASED LEUKOCYTE ALKALINE PHOSPHATASE IN SICK NEONATES

Alkaline phosphatase (LAP) and myeloperoxidase (MPO) were evaluated in polymorphonuclear leukocytes (PMNs) from neonates using a computer-assisted cytospectrophotometer. This method allowed quantitative enzyme determinations within each PMN. Population distributions were determined for 100 individual cells. Of the 10 infants studied, 8 were ill, 3 with congenital pneumonia and 5 without proven infection, and 2 were well pre-term infants. PMNs were obtained from all during the first week of life and from 5 during the third week, as well. The 8 ill infants showed a broad distribution of LAP activity in their PMNs in the first week of life, with 75.4±25.2% ([xmacr ;[plusmn;S.D.) of PMNs having LAP activity greater than the 95th percentile of our adult controls. By the third week of life, the percent of PMNs had significantly diminished to 35.6±33.9% ([xmacr ;[plusmn;S.D.) (p < .05). The 2 well pre-term infants in the first week of life had only 7±4% ([xmacr ;[plusmn;S.D.) of PMNs with activity greater than the 95th percentile for adult controls. The distribution of MPO activity did not differ from adult controls in any infant.In summary, we found increased PMN LAP activity in the first week of life in sick neonates with and without infection as compared with adults and well neonates. Decreasing LAP activity in the third week of life corresponded with clinical improvement. Thus, differences in intracellular LAP activity appear to be due to pathologic rather than maturational processes.