Morphologic and Functional Characteristics of AM Recovered from Patients with Pulmonary Alveolar Proteinosis
Light microscopy disclosed that the structure of AM from patients with pulmonary alveolar proteinosis was strikingly different from that of AM from healthy nonsmoking control subjects. The AM from patients with pulmonary alveolar proteinosis were generally two to three times the size of normal AM when viewed at equal magnification (Fig 1A and 1B). The cytoplasm of macrophages from all patients with pulmonary alveolar proteinosis contained large globular, as well as amorphous inclusions, often giving the cytoplasm a foamy appearance. Interestingly, when cultured with medium containing either or P2, AM from healthy control subjects acquired morphologic characteristics similar to those of AM from the patients with pulmonary alveolar proteinosis (Fig 1C and 1D).
Although the number of AM phagocytizing yeast at two hours did not differ significantly between the patients with pulmonary alveolar proteinosis and the normal volunteers, the mean number of yeast cells ingested per macrophage was decreased in AM from patients with pulmonary alveolar proteinosis compared with normal control subjects (Table 1). Possibly the most important finding from these experiments was that AM from patients with pulmonary alveolar proteinosis uniformly exhibited decreased phagolysosome fusion (Table 1).
In Vitro Effects of Cell-Free Fractions of Lavage from Pulmonary Alveolar Proteinosis on Normal Human AM
Of adherent AM remaining in culture at 48 hours, both trypan blue exclusion and acridine orange staining showed that more than 90 percent were viable, and there was no difference in the viability of AM from pulmonary alveolar proteinosis and AM from normal volunteers. When AM from normal volunteers were cultured with a 1:20 or 1:10 (v/v) concentration of Px for 48 hours, 90 percent or more of the AM were nonvia-ble and had detached from the coverslip. This demonstrated that at concentrations much below the ratio of Pt to cell pellet in the lavage material of patients with pulmonary alveolar proteinosis, was markedly toxic to normal human AM. To ascertain that we had a comparably viable and adherent cell population, we serially diluted Px concentration in subsequent experiments until most of the AM in culture remained adherent and viable at 48 hours. This concentration was one volume of Px to 75 volumes of culture medium. Macrophages of normal volunteers that were cultured in the presence of P2 (1 mg of protein per milliliter) for 48 hours showed no differences in either adherence or viability when compared with normal AM established in culture medium alone. More for read is on Canadian Neighbor Pharmacy website.
When normal human AM were incubated for 48 hours with a concentration of P2 (1:75 dilution) that did not affect adherence or viability, there was no effect on either the numbers of AM phagocytizing yeast or the number of yeast cells ingested per AM; but phagolysosome fusion was significantly decreased (Table 2). In contrast, P2 decreased both the number of AM phagocytizing yeast and the number of yeast cells ingested per AM in macrophages from normal human subjects without affecting phagolysosome fusion. This demonstrates that both Fl and P2 have detrimental but different effects on the phagocytic process in normal AM.
After determining that the phagocytic process, including phagolysosome fusion of normal rat AM, was affected in the same way as normal human AM when incubated with Px and P2 fraction of lavage from patients with pulmonary alveolar proteinosis, we used rat AM to evaluate the effect of P2 fraction of lavage from normal human subjects on phagocytosis. Phagolysosome fusion was not evaluated in AM treated with normal P2 fraction because phagolysosome fusion was not decreased by P2 from patients with pulmonary alveolar proteinosis. Treatment of normal AM with low concentrations (100 μg and 200μg of protein per milliliter) of P2 from patients with pulmonary alveolar proteinosis did not affect phagocytosis, but phagocytosis was decreased by 75 percent and 50 percent with high concentrations of P2 (500 μg and 1,000μg of protein per milliliter, respectively). The treatment of normal AM with concentrations of P2 fraction from normal subjects in excess of 1,000μg of protein per milliliter had no effect on phagocytosis. Because of the small quantity of P2 fraction available, these data are from a single experiment, and statistical analysis was not done; however, the results from this single experiment suggest that P2 from normal lavage does not affect phagocytosis in normal rat AM, and the depressant effect of P2 from patients with pulmonary alveolar proteinosis is abnormal and related to the disease process.
Figure 1. Alveolar macrophages under oil-immersion fluorescence microscopy (all at same magnification) after cells have been labeled with acridine orange. A, Normal human AM. B, Alveolar macrophage from patient with pulmonary alveolar proteinosis. C, Normal human AM after incubation with Pj fraction of pulmonary lavage material from patient with pulmonary alveolar proteinosis. D, Normal human AM after incubation with P2 fraction of pulmonary lavage material from patient with pulmonary alveolar proteinosis.
Table 1—Studies of AM Function: Phagocytic Activity and Phagolysosome Fusion
|Subjects||Macrophages Phagocytizing Yeast, percent*||No. of Yeast Cells per Macrophage*||Phagolysosome Fusion, percent*|
|Mean ± SE||30±6||3.3 ±0.2+||64 ± 1$|
|Mean ± SE||32 ±5||2.3±0.3t||33 ±6*|
Table 2—Culture qf Normal AM for 48 Hours in Vitro in the Presence qfDifferent Fractions of Cell-Free Lavage Material from Pulmonary Alveolar Proteinosis
|No. of Yeast Cells per Macrophage||PhagolysosomeFusion,|
|Untreated controls controls|
|Treated with Pj*|
|Differences vs controlst||3.67 ±2.33||0.50 ±0.40||18 ±2.52|
|Treated with PJI|
|Difference vs controlst Significance§||13.33±0.33p<0.001||1.37 ±0.29 p<0.005||7 ±2.0 NS|