INTRODUCTION

TOP ABSTRACT INTRODUCTION DISCUSSION REFERENCES

Inhaled particles in cigarette smoke are phagocytosed by alveolar macrophages (AM) and lead to characteristic morphologic changes. AM from cigarette smokers are more numerous, larger in size, frequently multinucleated, and contain characteristic dark basophilic smoker's inclusion bodies within the cytoplasm as a result of ingestion of inhaled smoke particulates (1).

After bone marrow transplantation, host AM disappear in a linear fashion, and their life span is approximately 80 d (6). However, the kinetics of material phagocytosed by AM and the length of time that morphologic changes of AM persist after smoking cessation is largely unknown. A previous cross-sectional study of nonsmokers, smokers, and ex-smokers demonstrated smoking-related inclusions in AM as long as 270 d after smoking had ceased. These investigators calculated that it takes about 3 yr after smoking cessation before the percentage of AM with inclusions approximates the values for nonsmokers (7). Another study showed that the autofluorescence of smoker's AM started to decrease after 6 mo and was still present at 15 mo after smoking cessation (8). Other investigators found, at autopsy, fluorescent material in AM from smokers for as long as 2 yr after smoking had ceased (9).

We investigated a never-smoker after single lung transplantation (TX) for 1-antitrypsin deficiency emphysema who received a donor's lung from a heavy cigarette smoker. The disappearance of AM with characteristic smoker's inclusions was analyzed by studying those cells in nine serial bronchoalveolar lavages (BAL) after TX for as long as 42 mo.

	CASE REPORT

A 51-yr-old male patient with lung emphysema caused by homozygote 1-antitrypsin deficiency was studied. He had never smoked. The patient had an uneventful course after right-sided single lung TX with no major episodes of infection or rejection. Before TX, VC was 1.9 L, FEV₁ was 0.5 L, and Pa_O2 was 72 mm Hg. Six months after TX, VC was 3.8 L, FEV₁ was 2.1 L, and Pa_O2 was 81 mm Hg. He received long-term immunosuppressive therapy with prednisone 10 mg/d, cyclosporine 250 mg/d, and azathioprine 150 mg/d.

BAL was performed in the donor's lung for transplant surveillance at 1, 2, 5, 7, 12, 18, 24, 36, and 42 mo after TX. Informed consent was obtained from the patient. Bronchoalveolar cells were collected by BAL using a subsegment of the middle lobe and instilling 100 or 200 ml sterile saline in 20-ml aliquots. The recovered BAL fluid was pooled and filtered through one layer of gauze, and the cells were separated by centrifugation for 10 min at 4° C. The total cell number was determined in a hemocytometer, and cytocentrifuge preparations were stained with May-Grünwald-Giemsa stain for differential cell counts. Immunocytology using a peroxidase-antiperoxidase method (10) was also performed.

May-Grünwald-Giemsa-stained slides were examined in a random sequence, and 600 AM were counted on each slide. Cells with two or more characteristic smoker's inclusions were considered as smoker's AM and expressed as a percentage of total AM.

The recovery of the instilled fluid was more than 50% in all lavages. There was a gradual decrease in the total number of cells. The CD4/CD8 quotient showed no significant changes and was below 1.0 in all lavages (see Table 1).

One month after TX the percentage of smoker's AM was 98%. BAL after 2, 5, 7, and 12 mo showed no change. After 18 mo a first decrease was seen, down to 78%, and after 24 mo it was down to 59%. After 36 and 42 mo, the smoker's AM had almost disappeared; only 3 and 5%, respectively, were still present (Figure 1).

View larger version (12K): [in this window] [in a new window]   Figure 1.   The percentage of smoker's alveolar macrophages (AM) in bronchoalveolar lavage fluid 2, 5, 7, 12, 18, 24, 36, and 42 mo after lung transplantation.

	DISCUSSION

TOP ABSTRACT INTRODUCTION DISCUSSION REFERENCES

This case study has shown that smoker's inclusions in AM can be detected for at least 2 yr after smoking has ceased. After 18 mo a first decrease was seen, down to 78%. After 3 yr, the smoker's AM had mostly disappeared.

Until now there has been no consensus on the criteria for identifying the smoker's AM by light microscopy. We adopted a simple classification based on two or more clear smoker's inclusions. A limitation of this method is that only cells with inclusions large enough to be seen by light microscopy are accurately counted. Also the number of particulates per AM cannot be assessed precisely. The percentage of AM containing smoker's inclusions remained relatively constant during the first 12 mo after transplantation. It is possible that the number of particles per AM decreased during this time, and that this was not detected by the method we used.

Good evidence indicates that the bone marrow is the source of tissue macrophages, including lung macrophages. Studies in animals and in humans have shown that the majority of AM are derived from peripheral blood monocytes (11- 13). The in vivo kinetics of human AM under normal steady-state conditions is not known. Thomas and coworkers (6) demonstrated that after bone marrow transplantation the AM have a life span of approximately 80 d. Although some studies have suggested that AM cannot replicate within the lungs (6, 11), several others have shown that human AM are capable of replication (14, 15). A small proportion (0.5% of AM from normal humans) incorporate thymidine, suggesting DNA synthesis. This percentage was shown to increase more than 10 times in smokers and in inflammatory states (16). Thus, to some extent the macrophage compartment in the human lung can be sustained by local cell proliferation. Along these lines, at least a proportion of the smoker's AM observed for as long as 2 yr after transplantation may be derived from AM that have divided in the lung and kept their smoker's inclusions. On the other hand, the persistence of smoking-induced changes in AM may also be explained by the fact that particles are released from old AM and rephagocytosed by young macrophages newly arrived from the recipient's bone marrow.

In conclusion, our findings have shown that smoker's inclusions in AM may persist for at least 2 yr after smoking cessation. This suggests that the time between cessation of smoking and the change of AM to a similar morphology to that of the nonsmoker is longer than the estimated life span of the these cells (6, 13). This again may indicate that the phagocytosed smoke particulates are recycled within the AM population for a considerable amount of time.