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Regional Differences in Emphysema Scores and BAL Glutathione Levels in HIV-Infected Individuals*

November 28, 2004

Study objectives: Evidence exists that HIV-seropositive individuals may be at increased risk for the development of precocious pulmonary emphysema. HIV infection is also associated with antioxidant deficiency in both the serum and lungs, and it is therefore possible that increased oxidant stress may contribute to parenchymal lung injury occurring in the setting of HIV. We sought to determine the regional distribution of emphysema and regional distribution of glutathione (GSH) concentrations among HIV- seropositive subjects with emphysema.

Design: Cross-sectional evaluation of a prospective, longitudinal study.

Setting: University teaching hospital.

Subjects/measurements: HIV-seropositive subjects without AIDS- related pulmonary complications participating in a descriptive study of lung biology in HIV-seropositive individuals. Emphysema scoring and evaluation of emphysema lobar distribution was performed among 40 subjects with emphysema. Eleven subjects underwent BAL of the right middle lobe (RML) and right upper lobe (RUL) with measurement of epithelial lining fluid (ELF) GSH in each lobe.

Results: We found that the mean emphysema scores were much higher in the upper lobes compared to the rest of the lung. Mean GSH levels were significantly greater in the RUL compared to the RML. The regional differences were present in both smokers and nonsmokers.

Conclusions: We conclude that in the setting of HIV, emphysema is more prominent and lung GSH concentrations are higher in the upper lobes. We hypothesize that the increased GSH may represent a compensatory response to increased oxidant stress in the upper lobes. (CHEST 2004; 126:1439-1442)

Key words: emphysema; glutathione; high-resolution chest CT; HIV; lung; oxidant stress

Abbreviations: ELF = epithelial lining fluid; GSH = glutathione; HRCT = high-resolution CT; RML = right middle lobe; RUL = right upper lobe

Evidence exists that HIV-seropositive individuals may be at increased risk for the development of precocious pulmonary emphysema.1-3 The precise pathophysiology underlying this phenomenon remains unclear. However, understanding the factors responsible for this accelerated process associated with HIV may he relevant to emphysema pathogenesis in general. Of note, HIV infection is associated with antioxidant deficiency in both the serum and lungs,4,5 which may make HIV-infected individuals more susceptible to oxidant stress. It is therefore possible that increased oxidant stress may contribute to parenchymal lung injury occurring in the setting of HIV.

In a study2 involving chest high-resolution CT (HRCT), we reported that HIV-seropositive individuals have higher total emphysema scores than age- and smoking-matched control subjects. In the current study, we reviewed this HRCT data to delineate the regional distribution of emphysema in the HIV-seropositive population. After finding a predilection for disease in the upper lobes, we then asked whether regional differences in oxidant stress might exist in the lungs of individuals with HIV. To address this question, we examined concentrations of glutathione (GSH), a ubiquitous antioxidant, in the epithelial lining fluid (ELF) of different lung regions.

MATERIALS AND METHODS

Subjects

All subjects were part of a cohort (n = 331) participating in a descriptive study of lung biology among HIV-seropositive individuals with no history of AIDS-related pulmonary complications. All subjects in the cohort had assessment of respiratory symptoms and pulmonary function. Some subjects, including those described in this report, participated in substudies involving more intensive testing with HRCT and/or BAL. The human subjects institutional review board approved the study, and informed consent was obtained on all individuals.

Distribution of Emphysema on HRCT

Clinical characteristics of individuals (n = 114) involved in the HRCT substudy have been previously described.2 Previously, we reported the incidence of emphysema among subjects with the most substantial changes of emphysema (n = 17).2 In the present study, we delineate the anatomic distribution of emphysema among individuals with any emphysema detected (n = 40). For comparison, we also evaluated the anatomic distribution of 14 HIV-seronegative subjects participating in HRCT substudy2 with any emphysema detected.

Chest CT was performed with a GE9800 CT scanner (GE Medical Systems: Milwaukee, WI) or a Picker PQ 2000 CT scanner (Picker International; Solon, OH) with 1.5-mm collimation at 10-mm intervals. Scans were obtained at total lung capacity. Images were reconstructed using the high-spatial-frequency algorithm and were photographed at a lung window width of 1,500 Hounsfield units (brightness level, – 700 Hounsfield units). Scans were read by two chest radiologists blinded to HIV status and clinical information. Emphysema was considered present if there was evidence of bullae, thin-walled cystic spaces, or abnormal decreases in attenuation, accompanied by vascular disruption. Emphysema severity was estimated by assigning an emphysema score (0 to 10) for each lobe according to the percentage of the lobe that was affected. For this analysis, the lingula was considered a separate lobe. The frequency of detection of emphysema in the various lobes as well as the total scores of the individual lobes were recorded.

Regional GSH Concentrations

We recruited 12 subjects for BAL of the RUL and RML. In one subject, lavage could not be analyzed because of insufficient return. This subject was excluded from analysis.

Table 1-Regional Distribution of Emphysema as Assessed by HRCT*

Bronchoalveolar Processing

Lavage fluid was processed as previously described.6 Five 20-mL aliquots of sterile saline solution were instilled into a distal segment of the RML and subsequently aspirated back into suction traps. The bronchoscope was then redirected into a distal segment of the RUL, where the procedure was repeated. Lavage fluid was immediately filtered through surgical gauze and centrifuged to separate cellular and noncellular components. Using 4 mol/L 50% 5- sulfosalicylic acid, the supernatant was acidified to a pH of 5.5 and immediately assayed for total GSH using the recycling assay of Sies and Akerboom.7 The volume of ELF was estimated by the urea dilution technique.8

Data Analysis

Differences in emphysema scores among the various lobes were analyzed by analysis of variance. Group mean differences in GSH levels between the upper and middle lobes were analyzed by paired t test. Correlations were performed using a Spearman correlation. Data represented are group means SE.

RESULTS

Regional Distribution of Emphysema

Table 1 demonstrates the frequency of emphysema detection and the mean emphysema scores among the various lobes in subjects with any detectable emphysema. As is demonstrated in Table 1, both the frequency of emphysema detection as well as the mean scores are much higher in the upper lobes compared to the rest of the lung. The upper lobe predominance is evident in both the right and left lung.

BAL GSH Levels

The clinical characteristics and ELF GSH results of the study population who underwent double-lobe lavage are shown in Table 2. As is demonstrated in Table 2, in all but one case GSH levels were higher in the RUL compared to the RML. Although not shown in Table 2, there was a slightly higher lavage return from the RML compared to the RUL (51.5 4.0 mL vs 41.6 2.6 mL; p = 0.022). However, within each lobe there was no significant correlation between lavage volume and GSH concentration.

Table 2-Baseline Clinical Characteristics and ELF GSH Levels Among HIV-Seropositive Subjects Undergoing Two-Lobe Lavage

Figure 1 demonstrates the difference in mean GSU levels in lavage samples obtained from the RML compared to those in the RUL, according to smoking history. As expected, smokers had higher GSH levels compared to nonsmokers. However, in the both smokers and nonsmokers there were higher GSH levels in the upper lobes compared to the middle lobes.

DISCUSSION

Insight into mechanisms responsible for COPD development are limited by the fact that only a minority of smokers get clinically significant disease, and by the long time course over which the disease develops. As such, examining an accelerated process as occurs in HIV may provide important insights into disease pathogenesis. Our data suggest that regional differences in oxidant stress occur in the lungs of HIV-seropositive individuals, a finding that may have relevance regarding the increased predilection for emphysema to develop in the upper lobes of both HIV- and non-HIV- seropositive smokers.

FIGURE 1. Regional ELF GSH concentrations among HIV-seropositive individuals according to smoking status. Data represented are group means SE. Values are nanomoles of GSH per milliliter of ELF.

A weakness of our study involves the relatively small number of subjects recruited for double-lobe lavage and measurement of ELF GSH. Nevertheless, BAL is an invasive procedure and was done entirely for research purposes. In addition, the differences between upper and middle lobes were highly significant, and similar changes were seen in both smokers and nonsmokers. Furthermore, 10 of 11 subjects had higher GSH levels in the upper lobes compared to the middle lobes. Thus, despite the relatively small numbers, \we believe the regional differences in GSH concentrations are real.

We hypothesize that the increased GSH concentrations in the upper lobes represents an adaptive response to a chronic increase in oxidant stress in this region of the lung, as both in vitro and in vivo data suggest that chronic exposure to oxidant stress increases lung GSH secondary to induction of GSH synthesis.9 For example, in vitro data have demonstrated that oxidant stress produced by a variety of sources, including hyperoxia, initially results in a decrease, followed by a sustained increase in GSH levels in alveolar and bronchial epithelial cells.10,11 This increase appears to be secondary to up-regulation of γ-glutamylcysteine messenger RNA.9 Similarly, 90-day exposure to ozone in an in vivo model results in a 64% increase in GSH in distal bronchioles in both rats and monkeys.12 Indeed, such an adaptive response to chronic oxidant stress has been suggested as a mechanism underlying the increase in GSH found in the ELF of long-term cigarette smokers.9

Our data do not address the etiology for increased GSH in the upper lung zones. However, it is interesting to note that both smokers and nonsmokers had increased GSH levels in the upper lobes compared to the middle lobes, suggesting that this difference is not merely a smoking-related phenomenon. One hypothesis may be related to higher oxygen tensions in the upper lobes resulting from higher ventilation/perfusion ratios. Alternatively, the upper zones may have greater exposure to inhaled toxins/pollutants causing relatively greater inflammatory cell trafficking with a resultant increase in oxidant stress.

The predilection for pulmonary emphysema to develop in the upper lobes in non-HIV-infected smokers is a well-recognized clinical phenomenon.13-15 Although the etiology is unknown, a number of hypotheses have been provided.13 For example, there may be less effective clearance of inhaled material from the upper lobes. Another hypothesis relates to slower transit time of leukocytes in the upper lobes compared to the lower lobes. This may allow a longer time for leukocytes to secrete inflammatory mediators. Each of these scenarios may be associated with increased oxidant stress in the upper lobes, and is consistent with our findings.

Whether our findings in the HIV population have relevance to the non-HIV population is not known. However, the similar tendency to develop upper lobe disease suggests that our findings among HIV- seropositive individuals may have relevance to the general population of smokers.

* From the Division of Pulmonary and Critical Care Medicine (Drs. Diaz, Wewers, Wade, and Clanton, and Ms. Hart), Department of Internal Medicine, and Department of Radiology (Mr. King), The Ohio State University, Columbus, OH.

Supported by National Institutes of Health, National Heart, Lung, and Blood Institute grants RO1 49730 and RO1 53229, and The Ohio State University General Clinical Research Center grant MO1 RR00034.

REFERENCES

1 Diaz PT, Clanton TL, Pacht ER. Emphysema-like pulmonary disease associated with human immunodeficiency virus infection. Ann Intern Med 1992; 116:124-128

2 Diaz PT, King MA, Pacht ER, et al. Inereased susceptibility to pulmonary emphysema among HIV-seropositive smokers. Ann Intern Med 2000; 132:369-372

3 Guillemi SA, Staples CA, Hogg JC, et al. Unexpected lung lesions in high resolution computed tomography (HRCT) among patients with advanced HIV disease. Eur Respir J 1996; 9:33-36

4 Pacht ER, Diaz P, Clanton T, et al. Serum vitamin E decreased in HIV-seropositive subjects over time. J Lab Clin Med 1997; 130:293- 296

5 Pacht ER, Diaz P, Clanton T, et al. Alveolar fluid glutathione decreases in asymptomatic HIV-seropositive subjects over time. Chest 1997; 112:785-788

6 Hunninghake GW, Gadek JE, Kawanami O, et al. Inflammatory and immune processes in the human lung in health and disease: evaluation by bronchoalveolar lavage. Am J Pathol 1979; 97:149-206

7 Sies H, Akerboom TPM. Glutathione disulfide (GSSG) efflux from cells and tissues. Methods Enzymol 1984; 105:445-451

8 Rennard SI, Basset G, Lecossier D, et al. Estimation of volume of epithelial lining fluid recovered by lavage using urea as marker of dilution. J Appl Physiol 1986; 60:532-538

9 Rahman I, MacNee W. Lung glutathione and oxidative stress: implications in cigarette smoke-induced airway disease. Am J Physiol 1999; 277:L1067-L1088

10 Hatcher EL, Chen Y, Kang A. Cadmium resistance in A549 cells correlates with elevated glutathione content but not antioxidant enzymatic activities. Free Radic Biol Med 1995; 19:805-812

11 Pietarinen-Runtti P, Raivio KO, Saksela M, et al. Antioxidant enzyme regulation and resistance to oxidants of human bronchial epithelial cells cultured under hypoxic conditions. Am J Respir Cell Mol Biol 1998; 19:286-292

12 Duan X, Buckpitt A, Pinkerton K, et al. Ozone induced alterations in glutathione in lung subcompartments of rats and monkeys. Am J Respir Cell Mol Biol 1996; 14:70-75

13 Fraser R, Muller N, Coleman N, et al. Diagnosis of diseases of the chest. Philadelphia, PA: W.B. Saunders, 1999

14 Anderson AE Jr, Foraker AG. Centrilobular emphysema and panlobular emphysema: two different diseases. Thorax 1973; 28:547- 550

15 Sweet HC, Wyatt JP, Fritsch AJ, et al. Panlobular and centrilobular emphysema: correlation of clinical findings with pathologic patterns. Ann Intern Med 1961; 55:565

Philip T. Diaz, MD; Mark D. Wewers, MD; Mark King; Joyce Wade, MD; Judy Hart; and Thomas L. Clanton, MD

Manuscript received December 11, 2003; revision accepted May 26, 2004.

Reproduction of this article is prohibited without written permission from the American College of Chest Physicians (e-mail: permissions@chestnet.org).

Correspondence to: Philip T. Diaz, MD, 201 Heart Lung Research Institute, 473 W. Twelfth Ave, Columbus, OH 43210; e-mail: diaz- 1@medctr.osu.edu

Copyright American College of Chest Physicians Nov 2004




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