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Imperial College London, Hammersmith Campus, London, United Kingdom
Correspondence and requests for reprints should be addressed to Hazel A. Jones, Ph.D., Imperial College London, Hammersmith Campus, London W12 0NN, UK. E-mail: hazel.jones{at}imperial.ac.uk
ABSTRACT
Acute and chronic lung diseases are almost invariably associated with some degree of inflammation. Cells that evolved as an effective mechanism to counter infection and heal lung tissue may, in some circumstances, themselves be partially responsible for the pathogenesis of chronic lung disease that leads to irreversible lung damage and loss of lung function. Although standard measurements of lung function can document the progression of disease, the contributions of the numerous interacting elements to the process are difficult to measure in life. The use of molecular imaging techniques allows the different components of the inflammatory response to be monitored in situ in humans. In particular, positron emission tomography of selected markers targeted to specific cells and biochemical pathways can provide accurate measurements of disease activity, enabling a better understanding of inflammatory processes at all stages of disease. The practicability of sequential measurements allows one to monitor the natural history of different lung diseases. More importantly, imaging provides a unique tool for quantification of the modulation of discrete and specific aspects of inflammatory lung disease by targeted interventions. This should facilitate the development of new treatment strategies with better specificity for key elements of each disease.
Key Words: positron emission tomography • neutrophils • macrophages • extracellular matrix
The pulmonary inflammatory response is a highly coordinated and effective mechanism to combat infection and normally resolving with little, if any, lasting damage. It is a complex process involving the very precise orchestration of the behavior of a number of different cell types. An inadequate inflammatory response to infection (e.g., in individuals with reduced immunity) may lead to acute lung injury or pneumonia requiring hospitalization. Poor regulation of the response can result in the persistence of inflammation, which is implicated in the pathogenesis of a number of lung diseases, including chronic obstructive pulmonary disease (1) and asthma (2), diseases that are becoming increasingly common in the developed world. Ongoing inflammation results in remodelling of the lung architecture with increased extracellular matrix deposition and the consequent compromise of the gas exchanging function of the lung. Although the development of chronic obstructive pulmonary disease is almost invariably associated with cigarette smoking, once established, the inflammatory processes continue even after smoking cessation. It is important to determine the key events driving the chronicity of each disease so that rational and specific interventions for treatments of inappropriate pulmonary inflammation may be developed and applied.
The many different and interacting processes contributing to pulmonary inflammation occur throughout the lung tissue. Although samples can be obtained from the blood and the airspaces for analysis of inflammatory mediators, the underlying mechanisms are still in question. External imaging of specific labeled markers offers methods for the measurement of cell behavior and biochemical events in situ. Markers can be developed for detection by both MRI and nuclear medicine imaging techniques, although the sheer chemical amounts of the labeled markers required to obtain a signal by MRI are a limitation to the variety of labels that can be used. External detection of tracer doses of intravenously injected compounds using a number of suitable isotopes is possible by 
-scintigraphy. Positron emission tomography (PET) of markers labeled with positron-emitting isotopes offers even greater sensitivity and the ability to obtain quantifiable data throughout the lung.
The contribution of a number of the components involved in inflammation in lung tissue can be measured by PET and other molecular imaging techniques. Further markers are being developed to targets that have been identified as important in determining the outcome of the inflammatory process.
ADHESION MOLECULE EXPRESSION
A prerequisite for recruiting immune cells from the blood is the expression of adhesion molecules by the vascular endothelium in response to inflammatory mediators, such as lipopolysaccharide. Antibody fragments to E-selectin and intercellular adhesion molecule 1 have been labeled with 111In and imaged by -scintigraphy. Unfortunately, significant uptake of the injected fragments by the liver and kidney limit the usefulness of the technique (3). It is to be hoped that improved markers, ideally labeled for PET, will be developed either from various selectin inhibitors or antibody fragments. These would enable monitoring and assessment of therapeutics directed toward selectins in lung disease.
WHITE CELL SEQUESTRATION AND EMIGRATION
White blood cells, principally neutrophils, are in the forefront of any inflammatory response. To track their movements, these cells can be obtained from samples of peripheral blood and labeled with radioisotopes in vitro. Mixed white cells can be further separated to obtain granulocytes and monocytes. The extreme radiosensitivity of lymphocytes renders them unsuitable for ex vivo labeling. On reinjection into the donor their distribution and kinetics can be monitored by imaging. The use of this technology has yielded a wealth of information about white cell kinetics in infection and chronic inflammation. Lung images obtained soon after injection show radioactivity localized to the lungs even in healthy individuals. These early images probably represent margination in lung capillaries because the cells are almost inevitably partially activated (primed) during the labeling procedure. In the absence of further stimulation from inflammatory mediators released from the infected site, the cells deprime (4), return to a quiescent state, and rejoin the circulation to be removed in due course by the liver and spleen. This inadvertent priming of cells labeled in vitro may enhance the clinical usefulness for localization of infection, because neutrophils require priming before they respond to a secondary stimulus to mount a further functional response, such as degranulation, phagocytosis, or respiratory burst activity (5). Positive lung images obtained on the day after injection reflect migration from the blood into the tissue or airspaces indicative of the ongoing neutrophil trafficking associated with inflammation, such as occurs in bronchiectasis.
Patients with lobar pneumonia seldom have a positive white cell scan (6), although neutrophils are abundant in the lung. Animal studies have shown that migration in response to Streptococcus pneumoniae is immense but transient, falling to baseline values by 12 hours postchallenge. Lack of a migration signal does not equate with absence of neutrophils in the lung (7).
Most white cell scans are carried out using -scintigraphy. The advent of positron emitters for cell labeling, however, improves sensitivity and renders possible the quantification of the regional signal from sequestered and migrated cells (8).
IN VIVO CELL LABELING
Antibodies or antibody fragments against cell surface antigens labeled with suitable isotopes have potential for in vivo measurement of different cell types and phenotypes. This technology is in its infancy, however, and results so far are disappointing: a study of an antibody to surface antigen on granulocytes showing that clearance from the blood into inflamed areas seemed to be nonspecific (9). Development of improved radiolabeled antibodies for tumor imaging and therapy should be adaptable to other fields of investigation including cells of the immune system (10).
NEUTROPHIL ACTIVATION
PET of intravenously injected 18F-labeled fluorodeoxyglucose gives an image of regional glucose metabolic requirements. This methodology is widely used in oncology to localize tumor activity and identify metastases. In inflammatory lung disease autoradiographic studies of tissues from animal models have shown the signal to be remarkably specific to activated neutrophils (11). This is probably because these cells are able to upregulate their glucose metabolism to a much greater extent than are other cells. This measurement of neutrophil behavior at the site of disease has been shown to be dissociated from neutrophil migration. Figure 1 (p. 513) shows very high levels of activation measured in human pneumonia in the absence of migration and lower levels of activation in bronchiectasis despite ongoing migration (12).
A summary of 18F-labeled fluorodeoxyglucose PET values from a number of studies is shown in Figure 2. Pulmonary 18F-labeled fluorodeoxyglucose PET scanning in humans has shown high 18F-labeled fluorodeoxyglucose uptake in infection (12), chronic obstructive pulmonary disease (13), sarcoidosis (14), and acute lung injury (15) but not in stable asthma (13), cystic fibrosis (16), or rejection following transplantation (17). Patients with head injury, who are at risk of developing acute respiratory distress syndrome, but who had no lung symptoms at the time of the scan had a surprisingly high pulmonary uptake of 18F-labeled fluorodeoxyglucose (18). This signal may reflect sequestration of primed neutrophils in lung capillaries. In vitro studies in isolated human neutrophils have demonstrated that the uptake of deoxyglucose is increased to the same extent in cells that are only primed or primed and stimulated (19). In the patients with head injury the absence of inflammatory stimuli in the lungs does not result in the additional activation, which leads to tissue damage. It does indicate the vulnerability of these patients while on ventilatory support in intensive care, however, because although the neutrophils remain in a primed state any additional stimulus precipitates actual tissue damage.
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Resident macrophages, which comprise most of the cells inhabiting the alveolar spaces, help initiate the inflammatory response to bacterial or toxic insult by the controlled release of mediators. Monocytes are subsequently recruited from the blood and differentiate into inflammatory macrophages, which orchestrate the course of the inflammation and are likely a pivotal point in determining resolution or progression to permanent lung damage. Interaction between macrophages with fibroblasts can lead to the production of extracellular matrix proteins and scarring.
The ligand PK11195 binds to receptors that are present in large numbers in macrophages (20). The 11C-labeled R-isomer of this ligand has been used in animal models to monitor accumulation and clearance from the lung of instilled particles by macrophages and has shown that clearance of nonscarring amorphous silica is efficient and rapid, whereas clearance of microcrystalline silica by macrophages is delayed (Figure 3 [p. 513]) (21). It may well be the compromised ability of the macrophage to clear particles efficiently from the lungs that results in permanent lung damage. Some patients with chronic obstructive pulmonary disease or asthma shown a higher than normal uptake of 11C-R-PK11195 (13), as do cigarette smokers with asthma (22).
Patients with lung fibrosis, a feature of which is the presence of interstitial macrophages (23), have unexpectedly low uptake (24). The signal detected by 11C-R-PK11195-PET scanning is, however, the product of cell numbers and receptor numbers per cell. Subsequent in vitro binding studies demonstrated that the Bmax of PK11195 to cells in bronchoalveolar lavage samples from lung fibrosis patients is much lower than that in samples from normal subjects (unpublished data). A single PK11195 scan obtained in patients with lung fibrosis is difficult to interpret, but repeat scans following therapeutic intervention would enable the effect of treatment to be assessed.
CELL DEATH
There are numerous ways in which a cell can die. Programmed cell death, such as apoptosis, is a mechanism by which the cells can be safely removed from an area of inflammation once they have fulfilled their function without releasing their histotoxic contents (7). This limitation of tissue damage is critical to the resolution phase of inflammation. It is an antiinflammatory mechanism and contrasts strongly with necrosis during which the cell breaks up and its toxic contents are released into the local environment causing potentiation of inflammation. Annexin V has been radiolabeled with 18F for PET imaging studies (25) and applied in inflammatory conditions to measure cell death. Unfortunately, it targets both apoptotic and necrotic cells. Specific markers are being developed that should allow specific identification of apoptosis in vivo. This will greatly aid the ability to monitor the critical balance point between resolution and progression to chronic inflammation. Interventions targeted to this process would be a major step forward in the control of pulmonary inflammatory disease.
CONTROL OF THE EXTRACELLULAR MATRIX
Disruption of the lung architecture is a major consequence of chronic inflammation. The maintenance of healthy lung architecture is accomplished by an active turnover of extracellular matrix proteins, with about 10% of the major component collagen being replaced every day (26). Any increase in synthesis relative to degradation results in the development of fibrosis, but the complex interactions involved are poorly understood (27). There is a pressing need for validated markers for these processes to advance the development of treatment in various forms of lung fibrosis. At present there are few treatments available that make any significant impact on these debilitating diseases (28, 29). Radiolabeled markers are now available that can be used to measure discrete components on both sides of the balance.
18F-labeled analogs of proline have enabled collagen synthesis to be monitored in vivo (30, 31) and uptake is increased during active scarring in animal models of lung fibrosis (32, 33). Figure 4 (p. 514) shows localization of 18F-labeled fluoroproline to fibrosing rabbit lung imaged by PET together with microautoradiography of lung tissue samples taken 1 hour after injection of tritiated proline, which shows localization to fibroblasts in the actively scarring region. The rate of 18F-proline uptake most likely reflects an active synthesis of collagen, although the 90-min scanning time is too short for the proline to be incorporated in the collagen itself.
The other side of the extracellular matrix balance is the degradation of proteins by metalloproteinase enzymes and their control by specific tissue inhibitors. The fibrosis that results from chronic inflammation is accompanied by increases in collagen synthesis but also an increase in the enzymes controlling degradation (34). Inhibitors to these enzymes are have been labeled with 18F (35) and 11C for oncology imaging with PET. Unfortunately, although the markers localized in tumors, this seemed to be related to nonspecific binding (36). The control of these enzymes involves a group of endogenous protein inhibitors known as tissue inhibitors of metalloproteinases, which have also been radiolabeled for imaging studies but not yet validated in vivo (37). The balance of these enzymes is a major determinant of extracellular matrix control (38).
CONCLUSIONS
Nuclear imaging techniques enable the noninvasive investigation of events occurring in tissue inaccessible by any other means. PET is especially useful because it has a high sensitivity and has the added advantage of being quantifiable. Currently, several markers are available for processes involved in pulmonary inflammation and others with considerable potential are being developed. The use of this technology will contribute to the increased understanding of the mechanisms involved in inflammatory lung disease and enable assessment of therapeutic interventions targeted at pivotal events in the inflammatory process.
FOOTNOTES
The color figures for this article are on pp. 513–514.
Conflict of Interest Statement: H.A.J. has received "no strings attached" research donations of £150,000 from GlaxoSmithKline (GSK) for research into pulmonary inflammation imaging in 2004 and a research grant of £637,295 from an Imperial College/GSK scheme in 2005 and a research grant of £193,228 from Pfizer Ltd. (jointly with other investigators).
(Received in original form July 27, 2005; accepted in final form August 9, 2005)
REFERENCES
) radioligand 11C-PK11195 measured by positron emission tomography (PET) in fibrosing alveolitis due to systemic sclerosis. Am J Respir Crit Care Med 2003;167:A474.Related articles in Proceedings of the American Thoracic Society:
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