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99mTc-Labeled Interleukin-8 for Scintigraphic Detection of Pulmonary Infections

99mTc-Labeled Interleukin-8 for Scintigraphic Detection of Pulmonary Infections

preliminary reports 99m Tc-Labeled Interleukin-8 for Scintigraphic Detection of Pulmonary Infections* Huub J. J. M. Rennen, PhD; Chantal P. Bleeker-...

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preliminary reports 99m

Tc-Labeled Interleukin-8 for Scintigraphic Detection of Pulmonary Infections*

Huub J. J. M. Rennen, PhD; Chantal P. Bleeker-Rovers, MD; Jullie¨tte E. M. van Eerd; Cathelijne Frielink; Wim J. G. Oyen, MD; Frans H. M. Corstens, MD; and Otto C. Boerman, PhD

Background: Interleukin (IL)-8 is a chemotactic cytokine that binds with high affinity to receptors on neutrophils. Previously we showed that 99mTc-labeled IL-8 is highly suitable for scintigraphic imaging in rabbit models of IM infection and of colitis. Study design: 99mTc-labeled IL-8 was tested for its potential to image pulmonary infection in three experimental rabbit models: aspergillosis in immunocompromised rabbits, pneumococcal (Gram-positive) pneumonia, and Escherichia coli-induced (Gram-negative) pneumonia in immunocompetent rabbits (four rabbits in each group). A derivative of hydrazinonicotinamide was used as bifunctional coupling agent to label IL-8 with 99mTc. Biodistribution of 99mTc IL-8 was determined both by gamma-camera imaging and by counting dissected tissues at 6 h after injection. Results: 99mTc IL-8 enabled early (within 2 h after injection) and excellent visualization of localization and extent of pulmonary infection in each of the three experimental models of pulmonary infection. Uptake of 99mTc IL-8 in the infected lung and the contralateral lung was (in percentage of the injected dose per gram of tissue ⴞ SEM) at 6 h after injection 0.63 ⴞ 0.12 and 0.12 ⴞ 0.02 (aspergillosis), 0.89 ⴞ 0.04 and 0.44 ⴞ 0.04 (pneumococcal pneumonia), and 1.53 ⴞ 0.12 and 0.36 ⴞ 0.06 (E coli pneumonia), respectively. In the E coli model, uptake of 99mTc IL-8 in the focus of infection even exceeded uptake in the kidneys, the main clearing organs. Conclusion: 99mTc IL-8 offers many advantages over the conventionally used radiopharmaceuticals to image pulmonary infection, 67Ga citrate and radiolabeled leukocytes, ie, rapid and easy preparation, short time span between injection and imaging, low radiation burden and, most importantly, clear delineation of the infectious foci. (CHEST 2004; 126:1954 –1961) Key words: experimental animal models; interleukin-8; pulmonary inflammation; radionuclide imaging;

99m

Tc

Abbreviations: HYNIC ⫽ hydrazinonicotinamide; IL ⫽ interleukin; MBq ⫽ megabecquerel; PBS ⫽ phosphate-buffered saline solution

diagnosis of infection presents a major A dequate challenge to the clinician. Early and accurate detection of infection is crucial to enable adequate

*From the Department of Nuclear Medicine, University Medical Center Nijmegen, Nijmegen, the Netherlands. Manuscript received January 27, 2004; revision accepted May 19, 2004. Reproduction of this article is prohibited without written permission from the American College of Chest Physicians (e-mail: [email protected]). Correspondence to: Huub J. J. M. Rennen, PhD, Department of Nuclear Medicine, University Medical Center Nijmegen, PO Box 9101, 6500 HB Nijmegen, the Netherlands; e-mail: [email protected] nucmed.umcn.nl 1954

treatment. In cases suspected of pulmonary infection, chest radiography and CT provide high-quality anatomic information. However, early stages of pulmonary infection, in the absence of structural changes in the lungs, are difficult to diagnose by these methods. Moreover, scar tissue formation may complicate the interpretation of the images. Also, in immunosuppressed patients with pneumonia, chest radiograph findings are often minimal or even absent. An alternative approach for detection of pulmonary infection is radionuclide imaging. Scintigraphic imaging does not depend on structural Preliminary Reports

changes in the lung induced by the presence of the invaded microorganisms, but on physiologic changes in the lung as induced by these microorganisms. Therefore, radionuclide imaging can be complementary to the conventional anatomic imaging modalities. In the United States, 67Ga citrate, 111In-labeled, and 99mTc-labeled leukocytes are the only approved radiopharmaceuticals for imaging of infection. 67Ga citrate binds, once injected IV, to circulating transferrin. This complex extravasates at the site of infection due to the locally enhanced vascular permeability.1 There are several shortcomings in the use of 67 Ga citrate that limit its clinical application. For instance, this radiopharmaceutical has unfavorable imaging characteristics (long physical half-life and high-energy ␥ photons), causing poor image quality and high-radiation absorbed doses.2 Moreover, the interval between injection and imaging is relatively long (several days). Techniques using isolated leukocytes, labeled either with 111In or 99mTc, have been introduced in the 1970s and 1980s,3– 6 and are generally adopted in nuclear medicine as techniques for infection imaging. Ex vivo labeling requires the withdrawal of blood from the patient, purification of leukocytes, labeling, and reinjection of the radiolabeled cells. Radiolabeled leukocytes can be used to image infection due to the fact that leukocytes, even after ex vivo labeling, have the capacity to migrate to the inflamed area. Disadvantages of this approach are, among others, a laborious preparation (3 h) and handling of potentially contaminated blood.7 These have stimulated investigators to search for alternative methods that would allow labeling of leukocytes in the circulation. Instead of isolating WBCs from a patient and labeling the cells ex vivo, new methods now aim to label WBCs in vivo. Radiolabeled interleukin (IL)-8 is an interesting candidate to replace radiolabeled leukocytes by in vivo labeling of neutrophils. Neutrophilic granulocytes are known to express two types of IL-8 receptors, CXCR1 and CXCR2, abundantly.8 IL-8 binds these receptors with high affinity (0.3 to 4 nmol/ L).9 –12 Recently, we described the development of a 99m Tc-labeled IL-8 preparation using hydrazinonicotinamide (HYNIC) as a bifunctional coupling agent.13–16 This preparation showed good characteristics for imaging of infection and inflammation in various experimental models including IM infection,13,16 colitis,14 and osteomyelitis.15 Specific uptake of 99mTc IL-8 in infected tissue was demonstrated by comparing its uptake to that of 99mTclabeled lysozyme, a control protein with similar size and charge but with no specific interaction with receptors on neutrophils.13 Abscess uptake of 99mTc www.chestjournal.org

IL-8 was ⬎ 10 times higher than abscess uptake of 99m Tc lysozyme. Furthermore, neutrophil-driven specificity was demonstrated by comparing abscess uptake of 99mTc IL-8 in neutropenic rabbits with that in rabbits with normal neutrophil counts.16 In neutropenic rabbits, uptake of 99mTc IL-8 in the abscess is only 10%, as compared to abscess uptake in normal rabbits. In the present study, 99mTc-labeled IL-8 was tested for its potential to image pulmonary infection. Three different experimental rabbit models were introduced in order to simulate a broad range of clinically relevant applications: aspergillosis in immunocompromised rabbits, pneumococcal pneumonia, and Escherichia coli-induced pneumonia in immunocompetent rabbits. Invasive pulmonary aspergillosis is an important cause of infectious morbidity and mortality in patients with granulocytopenia following chemotherapy. Streptococcus pneumoniae is a representative of the group of Gram-positive bacteria, and is the leading cause of community-acquired pneumonia. E coli-induced pneumonia is an example of a pneumonia caused by Gram-negative bacteria, and is frequently encountered as a nosocomial infection. Severe S pneumoniae and E coli pneumonia infections have significant morbidity and mortality rates.

Materials and Methods Human recombinant IL-8 was kindly provided by Dr. I. Lindley (Novartis; Vienna, Austria). Tricine (N-[Tris(hydroxymethyl)-methyl]glycine) was purchased from Fluka (Buchs, Switzerland), and nicotinic acid was purchased from Sigma-Aldrich (St. Louis, MO). Conjugation of HYNIC to IL-8 HYNIC was synthesized essentially as described previously.17,18 The IL-8 HYNIC conjugate was prepared as described previously.13,19 Briefly, in a 1.5-mL vial, 5 ␮L 1 mol/L NaHCO3, pH 8.2, was added to 35 ␮L IL-8 (5 mg/mL). Subsequently, a threefold molar excess of HYNIC in 5 ␮L dry dimethyl sulfoxide was added drop-wise to the mixture. After incubation for 5 min at room temperature, the reaction was stopped by adding an excess of glycine (200 ␮L, 1 mol/L in phosphate-buffered saline solution [PBS]). To remove excess unbound HYNIC, the mixture was extensively dialyzed against PBS (0.1- to 0.5-mL dialysis cell, 3,500 molecular weight cut-off; Pierce; Rockford, IL). Dialyzed samples of circa 8 ␮g IL-8 HYNIC were stored at – 20°C. 99m

Tc Labeling of HYNIC-Conjugated IL-8

To a thawed sample of 8 ␮g IL-8 HYNIC was added 0.2 mL solution of tricine and nicotinic acid (125 mg/mL and 12.5 mg/mL, respectively, in PBS, adjusted to pH 7.0 with 2 mol/L NaOH), 20 ␮L freshly prepared tin (II) solution (10 mg SnSO4 in 10 mL nitrogen-purged 0.1 N HCl), and 400 megabecquerels (MBq) 99mTcO4- in saline solution. The mixture was incubated at CHEST / 126 / 6 / DECEMBER, 2004

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70°C for 30 min. The radiochemical purity was determined by instant thin-layer chromatography on ITLC-SG strips (Gelman Laboratories; Ann Arbor, MI) with 0.1 mol/L citrate, pH 6.0, as the mobile phase. Following the labeling reaction, the reaction mixture was diluted with PBS, 0.5% bovine serum albumin to a concentration of 40 MBq/mL, ready for IV administration in the rabbits. A description of an extensive in vitro characterization (high-performance liquid chromatography, stability, receptor binding assays) has been given elsewhere.19 Animal Studies All animal experiments were carried out in accordance with the guidelines of the local animal welfare committee. Female New Zealand White rabbits weighing 2.3 to 2.8 kg were used in these experiments. Animals were housed individually and fed standard laboratory chow and water ad libitum. Pulmonary Infection Models Pulmonary Aspergillosis: Induction of aspergillosis was based on the animal model described by Groll et al,20 with a few modifications. Four female New Zealand White rabbits received injections of cytarabine to reduce their neutrophil counts. Cytarabine was administered daily IV (25 mg/kg at approximately 525 mg/m2) on day – 4 through day – 1 prior to the injection of 99m Tc IL-8. At day – 3, after the second dose of cytarabine, the inoculation of Aspergillus fumigatus was performed. A fumigatus was isolated from an immunocompromised patient with a diagnosis of aspergillosis. Before inoculation, the animals were sedated with a subcutaneous injection of 0.7 mL fentanyl, 0.315 mg/mL, and fluanisone, 10 mg/mL. The rabbits were anesthetized with a mixture of isoflurane, nitrous oxide, and oxygen, and were placed on the surgical table. The inoculum of 1 ⫻ 108 conidia in 250 ␮L PBS was administered intratracheally into the left lung, together with 50 ␮L 5% Evans blue dye (SigmaAldrich; Zwijndrecht, the Netherlands) via a syringe attached to a polyethylene 0.76- by 1.22-mm catheter (Maxxim Medical; Oss, the Netherlands). The use of Evans blue dye enabled, for dissection purposes, the localization of the spot where the infection was initiated. Pneumococcal pneumonia and E coli-Induced Pneumonia: Pneumonia was initiated in female New Zealand White rabbits 1 day before injection of 99mTc IL-8. The animals were sedated and anesthetized as described above. Four rabbits were inoculated intratracheally with 1 ⫻ 109 cfu of S pneumoniae and four rabbits with 1 ⫻ 109 E coli together with 50 ␮L 5% Evans blue dye as described above. Scintigraphic Imaging of Rabbits with Pulmonary Infection Twenty MBq 99mTc-labeled IL-8 (0.4 ␮g protein, 0.5 mL) were injected via the ear vein in rabbits with aspergillosis 2 days after installation of pulmonary aspergillosis, and in rabbits with S pneumoniae and E coli pulmonary infection 1 day after induction of pulmonary infection (four rabbits in each group). Three rabbits in each group were randomly selected and used for gammacamera imaging. These rabbits were immobilized in a mold, placed prone on the gamma-camera, and images were acquired at 1 to 5 min, 2 h, 4 h, and 6 h after injection with a single-head gamma-camera (Orbiter; Siemens Medical Systems; Hoffman Estates, IL) equipped with a parallel-hole, low-energy, allpurpose collimator. Images were obtained with a 15% symmetrical window over the 140 keV energy peak of 99mTc. After acquisition of 200,000 to 300,000 counts, the images were digitally stored in a 256 ⫻ 256 matrix. Scintigraphic images were 1956

analyzed quantitatively by drawing regions of interest over the affected region of the left lung (target) and a similar region over the contralateral unaffected right lung (background). Target-tobackground ratios were calculated. After completion of the final imaging session (6 h after injection), all rabbits were killed with a lethal dose of pentobarbital. Samples of blood, muscle, affected lung, unaffected contralateral lung, spleen, liver, kidneys, and intestines were collected. The dissected tissues were weighed, and the activity in the samples was measured in a ␥-counter. To correct for radioactive decay, injection standards were counted simultaneously. The measured activity in samples was expressed as a percentage of injected dose per gram of tissue. Affected lung-to-control lung ratios and affected-lung-to-blood ratios were calculated. For comparison, two rabbits with pneumococcal pneumonia were imaged with 67Ga citrate (Mallinckrodt; Petten, the Netherlands), using the same gamma-camera as above, equipped with a parallel-hole, medium-energy collimator. Images were acquired at 1 to 5 min, 2 h, 4 h, 6 h and, additionally, 24 h after injection of 18 MBq 67Ga citrate. For histologic examination, samples of affected and unaffected lung were fixed in formalin and embedded in paraffin; 5 ␮m sections were cut and stained with hematoxylin-eosin and/or Grocott methenamine silver (aspergillosis only) for light microscopic examination. Statistical Analysis All mean values are expressed as percentage of injected dose per gram of tissue, percentage of injected dose, or ratios ⫾ 1 SEM. Data were analyzed statistically using the one-way analysis of variance test with Tukey-Kramer multiple comparisons posttest (GraphPad Instat 3.00 Win 95; GraphPad; San Diego, CA).

Results 99m

Tc Labeling of IL-8

The radiochemical purity of the 99mTc IL-8 preparation exceeded 98%, as determined by instant thin-layer chromatography, excluding the need for further purification, in line with our previous findings.18 The specific activity of the 99mTc IL-8 preparations used in these studies was high, approximately 50 MBq/␮g IL-8. Animal Studies Pulmonary Infection Models: Cytarabine treatment of rabbits in the aspergillosis group resulted in an average reduction of total WBC counts of 61 ⫾ 7%. Histologic examination revealed the presence of abundant A fumigatus conidia in lung tissue, and a moderate leukocyte infiltration similar to what was found in our rat aspergillosis model.21 Massive leukocyte infiltration in infected lung tissue in both pneumococcal pneumonia and E coli-induced pneumonia was observed. Scintigraphic Imaging of Pulmonary Infection in Rabbits: Figure 1 shows the scintigrams acquired between 0 h and 6 h after injection of 99mTc IL-8. The Preliminary Reports

Figure 1. Images of rabbits with pulmonary infection in the left lung at 1 to 5 min, 2 h, 4 h, and 6 h after injection of 99mTc IL-8, showing a typical example of aspergillosis (top row), pneumococcal pneumonia (middle row), and E coli-induced pneumonia (bottom row). An arrow in the 1- to 5-min E coli infection image indicates a remarkable photopenic area in the affected part of the lung.

pulmonary infections were clearly delineated in all three models. Images acquired immediately after injection occasionally showed the affected lung area as a photopenic area: high uptake of 99mTc IL-8 in the contralateral unaffected lung and relatively low uptake in the affected part of the lung, resulting in a photopenic area (arrow, Fig 1), suggesting that the affected area is less well perfused, and accumulation of 99mTc IL-8 in the focus of infection occurs in the first hours after injection. Quantitative analysis of the images shown in Figure 2 showed that the target-to background (affected lung to nonaffected lung) ratios improved with time in all models of pulmonary infection up to 3.4 ⫾ 0.3 in the aspergillosis model, 3.2 ⫾ 0.5 in the S pneumoniae model, and 4.5 ⫾ 0.25 in the E coli model. These values were not significantly different. www.chestjournal.org

The biodistribution of 99mTc IL-8 in the three rabbit models of pulmonary infection is presented in Table 1. 99mTc IL-8 cleared faster from the blood in rabbits with aspergillosis as compared to rabbits with S pneumoniae or E coli infection, resulting in a significantly lower percentage of residual activity in the blood at 6 h after injection (p ⬍ 0.01). Uptake of 99m Tc IL-8 in the contralateral healthy lung lobe was lowest in the aspergillosis model (p ⬍ 0.01). Uptake of the radiolabel in the infected lung was similar in the aspergillosis and the S pneumoniae models. The uptake in the infected lung in the E coli model surpassed the uptake in the other models in this area (p ⬍ 0.01). In the aspergillosis model, the uptake in the spleen was significantly lower than in the S pneumoniae model (p ⬍ 0.05). Kidneys were the main clearing organs in all three models, and kidney CHEST / 126 / 6 / DECEMBER, 2004

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Figure 2. Target-to-background (affected lung to nonaffected lung) ratios from quantitative analysis of the images of rabbits with pulmonary infection at 1 to 5 min, 2 h, 4 h, and 6 h after injection of 99mTc IL-8.

uptake was highest in the aspergillosis model (p ⬍ 0.05). In the E coli model, the uptake in the infected lung was even higher than uptake in the kidneys. Figure 3 summarizes uptake of 99mTc IL-8 in (affected and nonaffected) lungs and spleen. Physiologic uptake of 99mTc IL-8 in nonaffected lungs was threefold lower in immunocompromised rabbits with aspergillosis than in immunocompetent rabbits with S pneumoniae or E coli infection. Spleen uptake was twofold higher in immunocompetent rabbits. Figure 4 shows for comparison scintigraphic images of a rabbit with pneumococcal pneumonia

Table 1—Biodistribution of 99mTc IL-8 in Three Rabbit Models of Pulmonary Infection (Four Rabbits per Model) at 6 h After Injection* Variables

Aspergillosis

S pneumoniae

E coli

Blood 0.0069 ⫾ 0.0022 0.041 ⫾ 0.008 0.035 ⫾ 0.007 Muscle 0.0010 ⫾ 0.0002 0.0032 ⫾ 0.0009 0.0020 ⫾ 0.0001 Lung abscess 0.63 ⫾ 0.12 0.89 ⫾ 0.04 1.53 ⫾ 0.12 Lung control 0.12 ⫾ 0.02 0.44 ⫾ 0.04 0.36 ⫾ 0.06 Spleen 0.56 ⫾ 0.18 1.31 ⫾ 0.18 1.16 ⫾ 0.20 Kidney 1.56 ⫾ 0.08 1.09 ⫾ 0.09 1.25 ⫾ 0.04 Liver 0.059 ⫾ 0.006 0.069 ⫾ 0.013 0.083 ⫾ 0.012 Intestine 0.010 ⫾ 0.001 0.013 ⫾ 0.001 0.015 ⫾ 0.001 Lung abscess/ 5.5 ⫾ 1.6 2.1 ⫾ 0.2 4.4 ⫾ 0.4 control Lung abscess/ 66 ⫾ 11 26 ⫾ 8 50 ⫾ 10 blood *Data are presented as percentage of injected dose per gram of tissue ⫾ SE or as ratios thereof. 1958

after injection of 67Ga citrate, illustrating the rather suboptimal characteristics of this radiopharmaceutical. Discussion 99m

Tc IL-8 has favorable characteristics as an infection imaging agent: rapid accumulation in target tissue and rapid clearance from blood and nontarget tissues. The activity is mainly cleared via the kidneys, which is an advantage over hepatobiliary clearance; high activity in liver and bowel would have made this agent less suitable for imaging of the abdominal region. This study in particular showed the suitability of 99mTc IL-8 to delineate infectious foci in rabbits with pulmonary infection. In previous studies with 99mTc IL-813,19 and with radioiodinated IL-8,22 a diffuse and initially high physiologic uptake of radiolabeled IL-8 was seen in the lungs. Therefore, there was concern about the suitability of 99mTc IL-8 to image foci of infection in the chest. High uptake in the lungs, immediately after IV administration of 99mTc IL-8, has been ascribed to the biological, agonistic effects of IL-8 on neutrophils.16,23–25 Circulating neutrophils pass through the narrow pulmonary capillary bed. Under nonstimulating conditions, there is already a delay in the pulmonary microvascular transit of neutrophils, relative to erythrocyte transit.26,27 Cell deformability plays a crucial role in transit time. IL-8 –treated neutrophils showed a transient increase in F-actin, Preliminary Reports

Figure 3. Uptake of 99mTc IL-8 at 6 h after injection in tissues with a high natural abundancy of neutrophils lung (infected and control noninfected) and spleen in three different rabbit models of pulmonary infection. Values are expressed as percentages of the injected dose per gram tissue (%ID/g) ⫾ 1 SEM.

which reduces cell deformability. A marked and rapid reduction of cell deformability could result in immediate retention of the neutrophils in the microvasculature of the lungs. It has been shown that cell deformability recovers within minutes following exposure to IL-8. The initially high physiologic uptake of 99mTc IL-8 in the lungs did not hamper the visualization of infectious foci in the lungs, for two reasons. In the first place, clearance of radioactivity from the lungs was relatively fast. Secondly, 99mTc IL-8 accumulated in the infected lung to a relatively high degree. The uptake in the lungs changed dramatically in the first hours after injection of 99mTc IL-8. Immediately after injection of 99mTc IL-8, uptake in the noninfected lung was often higher than in the infected lung, resulting in a relatively photopenic area in the affected part of the lung on the scintigrams. In contrast, images acquired at 2 h after injection showed a reversal: a clearly positive image of the

affected area and a strongly reduced uptake in noninfected lung tissue. Although the activity in the background was higher in the pulmonary infection models than in our previous studies in the muscular infection model, this is compensated by a stronger signal from the target. Residual activity (as expressed in percentage of injected dose per gram of tissue) in infected tissue ranged from 0.63 ⫾ 0.12 to 1.53 ⫾ 0.12 in pulmonary infection, as compared to 0.55 ⫾ 0.13 in IM infection.19 In the aspergillosis model, the relatively modest uptake in the affected lung was balanced by low uptake of the radiolabel in the unaffected lung, being approximately one third of the lung background activity in the other pulmonary infection models and the models of IM infection as well. We have selected three different experimental models of pulmonary infection in order to cover a broad range of (clinical) applications of 99mTc IL-8 in detection of pulmonary infection. First, we studied a

Figure 4. Scintigraphic images of a rabbit with pneumococcal pneumonia in the left lung at 1 to 5 min, 2 h, 4 h, 6 h, and 24 h after injection of 67Ga citrate. www.chestjournal.org

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rabbit model of invasive pulmonary aspergillosis that would reflect conditions encountered in patients with profound granulocytopenia after, for example, chemotherapeutic treatment. Even if the underlying disorder (eg, tumor) has resolved completely, the aspergillosis may persist. Despite aggressive antifungal therapy, the mortality from pulmonary aspergillosis in these patients remains high, illustrating the need for early and accurate diagnosis. In this study, 99m Tc IL-8 proved to be a useful diagnostic tool for pulmonary aspergillosis. As 99mTc IL-8 binds to receptors expressed on neutrophils, the use of this radiopharmaceutical might encounter its limitations in diagnosing pulmonary aspergillosis in deeply neutropenic conditions. We found in our aspergillosis model lower physiologic uptake of 99mTc IL-8 in neutrophil abundant organs such as lung and spleen as compared to infection models with normal neutrophil counts. A significant reduction of background signal in unaffected lung tissue resulted in a good visualization of the infectious foci in the affected lung. However, in persistent, deeply neutropenic conditions, that is, in a severe paucity of target cells for 99mTc IL-8, this new tool may meet its limitations as an infection imaging agent.16 Clinical trials with this new radiopharmaceutical are planned and will reveal whether 99mTc IL-8 can be used to image patients with deep neutropenia. Two more experimental models of pulmonary infection were included in this study. Pneumococcal and also E coli-induced pneumonia in previously healthy individuals is in most cases easily detected by chest radiography. Diagnosing these infections in patients with preexisting lung disease is more difficult. Conventional techniques such as chest radiography and CT have difficulty in distinguishing residual anatomic changes due to cured processes from active infection. Since IL-8 scintigraphy shows physiologic changes only, it should be able to differentiate scar tissue from active infection in such patients. Early diagnosis and prompt treatment of active infection, especially in patients with preexisting lung disease, will result in earlier recovery and may reduce mortality in these patients. 99mTc IL-8 proved to be very efficacious in visualizing foci of pulmonary infection in experimental models of pneumococcal- and E coli-induced pneumonia. Uptake of 99mTc IL-8 in affected and nonaffected lungs and in the spleen was higher in immunocompetent rabbits in these two models than in immunocompromised rabbits in the aspergillosis model. These results support the possible relationship between numbers of neutrophils in tissues and uptake of 99mTc IL-8. IL-8 receptors are preferentially expressed on neutrophils, and high uptake of 99mTc IL-8 is seen in neutrophil abundant organs as lung and spleen. As a 1960

result, in immunocompromised rabbits, with lower levels of IL-8 receptor-expressing cells, physiologic uptake of 99mTc IL-8 in these organs is lower. Higher uptake of 99mTc IL-8 by the kidneys and a lower level of residual activity in the blood might be explained by the fact that a larger percentage of 99m Tc IL-8 in immunocompromised rabbits is not bound to target cells (neutrophils), and unbound 99m Tc IL-8 is rapidly cleared by the kidneys. 99m Tc IL-8 offers several advantages over the most commonly used radiopharmaceuticals for infection imaging, 67Ga citrate and radiolabeled leukocytes.28 The radionuclide 99mTc is preferred over 67Ga because of its virtually ideal physical characteristics (short half-life, ideal energy, low radiation burden), its cost-effectiveness, and general availability.2 67Ga has a longer physical half-life and high-energy ␥ radiations, causing high-radiation absorbed doses and generating images of lower resolution. Generally, 67Ga citrate shows relatively slow pharmacokinetics. As a consequence, a long interval between injection of the radiopharmaceutical and imaging is required. Typically, 67Ga imaging is performed between 48 h and 72 h after injection. In imaging rabbits using 99mTc IL-8, a 2-h interval between injection and imaging was sufficient. In humans, slower pharmacokinetics can be expected, but diagnostic images within 4 h after injection in patients should be possible. 99m Tc IL-8 offers many advantages over radiolabeled leukocytes. Preparation of 99mTc IL-8 is easy and rapid, ready within 30 min, and with no need for further purification, whereas preparation of labeled leukocytes, be it with either 111In or 99mTc, is cumbersome, time-consuming, and not possible in granulocytopenic patients.2 The procedure of taking blood from a patient, purification of the leukocytes, and labeling of these cells takes a trained technician approximately 3 h. Cells should be handled cautiously in order to preserve their capacity to migrate to the inflamed area on reinjection. In addition, the need to handle potentially contaminated blood could lead to transmission of blood-borne pathogens such as HIV,7 and presents serious risks to both personnel and patient. In conclusion, imaging of a variety of pulmonary infections with 99mTc IL-8 proved to be feasible, and offers many advantages over the conventionally used tools in nuclear medicine, 67Ga citrate and radiolabeled leukocytes. 99mTc IL-8 allowed excellent visualization of localization and extent of pulmonary infection in three experimental models simulating immunocompromised conditions (aspergillosis), as well as models simulating immunocompetent conditions with Gram-positive (S pneumoniae) as well as Gram-negative (E coli) bacterial infections. Preliminary Reports

ACKNOWLEDGMENT: The authors thank Gerry Grutters and Hennie Eikholt (University Medical Center Nijmegen, Central Animal Laboratory) for technical assistance.

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