Synthesis and Characterization of ¹8F-Interleukin-8 Using a Cell-Free Translation System and 4-¹8F-Fluoro-L-Proline.

UNLABELLED: Macromolecules such as proteins are attracting increasing interest for molecular imaging. We previously proposed a novel strategy for preparing macromolecules labeled with a PET radionuclide, (11)C, using a cell-free translation system with (11)C-methionine. However, macromolecules tend to exhibit slower kinetics, thus requiring a longer scanning time. Here, we expand our strategy using (18)F, which has a longer half-life, with the cell-free translation system with 4-(18)F-fluoro-L-proline ((18)F-FPro). We evaluated (18)F-interleukin-8 ((18)F-IL-8) produced by this method in vitro and in vivo to provide a proof of concept of our strategy. METHODS: We tested some fluorinated amino acids to be incorporated into a protein. Trans-(18)F-FPro was radiolabeled from the corresponding precursor. (18)F-IL-8 was produced using the cell-free translation system with trans-(18)F-FPro instead of natural L-proline with incubation at 37°C for 120 min. An in vitro binding assay of (18)F-IL-8 was performed using IL-8 receptor-expressing cells. After intravenous administration of (18)F-IL-8, in vivo PET imaging of IL-8 receptor-expressing xenograft-bearing mice was performed using a small-animal PET system. RESULTS: FPro was identified as an amino acid incorporated into the protein. (18)F-IL-8 was successfully prepared using the cell-free translation system and trans-(18)F-FPro with the radiochemical yield of 1.5% (decay-corrected) based on trans-(18)F-FPro. In vitro binding assays of (18)F-IL-8 demonstrated its binding to IL-8 receptor. In vivo PET imaging demonstrated that (18)F-IL-8 clearly accumulated in IL-8 receptor-expressing xenografts in mice, unlike trans-(18)F-FPro. CONCLUSION: (18)F-IL-8 produced by this method binds to IL-8 receptors in vitro, and (18)F-IL-8 PET clearly visualizes its target receptor-expressing xenograft in vivo. Therefore, this technique might be useful for labeling macromolecules and performing preclinical evaluations of proteins of interest in vitro and in vivo.

Preclinical evaluation of technetium 99m-labeled P1827DS for infection imaging and comparison with technetium 99m IL-8.

BACKGROUND: The technetium 99 m (99mTc)-radiolabeled, leukocyte-avid peptide-glycoseaminoglycan complex, [99mTc]P1827DS, has been synthesized as an improved infection/inflammation imaging agent to [99mTc]P483H (LeukoTect, Diatide). In a phase I/II clinical trail, [99mTc]P483H images were equivalent to those obtained with 111In ex vivo labeled leukocytes. However, there was physiologic accumulation of radioactivity in the body that could hamper interpretation of the images. In this study, the potential of [99mTc]P1827DS for infection imaging was assessed in comparison with [99mTc]P483H and the well-described imaging agent [99mTc] hydrazinonicotinamide (HYNIC)-interleukin 8 (IL-8). METHODS: The binding of [99mTc]P1827DS to human blood cell was studied in vitro. A rabbit Escherichia coli infection model was used to perform the biodistribution and imaging studies with [99mTc]P1827DS, [99mTc]P483H and [99mTc]HYNIC-IL-8. RESULTS: [99mTc]P1827DS binds to leukocytes but not to erythrocytes. The leukocyte binding was not saturable up to an investigated concentration of 10 microM. The accumulation of [99mTc]P1827/DS at the infection site strongly depends on the P1827/DS ratio and was optimal at a molar ratio of 10:1. [99mTc]P1827DS shows improved biodistribution over [99mTc]P483H with similar uptake at the infection site. Abscess uptake of [99mTc]HYNIC-IL-8 was approximately three times higher than that of [99mTc]P1827DS. [99mTc]HYNIC-IL-8 showed high accumulation in the kidneys, whereas [99mTc]P1827DS showed high lung uptake and slightly higher accumulation in the liver and spleen. CONCLUSION: [99mTc]P1827DS is a potential new inflammation imaging agent, which clearly visualized the abscess in the rabbit E. coli infection model and showed improved biodistribution compared to [99mTc]P483H. However, the infection uptake and biodistribution of [99mTc]P1827DS is not superior to that of [99mTc]HYNIC-IL-8 in this animal model.

99mTc-labeled interleukin 8 for the scintigraphic detection of infection and inflammation: first clinical evaluation.

UNLABELLED: Interleukin 8 (IL-8) is a chemotactic cytokine that binds with a high affinity to receptors expressed on neutrophils. Previous studies with various animal models showed that (99m)Tc-labeled IL-8 accumulates specifically and rapidly in infectious and inflammatory foci. The aims of the present study were to evaluate the safety of IL-8 in humans and to assess the value of (99m)Tc-IL-8 scintigraphy in patients with suspected localized infections. METHODS: (99m)Tc-IL-8 was intravenously injected at 400 MBq into 20 patients with various suspected localized infections. Patients were monitored for IL-8-related side effects for 4 h. Whole-body imaging was performed directly after injection and at 4 h after injection. Imaging after 24 h was performed for the first 7 patients and for subsequent patients when the results of (99m)Tc-IL-8 scintigraphy at 4 h after injection were normal or equivocal. Blood was drawn at several time points to determine the total number of leukocytes and leukocyte differentiation (all patients) and to determine pharmacokinetics (6 patients). RESULTS: (99m)Tc-IL-8 scintigraphy was performed for 20 patients (13 men and 7 women) with a mean age of 60 y (range, 21-76 y). No significant side effects were noted. Patients had suspected joint prosthesis infections (n = 9), osteomyelitis (n = 8), liver abscess (n = 1), and soft-tissue infections (n = 2). (99m)Tc-IL-8 was rapidly cleared from the blood and most other organs. In 10 of 12 patients with infections, (99m)Tc-IL-8 localized the infection at 4 h after injection. In 1 patient with vertebral osteomyelitis and in 1 patient with an infected knee prosthesis, (99m)Tc-IL-8 scintigraphy results were false-negative. In 8 patients with noninfectious disorders, no focal accumulation of (99m)Tc-IL-8 was found. CONCLUSION: Injection of (99m)Tc-IL-8 is well tolerated. (99m)Tc-IL-8 scintigraphy is a promising new tool for the detection of infections in patients as early as 4 h after injection.

Prosthetic radioiodination of interleukin-8 ([(123/131)I]-IL-8): biological behavior in a mouse infection model.

Numerous molecular entities with diverse structures have been radiolabeled and investigated as potential infection and inflammation detection agents. However, none of these molecules have gained the acceptance of gallium citrate or radiolabeled autologous white blood cells. We have radioiodinated interleukin-8 using two different methods and tested the biological behavior of the products in mice. As expected, the direct radioiodinated material displayed extensive in vivo deiodination. The use of pyridine-based prosthetic label yielded a product with better kinetics than the direct radioiodination method and showed a better target to non-target ratio. Nonetheless, this method is not suited for labeling of bioactive peptides such as the title peptide because of the very high specific activity required to prevent cytotoxic effects in a human application.

Kinetics of 99mTc-labeled interleukin-8 in experimental inflammation and infection.

UNLABELLED: The cytokine interleukin-8 (IL-8) binds with high affinity to the CXCR1 and CXCR2 receptors on neutrophils. In previous studies, we showed that (99m)Tc-IL-8 could rapidly and effectively delineate foci of infection and inflammation in rabbit models of intramuscular infection, colitis, and osteomyelitis. Here, the in vivo kinetics and pharmacodynamics of (99m)Tc-IL-8 are studied in detail. A derivative of hydrazinonicotinamide (HYNIC) was used as a bifunctional coupling agent to label the protein with (99m)Tc. METHODS: To address specificity of uptake of (99m)Tc-IL-8 in the abscess, uptake in turpentine-induced abscesses in neutropenic rabbits was compared with uptake in turpentine-induced abscesses in normal rabbits. The pharmacokinetics of (99m)Tc-IL-8 were studied in neutropenic rabbits and compared with those in normal rabbits. To investigate the interaction of (99m)Tc-IL-8 with blood cells in circulation in normal rabbits, the distribution of the radiolabel over circulating white and red blood cells and plasma was determined. The in vivo kinetics of (99m)Tc-IL-8 were studied by quantitative analysis of whole-body images acquired between 0 and 6 h after injection. The results of this analysis (in vivo biodistribution) were validated by ex vivo counting of radioactivity in dissected tissues. RESULTS: The abscess uptake (percentage of injected dose per gram of tissue [%ID/g] +/- SEM) in immunocompetent rabbits (0.41 +/- 0.05) was 10 times higher than that in neutropenic rabbits (0.038 +/- 0.014), demonstrating specificity of the target uptake of (99m)Tc-IL-8. Abscess-to-muscle ratios +/- SEM were also 10 times higher (110 +/- 10 vs. 10 +/- 5). Lung and spleen uptake in normal rabbits was 3 times higher than that in neutropenic rabbits. The blood clearance of the radiolabel in neutropenic rabbits was similar to that in normal rabbits. In circulation, most of (99m)Tc-IL-8 (70%) was found in the plasma fraction. Less than one third was associated with red blood cells, and only a very low percentage (<2.5%) was associated with white blood cells. Image analysis revealed a gradually increasing abscess uptake over time up to >15%ID, which was confirmed by ex vivo gamma-counting of the infected muscle. The highest increase in uptake in the abscess was observed after 2 h following injection, when most of (99m)Tc-IL-8 was cleared from the blood, suggesting specific neutrophil-mediated accumulation of (99m)Tc-IL-8 in the abscess. Furthermore, region-of-interest analysis revealed that gradual accumulation of (99m)Tc-IL-8 in the abscess was accompanied by a simultaneous clearance of activity from the lungs, suggesting that neutrophil-associated (99m)Tc-IL-8 that was initially trapped in the lungs migrates to the abscess at later time points, favoring neutrophil-bound transportation from the lungs to the abscess. CONCLUSION: Substantial support is given for the hypothesis that (99m)Tc-IL-8 localizes in the abscess, mainly bound to peripheral neutrophils. Accumulation in the abscess is a highly specific, neutrophil-driven process. As assessed by in vivo and ex vivo analysis, the total fraction that accumulates in the inflamed tissue is extremely high (up to >15 %ID) compared with that of other agents used for imaging infection and inflammation.

[Study of 125I-interleukin-8 distribution in mice].

IL-8 was radiolabeled with 125I via Bolten-Hunter agent and the distribution of 125I-IL-8 in mice was determined in order to understand IL-8 behaviors in vivo. The percentage of 125I-IL-8 in blood, heart, liver, lung, kidneys, bone and spleen was obtained. The half-clearance of fast phase T1/2 alpha and slow phase T1/2 beta were 0.32 h and 8.01 h respectively. Most of 125I-IL-8 was excluded by kidneys.

Effects of coligand variation on the in vivo characteristics of Tc-99m-labeled interleukin-8 in detection of infection.

In our previous studies, interleukin-8 (IL-8) was labeled with (99m)Tc using hydrazinonicotinamide (HYNIC) as bifunctional coupling agent and tricine as coligand. This preparation showed excellent characteristics for imaging of infection in a rabbit model of soft-tissue infection. In the present study, the propylaldehyde hydrazone formulation of HYNIC was introduced to stabilize HYNIC-IL-8. (99m)Tc-HYNIC-IL-8 was prepared using 5 different coligand formulations. The effect of these coligand formulations on the in vitro characteristics and in vivo behavior of (99m)Tc-HYNIC-IL-8 was investigated. HYNIC-conjugated IL-8 was labeled with (99m)Tc in the presence of either (A) tricine, (B) ethylenediaminediacetic acid (EDDA), (C) tricine and trisodium triphenylphosphinetrisulfonate (TPPTS), (D) tricine and nicotinic acid (NIC), or (E) tricine and isonicotinic acid (ISONIC). These preparations were characterized in vitro by RP-HPLC, determination of the octanol/water partition coefficient, stability studies, and receptor binding assays. The in vivo biodistribution of the radiolabel in rabbits with E. coli-induced soft-tissue infection was determined both by gamma-camera imaging as well as by tissue counting at 6 h pi. Specific activity (MBq/microg) was highest for (ISO)NIC (up to 80) > TPPTS (40) > tricine (15) > EDDA (7). RP-HPLC and octanol/water partition coefficients showed a shift toward higher lipophilicity for the TPPTS preparation. The leukocyte receptor binding fractions were around 40-55% for all preparations except for TPPTS, which showed predominantly nonspecific binding. All preparations were stabilized in serum, but the stability in PBS was highest for NIC and TPPTS > EDDA > ISONIC > tricine. The in vivo biodistribution showed highest abscess/muscle for NIC and ISONIC (>200) > EDDA and tricine (approximately 100) > TPPTS (<40). Gamma camera imaging rapidly visualized the abscess from 2 h pi onward for all formulations. The abscess/background (A/B) at 6 h pi for ISONIC was significantly higher (P < 0.05) than that of tricine and the A/B of TPPTS was significantly lower (P < 0.05). IL-8 can be rapidly and easily labeled with (99m)Tc using HYNIC as a chelator in combination with various coligands. The most optimal infection imaging characteristics were found for formulations using nicotinic acid/tricine as coligand system combining a high specific activity and high in vitro stability with high abscess/muscle ratios (>200) and high abscess/background ratios (>20). Protein doses to be administered were as low as 70 ng/kg bodyweight. At these low protein doses, side effects are not to be expected in the human system. This paves the way for infection imaging studies in patients.

Tissue-specific mechanisms control the retention of IL-8 in lungs and skin.

Chemokines are a group of structurally related peptides that promote the directed migration of leukocytes in tissue. Mechanisms controlling the retention of chemokines in tissue are not well understood. In this study we present evidence that two different mechanisms control the persistence of the CXC chemokine, IL-8, in lungs and skin. (125)I-labeled IL-8 was injected into the airspaces of the lungs and the dermis of the skin and the amount of (125)I-labeled IL-8 that remained at specified times was measured by scintillation counting. The (125)I-labeled IL-8 was cleared much more rapidly from skin than lungs, as only 2% of the (125)I-labeled IL-8 remained in skin at 4 h whereas 50% of the (125)I-labeled IL-8 remained in lungs at 4 h. Studies in neutropenic rabbits showed that neutrophils shortened the retention of (125)I-labeled IL-8 in skin but not lungs. A monomeric form of IL-8, N-methyl-leucine 25 IL-8, was not retained as long in lungs as recombinant human IL-8, indicating that dimerization of IL-8 is a mechanism that increases the local concentration and prolongs the retention of (125)I-labeled IL-8 in lungs. These observations show that the mechanisms that control the retention of IL-8 in tissue include neutrophil migration and dimerization, and that the importance of these varies in different tissues.

Adapting pharmacokinetic properties of a humanized anti-interleukin-8 antibody for therapeutic applications using site-specific pegylation.

A neutralizing anti-interleukin-(IL-)8 monoclonal antibody was humanized by grafting the complementary determining regions onto the human IgG framework. Subsequent alanine scanning mutagenesis and phage display enabled the production of an affinity matured antibody with a >100-fold improvement in IL-8 binding. Antibody fragments can be efficiently produced in Escherichia coli but have the limitation of rapid clearance rates in vivo. The Fab' fragment of the antibody was therefore modified with polyethylene glycol (PEG) in order to obtain a more desirable pharmacokinetic profile. PEG (5-40 kDa) was site-specifically conjugated to the Fab' via the single free cysteine residue in the hinge region. In vitro binding and bioassays showed little or no loss of activity. The pharmacokinetic profiles of the 20 kDa, 30 kDa, 40 kDa, and 40 kDa branched PEG-Fab' molecules were evaluated in rabbits. Relative to the native Fab', the clearance rates of the PEGylated molecules were decreased by 44-175-fold. In a rabbit ear model of ischemia/reperfusion injury, all PEGylated Fab' molecules were as efficacious in reducing oedema as the original monoclonal antibody. These studies demonstrate that it is possible to customize the pharmacokinetic properties of a Fab' while retaining its antigen binding activity.

Imaging of human infection with (131)I-labeled recombinant human interleukin-8.

UNLABELLED: The chemotactic cytokine interleukin-8 (IL-8) plays an important role in attraction and activation of polymorphonuclear leukocytes in infection and inflammation. A pilot study was conducted to determine if radiolabeled IL-8 would depict infection in humans. METHODS: Human recombinant IL-8 (rhIL-8) labeled with (131)I (specific activity, 0.4-0.7 MBq [11-18 microCi] (131)I/microg IL-8) was injected intravenously into 8 diabetic patients with active foot infections and evidence of osteomyelitis, 2 patients with successfully treated osteomyelitis, and 1 patient with cellulitis of the thumb. RESULTS: Focal accumulation of (131)I-rhIL-8 was seen in 8 of 8 patients with active foot infection and diffuse uptake was seen in the thumb of the 1 patient with cellulitis. In the 2 patients with successfully treated bone infection, multiphase (99m)Tc-hydroxyethylene diphosphonate bone scans were negative early, but late-phase (>3 h) uptake depicted degenerative lesions that did not image with (131)I-rhIL-8. CONCLUSION: (131)I-rhIL-8 accumulates rapidly within infected foci in osteomyelitis and cellulitis but not in successfully treated infections or degenerative joint disease.

Interleukin-8 administration enhances venous thrombosis resolution in a rat model.

BACKGROUND: Therapy for deep vein thrombosis (DVT) resolution in those patients in whom a complication or contraindication to anticoagulation occurs is limited. As prior work suggests that thrombus maturation involves early influx of neutrophils (PMN) and neovascularization, we hypothesized that administering the proinflammatory/proangiogenic chemokine interleukin (IL)-8 might accelerate thrombus resolution. MATERIALS AND METHODS: An established rodent model of DVT (inferior vena cava [IVC] ligation) was used whereby daily intravenous recombinant human IL-8 (1 microg) or vehicle control was administered, with sacrifice at 4 and 8 days. Prior to sacrifice and at harvest, duplex ultrasound of the DVT and femoral venous pressure measurements were performed. Thrombi were analyzed by immunohistochemical techniques for PMN, monocytes, and neovascularization; for chemokines, by enzyme-linked immunoassay; and fibrosis, by hydroxyproline assay and trichrome staining. RESULTS: IL-8 accelerated thrombus dissolution 4 days after IVC ligation, with 6-fold increased thrombus blood flow by duplex ultrasound and a 23% increased absolute femoral venous pressure compared with controls (both P < 0.05). These findings may be partially explained by the fact that animals receiving IL-8, as compared with controls, had 2.5-fold greater thrombus neovascularization (with a trend continuing to 8 days) and increased PMN at 4 days. Thrombus vascular endothelial growth factor was significantly reduced at 8 days postligation, while monocyte chemotactic protein-1 and macrophage inflammatory protein-1alpha were not altered by IL-8 administration. At 8 days post-IVC-ligation, fibrosis was 12-fold greater with IL-8 treatment compared with controls. CONCLUSIONS: A proinflammatory/proangiogenic thrombus milieu, as conferred by IL-8, enhances thrombus resolution and underscores the important relationship between neovascularity and inflammation.

Rapid imaging of experimental colitis with (99m)Tc-interleukin-8 in rabbits.

UNLABELLED: Radiolabeled autologous leukocytes (WBCs) are the gold standard for imaging inflammatory bowel disease (IBD). For the rapid and adequate management of patients with IBD, there is need for a new agent at least as good as radiolabeled WBCs, but easier to prepare and without its inherent risks. In this study, the potential of interleukin-8 (IL-8) labeled with (99m)Tc using hydrazinonicotinamide (HYNIC) to image IBD was investigated in a rabbit model of acute colitis and compared with that of (99m)Tc-HMPAO-labeled granulocytes. METHODS: In rabbits with chemically induced acute colitis, inflammatory lesions were scintigraphically visualized after injection of either IL-8 or purified granulocytes, both labeled with (99m)Tc. Gamma camera images were acquired at 2 min and at 1, 2, and 4 h after injection. Four hours after injection, the rabbits were killed, and the uptake of the radiolabel in the dissected tissues was determined. The dissected colon was imaged and the inflammatory lesions were scored macroscopically. For each affected colon segment, the colitis index (affected colon-to-normal colon uptake ratio, CI) was calculated and correlated with the macroscopically scored severity of inflammation. RESULTS: Both agents visualized the colitis within 1 h after injection. (99m)Tc-HYNIC-IL-8 images of the colonic abnormalities were more accurate and the intensity of uptake in the affected colon continuously increased until 4 h after injection, whereas no further increase 1 h after injection was noticed scintigraphically for (99m)Tc-HMPAO-granulocytes. The absolute uptake in the affected colon was much higher for IL-8 than for the radiolabeled granulocytes with the percentage injected dose per gram (%ID/g) 0.41 +/- 0.04 %ID/g and 0.09 +/- 0.05 4 %ID/g h after injection, respectively. With increasing severity, the CI at 4 h after injection for (99m)Tc-HYNIC-IL-8 was 4.4 +/- 0.6, 13.5 +/- 0.5, and 25.8 +/- 1.0; for granulocytes, the CI at 4 h after injection was 1.5 +/- 0.1, 3.4 +/- 0.2, and 6.4 +/- 0.5, respectively. The CI correlated with the severity of the inflammation (r = 0.95, P < 0.0001 for IL-8; r = 0.95, P < 0.0001 for granulocytes). CONCLUSION: Within 1 h after injection, visualization of the extent of colonic inflammation in vivo was possible with (99m)Tc-HYNIC-IL-8 and (99m)Tc-HMPAO-granulocytes. Within 2 h after injection, (99m)Tc-IL-8 allowed a good evaluation, and within 4 h after injection, a meticulous evaluation of the severity of IBD. Although (99m)Tc-HMPAO-granulocytes were able to delineate the extent of IBD within 2 h after injection, an accurate estimation of severity of inflammation was not possible. (99m)Tc-HYNIC-IL-8 is an inflammation-imaging agent that showed promising results in this study. (99m)Tc-IL-8 can be prepared off-the-shelf and yields excellent imaging with high target-to-background ratios.

Interleukin-8 and leukotriene-B(4), but not formylmethionyl leucylphenylalanine, stimulate CD18-independent migration of neutrophils across human pulmonary endothelial cells in vitro.

Although neutrophil migration from the systemic circulation involves the beta2- (or CD18) integrin family, the existence of an alternative, CD18-independent route of neutrophil extravasation to tissues has been demonstrated in animal models. The molecular interactions involved in this alternative migratory route have not yet been characterized. The objective of this study was to assess the CD18-dependency of neutrophil migration across human endothelial cells from an organ known to support CD18-independent migration, the lung, with a view to establishing an in vitro model to facilitate study of CD18-independent migration. Neutrophil migration across human pulmonary artery endothelial cells (HPAECs) in response to three different chemoattractants, formylmethionyl leucylphenyl-alanine (FMLP), interleukin (IL)-8, and leukotriene (LT) B(4), was examined. Results demonstrated that a function-blocking antibody to CD18 decreased FMLP-stimulated migration by 71.7 +/- 4.4% (P < 0.001). In contrast, migration in response to LTB(4) was decreased by only 20.5 +/- 10.2% (P < 0.01), and no significant decrease was observed with migration to IL-8. Neutrophils that migrated to FMLP had 1.7-fold more surface CD11b/CD18 compared with nonmigrated neutrophils (P < 0.01), whereas this integrin complex was not significantly upregulated on neutrophils that had migrated to IL-8 or LTB(4). Further investigation of this migratory route indicated that it did not involve the beta1 integrins (CD29) or the endothelial selectins, E- or P-selectin, nor did it require the activity of either metalloproteinases or neutrophil elastase. These results indicate that neutrophil migration across HPAECs in vitro to IL-8 and LTB(4) is predominantly CD18-independent and provides a much-needed in vitro system for examination of the neutrophil-endothelial interactions involved in this alternative migratory route.

Involvement of alpha-2-macroglobulin receptor in clearance of interleukin 8-alpha-2-macroglobulin complexes by human alveolar macrophages.

The purpose of this study was to determine if interleukin 8 (IL-8) in complex with alpha2-macroglobulin (alpha-2-M) can be taken up by human alveolar macrophages. First, we demonstrated that human alveolar macrophages have receptors for alpha-2-M but not IL-8. The binding of(125)I-labeled alpha-2-M to the cells was specific and saturable, whereas(125)I-labeled recombinant human IL-8 (rhIL-8) did not bind to macrophages. However,(125)I-rhIL-8-alpha-2-M complexes bound to macrophages, and unlabeled alpha-2-M competed for the binding. We then cultured the cells in the presence of(125)I-rhIL-8-alpha-2-M complexes,(125)I-rhIL-8 alone or buffer for 24 h. Macrophages were lysed, and the released radioactivity measured. IL-8 concentrations in supernatants and cells were also measured using an IL-8 ELISA. When the macrophages were incubated with(125)I-rhIL-8-alpha-2-M complexes there was a significant amount of IL-8 associated with the cells. However, this was not the case when the cells were incubated with(125)I- rhIL-8 alone suggesting that only these complexes were taken-up by human alveolar macrophages. Furthermore, the clearance of complexes was specifically inhibited by a monoclonal antibody against the 515-kDa subunit of the alpha-2-M receptor (alpha-2-MR) but not by an isotopic mouse IgG1. The study shows an important clearance mechanism for IL-8 in the lung.

The kinetics of radiolabelled interleukin-8 in infection and sterile inflammation.

Radiolabelled interleukin-8 (IL-8) is a promising agent for the imaging of infection and inflammation. Several experiments were performed to explore further the imaging potential of radiolabelled IL-8. IL-8 was radioiodinated via the Bolton-Hunter method. Rabbits with focal infection (Escherichia coli, Staphylococcus aureus) or sterile inflammation (zymosan) were injected intravenously with 18.5 MBq (0.5 mCi) of 123I-IL-8. In separate studies, rabbits were injected intravenously with 111In-granulocytes with or without 125I-IL-8. Gamma camera images were obtained at 5 min, 1, 4 and 8 h post-injection (p.i.). Biodistribution was determined at 8 h p.i. In all models, the biodistribution of 123I-IL-8 was characterized by rapid blood clearance and high uptake in infection and sterile inflammation. All foci could be clearly visualized within 4 h p.i. Ex vivo abscess-to-contralateral muscle ratios increased to 114.7+/-23.0 (E. coli), 52.3+/-24.5 (S. aureus) and 49.8+/-8.3 (zymosan) at 8 h p.i. In the circulation, most 123I-IL-8 was bound to erythrocytes. The abscess uptake of 125I-IL-8 reached high levels despite reduced migration of granulocytes towards the site of infection due to the anti-inflammatory activity of intravenously injected IL-8. IL-8 could be injected without induction of neutropenia at a dosage of 2 ng kg(-1). In conclusion, the characteristics of radiolabelled IL-8 for imaging of infection and sterile inflammation are highly encouraging and warrant further optimization for clinical application.

Scintigraphy of acute inflammatory lesions in rats with radiolabelled recombinant human interleukin-8.

We compared 125I-labelled recombinant human interleukin-8 (125I-IL-8) with 111In-labelled human leukocytes (111In-WBC) and 67Ga-citrate for scintigraphic depiction of acute sterile inflammatory lesions in rats. Radioiodination of IL-8 was catalysed by chloramine-T, and human leukocytes were radiolabelled with 111In-oxine. Inflammatory lesions were induced in male rats by subcutaneous injection of 2% carrageenan suspension into their left hindlimbs. Twenty-four hours later, each rat received 1.8-3.7 MBq (50-100 microCi) of a single agent by intravenous injection. Sequential whole-body scintigrams were obtained between 0 and 96 h post-injection. Activities in the lesion-bearing and control hindlimbs were expressed as regional percent injected activity corrected for physical decay (%IA) by reference to concurrently imaged standards, and for 125I-IL-8 by direct tissue counting at necropsy as well. 125I-IL-8 displayed appropriate electrophoretic mobility, retained chemotactic and high-affinity receptor-binding activity in vitro, and exhibited exponentially decreasing activity in most tissues beginning shortly after intravenous injection. Scintigrams showed asymmetrically increased activity in the lesion-bearing hindlimb for all three agents. By scintigraphy, 125I-IL-8 activity in the lesion-bearing hindlimb reached a zenith 1-3 h post-injection at 4.8 +/- 0.5 %IA and decreased exponentially thereafter, with little change in lesioned-to-control limb ratios (mean L/C = 3.0 +/- 0.7) over the imaging period. By direct tissue counting, abscess-associated mean IL-8 activity per gram of tissue increased to four times that of adjacent muscle and nearly seven times that of contralateral muscle by 24 h post-injection. Lesion-bearing hindlimb 111In-WBC activity also rose rapidly, reaching 4.2 +/- 0.6 %IA by scintigraphy at 3 h and an eventual plateau (maximum of 4.5 +/- 0.4 %IA) by 24 h. 67Ga scintigraphic activity in the lesion-bearing hindlimb peaked briefly at 3-6 h post-injection (9.2 +/- 0.5 %IA) and subsequently declined to a constant level of about 7.5 %IA. However, L/C for 111In-WBC and for 67Ga-citrate each averaged only 1.5 +/- 0.3 over the imaging period, compared with a mean L/C of 1.2 +/- 0.2 for a blood pool radiotracer. We conclude that 125I-IL-8 is rapidly and selectively concentrated in regions of acute inflammation, presumably by high-affinity binding to IL-8 receptors on neutrophils within the inflammatory focus. Radioiodinated IL-8 offers an attractive alternative to 67Ga-citrate and 111In-WBC for early imaging of acute inflammatory lesions, and demonstrates significantly higher target-to-nontarget activity ratios in this model. The potential usefulness of radiolabelled IL-8 for clinical scintigraphy should be evaluated.

Rapid mobilization of hematopoietic progenitor cells in rhesus monkeys by a single intravenous injection of interleukin-8.

Interleukin-8 (IL-8) is a chemoattractant cytokine involved in chemotaxis and activation of neutrophils. Because in vivo administration of IL-8 induces mobilization of hematopoietic stem cells in mice, we assessed the mobilizing properties of IL-8 in rhesus monkeys. Recombinant human IL-8 was administered as a single intravenous injection at doses of 10, 30, and 100 micrograms/kg to rhesus monkeys (age, 2 to 3 years; weight, 2.5 to 4.5 kg). Venous blood samples were obtained at time intervals ranging from 1 to 480 minutes after IL-8 administration. Cell counts, colony-forming unit-Mix assays, and fluorescence-activated cell sorter analysis were performed. Plasma was harvested to assess IL-8 levels. A time-controlled bolus intravenous injection of 100 micrograms IL-8 per kilogram of body weight resulted in peak IL-8 plasma levels up to 5 micrograms/mL. The calculated half-time life of free IL-8 was 9.9 +/- 2.2 minutes. IL-8 injection resulted in instant neutropenia that was due to pulmonary sequestration, as shown using 99mTc-labeled leukocytes. Within 30 minutes after IL-8 injection, neutrophilia developed with counts up to 10-fold greater than baseline levels. The numbers of hematopoietic progenitor cells (HPCs) increased from 45 +/- 48/mL to 1,382 +/- 599/mL of blood at 30 minutes after injection of 100 micrograms IL-8 per kilogram of bodyweight (mean +/- SD, n = 8). Individual animals showed 10- to 100-fold increase in numbers of circulating HPCs that returned to almost pretreatment values (92 +/- 52 CFU/mL) at 240 minutes after the injection of IL-8. Immunophenotyping showed no significant changes in lymphocyte (sub)populations. A second bolus injection of IL-8 with an interval of 72 hours resulted in similar numbers of mobilized stem cells as observed after the first injection, showing that no tachyphylaxis had occurred. We conclude that IL-8 induces mobilization of HPCs from the bone marrow of rhesus monkeys in a rapid and reproducible fashion. Therefore, IL-8 may be a potentially useful cytokine in the setting of blood stem cell transplantation.

Site-specific attachment of functionalized poly(ethylene glycol) to the amino terminus of proteins.

A convenient method for the construction of site-specifically modified poly(ethylene glycol)-protein conjugates is described. This method relies on the ability to generate a reactive carbonyl group in place of the terminal amino group. If the protein has N-terminal serine or threonine, this can be done by very mild periodate oxidation and generates a glyoxylyl group. A method less restricted by the nature of the N-terminal residue, but which requires somewhat harsher conditions, is metal-catalyzed transamination, which gives a keto group. The N-terminal-introduced reactive carbonyl group specifically reacts, under mild acidic conditions, with an aminooxy-functionalized poly(ethylene glycol) to form a stable oxime bond. Using polymers of different size and shape (linear or multibranched), various conjugates of IL-8, G-CSF, and IL-1ra were constructed and further characterized with respect to their biological activity and pharmacokinetic behavior in rats. Unlike most previous methods, this approach places a single PEG chain at a defined site on the protein. It should therefore be more likely to conserve biological activity when the latter depends on interaction with another macromolecule (unlike enzymic activity which often survives multiple PEGylation).

Effects of intravenous IL-8 administration in nonhuman primates.

IL-8, a cytokine known for its potent and specific neutrophil activation and chemoattractant properties, has been recently detected in the circulation during septic shock, endotoxemia, and after IL-1 alpha administration. Because of its observed in vitro actions, it has been hypothesized that IL-8 may contribute to the dynamics of circulating granulocytes and to the pathologic sequelae seen in sepsis. Here, human rIL-8 is administered to healthy nonhuman primates as a single i.v. injection or as a continuous 8-h i.v. infusion. We demonstrate that both methods of i.v. administration result in a rapid but transient, severe granulocytopenia, followed by a granulocytosis that persists as long as IL-8 levels are detectable in the circulation. There were no hemodynamic changes after IL-8 administration, and animals remained clinically stable during the 24-h observation period. No detectable circulating TNF-alpha, IL-1 beta, or IL-6 response was induced by either IL-8 administration regimen. Histopathologic examination revealed mild to moderate neutrophilic margination in lung, liver, and spleen, of greater severity in baboons receiving the 8-h infusion. There was no associated neutrophilic infiltration or tissue injury. Thus, IL-8 modulates circulating granulocyte dynamics and likely directs their actions, but when administered i.v. to healthy animals, either as a bolus dose or as a continuous infusion for up to 8 h, does not induce the hemodynamic and metabolic aberrations or the acute organ damage seen during sepsis.