A role of the Duffy antigen for the maintenance of plasma chemokine concentrations.

We examined plasma chemokine concentrations and chemokine clearance rates in Duffy antigen knockout mice. The plasma concentrations of eotaxin and MCP-1 in Duffy antigen knockout mice were less than one-third of those in wild-type mice. When eotaxin or hMGSA was intravenously injected, the chemokine disappeared more rapidly from the plasma of Duffy antigen knockout mice than from the plasma of wild-type mice. The half-lives of hIP-10 and interferon-gamma, which do not have an affinity for the Duffy antigen, in plasma were indistinguishable between Duffy antigen knockout mice and wild-type mice. These results suggest that the Duffy antigen delays the disappearance of chemokines from the plasma, resulting in the maintenance of plasma chemokine concentrations.

Radiolabeled chemotactic cytokines: new agents for scintigraphic imaging of infection and inflammation.

Several radiopharmaceuticals are currently used for diagnosis of inflammatory and infectious diseases in patients. Most inflammatory and infectious processes can be visualized with radiolabeled autologous leukocytes, currently considered to be the most appropriate radiopharmaceutical for this purpose. This agent is very well capable to delineate most inflammatory and infectious foci in a relatively short time after injection. The time-consuming and intricate labeling procedure and the handling of potentially contaminated blood, however cause that there is a great interest in the development of new radiopharmaceuticals comprising the same imaging qualities but without these disadvantages. Besides radiolabeled leukocytes several other radiopharmaceuticals, such as (67)Ga-citrate, radiolabeled anti-granulocyte antibodies and FDG are used to image infection and inflammation. These agents accumulate in infectious and inflammatory lesions in a non-specific manner or have suboptimal diagnostic characteristics. Nowadays, there is a great interest in the development of radiolabeled chemotactic and chemokinetic cytokines that accumulate and are retained in infectious and inflammatory foci by specific interaction with infiltrated inflammatory cells. In this review we describe the specific characteristics of the chemotactic and chemokinetic compounds that are currently studied as potential radiopharmaceutical to visualize infectious and inflammatory foci. The characteristics of a series of cytokines (IL-1, IL-2), chemokines (IL-8, PF-4, MCP-1, NAP-2), complement factors (C5a, C5adR), chemotactic peptides (fMLF) and other chemotactic factors (LTB4) are described. The potentials of these compounds to serve as an imaging agent are discussed.

Pharmacokinetics and tissue distribution of SB-251353, a novel human CXC chemokine, after intravenous administration to mice.

The pharmacokinetics and tissue distribution of SB-251353, a novel truncated form of the human CXC chemokine growth-related gene product beta, were studied after intravenous administration to the mouse (0.1--250 mg/kg). At the lowest dose, the clearance exceeded blood flow to the kidney. As the dose increased, clearance approached the glomerular filtration rate in the mouse. Clearance of this chemokine may be mediated by its pharmacologic receptor, CXCR2, via endocytosis with subsequent lysosomal degradation, as has been observed for several growth and hematopoietic factors. Apparent distribution volumes were high (> or =1 l/kg). Moderate binding to the Duffy antigen/receptor for chemokines on erythrocytes was observed. Consistent with the pharmacokinetic analysis, microscopic autoradiography showed uptake into renal proximal tubule epithelial cells. Limited excretion of SB-251353 in the urine (<2%) was consistent with catabolism of the chemokine in the tubules. Binding to hepatic sinusoids and connective tissue in the dermis was observed. This possibly reflected interaction of SB-251353 with heparin sulfate proteoglycan and may explain the large distribution volumes. This first study of the disposition of a chemokine provides insight into mechanism of action and physiological factors that may influence chemokine pharmacodynamics.

Changing the chemokine gradient: CINC1 crosses the blood-brain barrier.

Chemokines are a large family of small, inducible, secreted, chemoattractant cytokines that are involved in inflammatory processes. It is well known that systemic and CNS infections cause disruption of the blood-brain barrier (BBB); however, it is not clear how chemokines are involved in this process. We studied the pharmacokinetics of the passage of the chemokine cytokine-induced neutrophil chemoattractant-1 (CINC1) from blood to brain after i.v. bolus injection and its efflux out of the brain after i.c.v. injection. Radiolabeled CINC1 was injected i.v. into mice, and the results were determined by multiple-time regression analysis. Using HPLC, we detected intact CINC1 in brain homogenate and blood after i.v. administration. CINC1 accumulated in the cerebral vasculature but also crossed the BBB completely and rapidly. No saturation of the influx was found, suggesting that either CINC1 crossed the BBB by simple diffusion or the dynamic interactions of binding and internalization precluded the self-inhibition typical of a transport system. Furthermore, there was no efflux system, with CINC1 exiting the brain at the same rate as reabsorption of CSF. The CINC1 injected into blood or CSF did not cause any breakdown of the BBB during the course of the experiments. Thus, the influx of CINC1 may alter the "chemokine gradient" across the BBB and therefore affect inflammatory reactions involving the CNS.

Pyrroloquinoline quinone: a novel vitamin?

Pyrroloquinoline quinone (PQQ), otherwise known as methoxatin, is a water-soluble, redox-cycling orthoquinone that was initially isolated from cultures of methylotropic bacteria. It has been found to be a cofactor of some bacterial alcohol dehydrogenases, and is present in many animal tissues. It may be a novel vitamin because it has been shown to be essential for normal growth and development. The redox-cycling ability of PQQ enables it to scavenge or generate superoxide. When fed to animals as a supplement, PQQ prevents oxidative changes that would ordinarily occur. It has been reported to inhibit glutamate decarboxylase activity and protect against N-methyl-D-aspartate (NMDA) receptor-mediated neurotoxicity in the brain. It appears that in the whole animal, however, PQQ does not cross the blood-brain barrier. Furthermore, it increases nerve growth factor (NGF) synthesis in mouse astroglial cells, but has to be bound to glycine to penetrate and exert this effect in whole brain. It may therefore be regarded as a "Janus faced" molecule, with its potential for a therapeutic role in the brain still in question.

Technetium-99m-labeled chemotactic peptides in acute infection and sterile inflammation.

UNLABELLED: Chemotactic peptides have been proposed as vehicles to image infection and inflammation. Previous studies have shown high uptake at the site of infection soon after injection, most likely because of specific binding to receptors on locally present leukocytes. To investigate this hypothesis, the in vivo behavior of a synthetic chemotactic peptide was compared to a control peptide of similar molecular weight with low receptor binding affinity. In addition, the potential to target to different infections and sterile inflammation was tested. METHODS: Twenty-four hours after induction of Escherichia coli, Staphylococcus aureus and zymosan abscesses, rabbits were i.v. injected with either 1 mCi of 99mTc-labeled formyl-methionyl-leucyl-phenylalanyl-lysine-hydrazinonicotinamid e (99mTc-fMLFK-HYNIC) or 99mTc-labeled hydrazinonicotinamide-methionyl-leucyl-phenylalanyl-OMe (99mTc-HYNIC-MLFOMe, control peptide). Gamma camera images were obtained at 5 min and 1, 4, 8 and 20 hr postinjection. Biodistribution was determined at 20 hr postinjection. RESULTS: The blood clearances of 99mTc-fMLFK-HYNIC and 99mTc-HYNIC-MLFOMe were similar. With time, 99mTc-fMLFK-HYNIC was retained in the abscess (E. coli), whereas the control agent 99mTc-HYNIC-MLFOMe was cleared from the abscess (0.049 +/- 0.011 versus 0.005 +/- 0.0003% 1D/g at 20 hr postinjection; p < 0.0005). Abscess-to-contralateral muscle ratios of 99mTc-fMLFK-HYNIC rose to 36.8 +/- 4.3 at 20 hr postinjection. E. coli, S. aureus and zymosan abscesses were clearly visualized from 4 hr postinjection onward. Abscess-to-background ratios increased to values varying from 4.4 +/- 0.2 (zymosan) to 7.1 +/- 0.6 (S. aureus) at 20 hr postinjection. The uptake in S. aureus and zymosan abscesses did not differ significantly from the uptake in E. coli abscesses. CONCLUSIONS: fMLFK-HYNIC is retained in both acute infection and sterile inflammation by means of specific receptor binding if sufficient cellular infiltration is present.

Localization of radiolabeled chemotactic peptide at focal sites of Escherichia coli infection in rabbits: evidence for a receptor-specific mechanism.

UNLABELLED: The infection imaging properties of a high-affinity 99mTc-labeled chemotactic peptide receptor agonist (N-formyl-methionyl-leucyl-phenylalanine-lysine; N-For-MLFK) were compared with a low-affinity agonist (N-Acetyl-MLFK; N-Ac-MLFK), a moderate-affinity antagonist (N-isobutyloxycarbonyl-MLFK; N-IBoc-MLFK) and non-specific inflammation imaging agents. METHODS: All peptides were prepared by solid-phase methods and purified by high-performance liquid chromatography. The products were assayed in vitro for N-formyl-methionyl-leucyl-phenylalanine receptor binding and superoxide production. Three types of studies were performed in rabbits with Escherichia coli infection: (Study A) Four groups of six animals were coinjected with 99mTc-N-For-MLFK-hydrazinonicotinamide (N-For-MLFK-HYNIC) plus 111In-immunoglobulin G, 111In-red blood cells or 111In-diethylene triamine pentaacetic acid. (Study B) Three groups of six rabbits were coinjected with 111In-leukocytes plus 99mTc-N-For-MLFK-HYNIC, 99mTc-N-Ac-MLFK-HYNIC or 99mTc-N-IBoc-MLFK-HYNIC. (Study C) Two groups of six rabbits were injected with 99mTc-N-For-MLFK-HYNIC and 111In-leukocytes with and without an excess of antagonist. In all three studies, the radiopharmaceuticals were injected 24 hr after infection and dual photon (99mTc and 111In) gamma camera images were acquired at 2-3 and 16-18 hr later. Target-to-background (T/B) ratios were calculated for regions of interest drawn over the infected and contralateral normal tissue. RESULTS: N-For-MLFK, N-Ac-MLFK and N-IBoc-MLFK had EC50s for receptor binding of 2.0, 830 and 150 nM, respectively. The corresponding EC50s for superoxide production were 20.0, approximately 10(3) and > 10(4). Study A demonstrated that the T/B for 99mTc-N-For-MLFK-HYNIC was higher than for any of the nonspecific imaging agents (p < 0.001), and 111In-immunoglobulin G had a higher T/B ratio than 111In-diethylenetriamine pentaacetic acid (p < 0.01) or 111In-red blood cells (p = NS). Study B showed that 99mTc-N-For-MLFK-HYNIC had a higher T/B ratio than the other peptides (p < 0.001). 111In-leukocytes and 99mTc-N-IBoc-MLFK-HYNIC had comparable T/B ratios, which were higher than for 99mTc-N-Ac-MLFK-HYNIC (p < 0.05). Study C demonstrated that coinjection with an antagonist resulted in a significant reduction in the T/B ratio for 99mTc-N-For-MLFK-HYNIC (p < 0.001), but did not affect the T/B ratio for 111In-leukocytes. CONCLUSION: Nonspecific mechanisms contribute minimally to the localization of 99mTc-chemotactic peptide analogs at sites of infection and the majority of the accumulation appears to be receptor mediated. Also, chemotactic peptide receptor antagonists can be used for infection imaging. These results provide important new insights for future radiopharmaceutical development.

Characteristics of the intestinal epithelial barrier during dietary manipulation and glucocorticoid stress.

OBJECTIVES: a) To determine the significance of stress-induced alterations in intestinal permeability by measuring the transmucosal flux of formyl-methionyl-leucyl-phenylalanine (f-MLP), a ubiquitous neutrophilic chemoattractant present in the human and rodent colon; and b) to determine whether stress and/or diet influence(s) bacterial adherence-induced changes in epithelial permeability by affecting the production of secretory immunoglobulin A (IgA), the main immune mechanism preventing bacterial adherence. DESIGN: Prospective, randomized, controlled study. SETTING: University animal research laboratory. SUBJECTS: Female Fischer rats. INTERVENTIONS: Rats were randomly assigned to four groups of seven animals each. Groups of animals were assigned to receive saline or dexamethasone (0.8 mg/kg ip) and were either starved (5% dextrose in water ad libitum) or fed (water and rat chow) for 48 hrs. MEASUREMENTS AND MAIN RESULTS: Mucosal barrier function was evaluated by measuring secretory IgA, bacterial adherence to the intestinal mucosa, and transepithelial electrical resistance, a measure of tight junction permeability. The f-MLP permeation across the mucosa was also determined in segments with significant permeability changes. Results indicate that starvation in dexamethasone-treated rats significantly impairs secretory IgA, promotes bacterial adherence to the mucosa, and results in increased intestinal permeability to f-MLP. These effects are significantly attenuated by the feeding of rat chow. CONCLUSIONS: Alterations in intestinal barrier function are characterized by depressed IgA, bacterial adherence to the intestinal mucosa, and permeation of clinically relevant proinflammatory luminal macromolecules (f-MLP). Enteral stimulation with foodstuffs is a necessary protective measure to prevent altered epithelial barrier function during glucocorticoid stress.

Comparison of the infection imaging properties of a 99mTc labeled chemotactic peptide with 111In IgG.

The biodistribution and infection imaging properties of a 99mTc labeled hydrazino nicotinamide (HYNIC) derivatized chemotactic peptide analog (For-Met-Leu-Phe-Lys-HYNIC) and 111In-DTPA-IgG were compared in rabbits with Escherichia coli infection. Six New Zealand white rabbits were injected in the left posterior thigh with a suspension of E. coli. Twenty four hours later, the animals were injected with: 1.0 mCi of 99mTc labeled peptide plus 0.1 mCi of 111In-DTPA-IgG. At 2-3 and 16-18 h, dual photon scintigrams were acquired and the images were corrected for crossover between the two windows. After recording the final images, the animals were sacrificed and biodistribution was determined. At both imaging times the biodistributions of the two reagents were markedly different. The highest concentrations of 111In-DTPA-IgG were detected in blood pool structures, liver and kidney. In contrast localization of 99mTc labeled peptide was greatest in spleen, lung and liver (consistent with binding to leukocytes). In general, the sites of infection were better visualized with the radiolabeled peptide and T/B ratios increased with time (P < 0.01). At both times, the T/Bs for 99mTc-peptide were higher (P < 0.01); 3.54 +/- 0.47 vs 2.52 +/- 0.38 at 2-3 h and 6.88 +/- 0.79 vs 3.78 +/- 0.36 at 16-18 h. These results indicate that although both radiopharmaceuticals localize at sites of infection, the radiolabeled peptide are superior reagents for the rapid detection of focal sites of infection. However, since the mechanisms of localization are different the combined use of both agents could have value in the general evaluation of infection/inflammation.

Effect of "co-ligand" on the biodistribution of 99mTc-labeled hydrazino nicotinic acid derivatized chemotactic peptides.

Hydrazinonicotinamide (HYNIC) derivatized chemotactic peptides radiolabeled with 99mTc- (via 99mTc-glucoheptonate) have been demonstrated to be useful for infection imaging [J. Nucl. Med. 34, 1964-1974 (1993)]. Since HYNIC can occupy only two sites of the technetium co-ordination sphere, the labeled product most probably contains additional ligands. Thus we hypothesized that glucoheptonate serves this role by acting as a "'co-ligand'". Due to the low molecular weight of the chemotactic peptides, the "co-ligand" used for technetium labeling could have profound effects on biodistribution. To evaluate this possibility, we measured the biodistribution of 99mTc-labeled For-MLFK-HYNIC radiolabeled using four different "co-ligand"s: glucarate, glucoheptonate, mannitol and glucamine, providing a small series of hydroxyl-backbone ligands which differ in the number and type of ionizable functional groups present. Each preparation was injected into groups of 6 rats (approximately 10 microCi/rat) and biodistribution was determined at 5, 30, 60 and 120 min. Although small differences in biodistribution were detected in most tissues, the most prominent differences (P < 0.01) were observed in lung (glucoheptonate, glucarate > mannitol >> glucamine), liver (glucarate, glucoheptonate, mannitol >> glucamine), kidney (mannitol > glucarate, glucoheptonate, glucamine), spleen (glucarate >> glucoheptonate, mannitol >> glucamine) and GI-tract (glucarate, glucamine >> gluco-heptonate >> mannitol). These results provide support for the "co-ligand" hypothesis and indicate that the nature of the "co-ligand" can have profound effects on biodistribution. Although radiolabeling using glucamine as the "co-ligand" results in the lowest concentrations of radioactivity in most organs, the extremely low concentration of mannitol-labeled peptide in the GI-tract suggests that this may be the "co-ligand" of choice for most applications.

Evidence of cardiac inflammation after open heart operations.

In this study, 6 anesthetized dogs underwent global cardiac arrest for 1 hour, followed by reperfusion on bypass for 45 minutes. The hearts were then weaned off cardiopulmonary bypass and monitored for an additional 2 hours. Using modified Boyden chambers, high levels of neutrophil chemotactic activity were detected (using a checkerboard analysis) in the coronary sinus effluents obtained during cardiac arrest. The activity tended to decline during reperfusion. Assay of myeloperoxidase (a marker for neutrophils) revealed an accumulation of large numbers of neutrophils in the right (14 +/- 1.1 x 10(4) cells/g wet weight) and left (16 +/- 1 x 10(4) cells/g wet weight) ventricles after 2 hours of reperfusion. Light microscopy evaluation confirmed the presence of neutrophils, not only in the ventricles, but also in a greater number in the right and left atria. Electron microscopy study of these hearts revealed the presence of mild reversible changes, indicating good preservation of the hearts during arrest. Results of this study provide evidence for an acute inflammatory reaction that takes place after cardiac operations and suggest a role for myocardial tissues in the initiation of such a response through their release of neutrophil chemotactic factors.

In vivo bioactivity and biodistribution of chemotactic peptide analogs in nonhuman primates.

The dose dependence of the effect of chemotactic peptide on peripheral leukocyte levels was measured in normal Rhesus monkeys. A 99mTc-labeled hydrazino nicotinamide (HYNIC) derivatized chemotactic peptide analog was used to study biodistribution and inflammation imaging in Rhesus monkeys. In normal animals the studies demonstrated that chemotactic peptide induced a clear dose-dependent reduction in peripheral leukocyte levels. The decrease in leukocyte number occurred almost immediately after injection and rapidly returned to baseline. Significant effects on differential WBC count, blood pressure, pulse rate or respiration rate were not detected. The lowest dose of peptide tested (10 ng/kg) had minimal effect on leukocyte level. The HYNIC derivatized peptide was prepared in excellent yield and purity, had biological activity similar to the native peptide and was readily labeled at specific activity of > 20,000 mCi/mumole. When approximately 0.5 mCi (< 2.0 ng/kg) of radiolabeled peptide was injected in monkeys with focal sites of mild sterile inflammation, a pattern of biodistribution similar to radiolabeled WBCs was observed and reductions in leukocyte levels were not detected. At 3 hr after injection, the site of inflammation was readily apparent with a target-to-background ratio of approximately 3:1. These studies demonstrate that radiolabeled chemotactic peptide analogs are effective agents for imaging sites of inflammation in monkeys. By radiolabeling at high specific activity, the effect of these reagents on peripheral leukocyte levels can be avoided.

Imaging focal sites of bacterial infection in rats with indium-111-labeled chemotactic peptide analogs.

Four DTPA-derivatized chemotactic peptide analogs: ForNleLFNleYK-DTPA (P1), ForMLFNH(CH2)6NH-DTPA (P2), ForNleLFK(NH2)-DTPA (P3), and ForNleLFK-DTPA (P4), were synthesized and evaluated for in vitro bioactivity and receptor binding. The peptides were radiolabeled with 111In by transchelation and their biodistribution determined in rats at 5, 30, 60 and 120 min after injection. Localization at sites of infection was determined by scintillation camera imaging in animals with deep-thigh infection due to Escherichia coli. Images were recorded from 5 min to 2 hr after injection. All peptides maintained biologic activity (EC50 for O2-production by human PMN's: 3-150 nM) and the ability to bind to the oligopeptide chemoattractant receptor on human PMN's (EC50 for binding: 7.5-50 nM); biologic activity and receptor binding were highly correlated (r = 0.99). For all the peptides, blood clearance was rapid (half-lives: 21.5, 33.1, 31.6, and 28.7 min for P1, P2, P3, and P4, respectively). Biodistributions of the individual peptides were similar with low levels of accumulation in the heart, lung, liver, spleen, and gastrointestinal tract. In the kidney, P1 had much greater accumulation than other organs. All peptides yielded high quality images of the infection sites within 1 hr of injection. This study demonstrates that 111In-labeled chemotactic peptide analogs were effective agents for the external imaging of focal sites of infection.