Immunopharmacology and InflammationAn α4β1 integrin antagonist decreases airway inflammation in ovalbumin-exposed mice
Introduction
Human asthma is a spectrum of diseases characterized by persistence of airway inflammation that contributes to structural airway changes and alterations in airway mechanics, including increased airway resistance and airway hyperreactivity. The key effector cells that dictate these changes in the airways include eosinophils, mast cells, and Th-2 CD4 lymphocytes. While we believe that the primary integrins, chemokines, and cytokines that govern the migration of these cells into the lung have been identified, it is not clear which targets in which pathways will lead to future asthma therapies.
Integrins are a family of cell surface receptors involved in numerous intercellular and cell-matrix trafficking functions. Integrins exist as αβ heterodimers; the α4 subunit has proven an attractive target for blocking organ-specific inflammation. Inhibition of the α4 subunit of both the α4β7 and the α4β1 (very late antigen 4, VLA-4) integrins has shown promise in decreasing airway inflammation and airway hyperresponsiveness. Whether such an inhibitor could improve the care of patients with asthma or other inflammatory airway disorders is now being studied (Ravensberg et al., 2006).
We have previously published a series of studies describing the synthesis and activity of LLP2A (Fig. 1), a novel, high-affinity α4β1 integrin peptidomimetic antagonist (Liu et al., 2006, Peng et al., 2008, Peng et al., 2006). For example, we have shown that LLP2A binds specifically to α4β1-expressing lymphomas (Peng et al., 2008, Peng et al., 2006). The purpose of the present study was to elucidate the role of the α4β1 integrin in the recruitment of eosinophils and lymphocytes to the lung in a common mouse model of allergic airway inflammation and hyperresponsiveness. We hypothesized that there is a direct linkage between the expression and activation of the α4β1 integrin on eosinophils and lymphocytes and increased airway inflammation and hyperresponsiveness caused by exposure to ovalbumin lbumin. To test this hypothesis, we administered LLP2A, or scrambled LLP2A (S-LLP2A, Fig. 1), prior to the ovalbumin exposures. We determined the response of this antagonist on the development of airway inflammation, airway hyperresponsiveness, and goblet cell hyperplasia. We further hypothesized that the bioavailability and efficacy of the synthetic α4β1 antagonist could be improved by constructing different PEGylated (PEG) formulations of the α4β1 antagonist, i.e., LLP2A conjugated to a linear 30 kDa polyethylene glycol (L-PEG-LLP2A), a branch 40 kDa PEG (B-PEG-LLP2A) and a tetravalent 4-arm 10 kDa PEG (T-PEG-LLP2A) (Fig. 1). LLP2A, S-LLP2A and the three PEGylated antagonists were tested in subsequent in vivo experiments.
Section snippets
Synthesis of LLP2A, S-LLP2A and PEGylated LLP2A conjugates
LLP2A and S-LLP2A were synthesized as previously reported (Peng et al., 2006). PEGylated LLP2A conjugates were designed to have PEG attached to the side chain of lysine (i.e., ɛ-amino group) on LLP2A-K (structure is shown in Scheme 1), and two hydrophilic linkers between LLP2A and K(PEG). The synthesis of LLP2A-K was performed on rink amide MBHA resin by a standard solid-phase peptide synthesis approach using Fmoc-tBu chemistry and HOBt/DIC coupling. The synthetic scheme is shown in Scheme 1.
Results
Balb/c mice were exposed to inhaled ovalbumin or to filtered air for seven to fourteen days and treated with LLP2A (experiments 1 and 2) or PEGylated (experiments 3 and 4) and LLP2A conjugates or scrambled LLP2A. Two mice treated with the L-PEG-LLP2A developed evidence of alveolar hemorrhage by gross observation and lung lavage, but this was not evident in subsequent experiments. The two animals were not specifically tested for evidence of acute bacterial or viral infection, but there were no
Discussion
We have previously demonstrated a sequence of inflammatory and structural airway changes in the lungs of BALB/c mice sensitized and exposed to ovalbumin aerosols for one to eight weeks (Kenyon et al., 2003a, Kenyon and Last, 2005). For this study, we hypothesized that the administration of the novel, small molecule α4β1 antagonist LLP2A would inhibit the migration of lymphocytes and eosinophils into the lung in mice exposed to ovalbumin for one to two weeks by affecting α4β1 integrin function.
Conflict of interest
The authors have no conflict of interest relevant to the contents of this manuscript.
Acknowledgements
We thank Lisa Franzi for technical assistance in performing these experiments and Dr. Jerold Last for his review of the manuscript. This work was supported by grants from the NIH (NHLBI (NJK) and NCI (KSL)).
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