The respiratory burst of neutrophils functions as a primary host-defence mechanism against invading micro-organisms. This microbicidal action occurs predominately inside the cell within the phagolysosome , and normally only a small portion of superoxide or its metabolites is released to the extracellular environment [24, 25] through the orifice formed by fusion of oxidant-producing compartments with the plasma membrane . However, the superoxide that is released extracellularly is transformed into H2O2 with the concurrent release of myeloperoxidase, which reacts with a halogen (e.g. Cl-) to form the highly toxic hypochlorous acid (HOCl). It is this extracellular generation of ROS that is believed to contribute to aggravated inflammation and cell damage in several diseases such as systemic inflammatory response syndrome , hypoxic injury followed by reoxygenation after transplantation and in myocardial, hepatic, intestinal, cerebral, renal, other ischemic diseases , and pulmonary inflammation .
The extracellular release of superoxide by circulating neutrophils and eosinophils is increased in patients with asthma [29–32] or cutaneous allergic reactions [33, 34]. The results of the current study show that an increase in the respiratory burst of circulating neutrophils also occurs with intestinal allergy, and may be a general feature of type I hypersensitivity reactions, although in our animal model it is predominately the generation of intracellular ROS within neutrophils that is increased by antigen challenge, whereas superoxide release is not altered. Normally, the NADPH oxidase complex in circulating leukocytes is unassembled and functionally inactive, a mechanism that prevents inappropriate generation of superoxide. However, upon exposure to a priming agent the NADPH oxidase complex is assembled so that after extravasating and migrating to the site of inflammation the phagocyte is functionally active . The results described herein suggest that an allergic reaction inappropriately primes the NADPH oxidase complex in circulating neutrophils, and although ideally the superoxide generated is directed into the phagolysosome a small portion of superoxide or its metabolites is released to the extracellular environment [24, 35]. This extracellular appearance of neutrophil-derived ROS that contributes to aggravated inflammation and cell damage. Interference with ROS production  may account for the therapeutic potential of some anti-asthmatic or anti-allergic drugs [37–39]. Similarly, the anti-allergic and anti-asthmatic properties of feG [6, 7] may be due, in part, to the reduction in the intracellular oxidative burst activity of neutrophils.
Several PKC isozymes (α, βII, δ and ζ) are involved in the regulation of NADPH oxidase and the respiratory burst of human and rat neutrophils [40–47], a process that involves phosphorylation by these four PKC isozymes of p47
[41, 43, 47]. This phosphorylation is a critical step for translocation of the cytosolic components and assembly of the active NADPH oxidase. Of particular relevance to PMA-stimulated generation of ROS in neutrophils are the PKC isozymes α, β, and δ. These isozymes require for their activation DAG, the endogenous ligand for PMA, whereas the PKCζ isoform, does not require DAG. Intracellular ROS production by circulating neutrophils is regulated predominately by PKCδ (Figure 3), and this result concords with reported role of PKCδ in regulating NADPH oxidase assembly for PMA-dependent generation of ROS in human neutrophils , monocytes [49, 50] and eosinophils. Generally, PKCδ is considered to positively regulate superoxide release from human eosinophils [51, 52], and the increase in PMA-stimulated release of superoxide from neutrophils of rats challenged with BSA (naïve antigen) in the presence of the PKCδ inhibitor, rottlerin (Figure 4) seems paradoxical. This potentiating action of rottlerin possibly reflects the positive and negative role of PKCδ in regulating cell function, as a similar increase in superoxide release was seen with zymosan-stimulated equine eosinophils , although data on neutrophils are lacking. It may be possible that PKCδ participates in shifting the direction of ROS production from intracellular accumulation to extracellular release, although this speculation requires confirmation. Given that eosinophils from atopic patients release superoxide predominately into the extracellular space, whereas that of neutrophils is directed more to the interior of the cell , it would be interest to determine if the directional differences reflect the different contributions of PKCδ to the Rac-dependent site of assembly of the NADPH oxidase complex in eosinophils and neutrophils, i.e. plasma membrane or phagolysosome, respectively .
In contrast, the release of superoxide from neutrophils is regulated predominately by PKCβ [43, 45], an observation that was corroborated in the present study (Figure 4). Our study also shows that antigen challenge of sensitized animals leads to loss of responsiveness to PKC inhibitors, as seen with the PKCδ inhibitor, rottlerin, on circulating neutrophils (Figure 3). This loss of responsiveness to rottlerin may reflect a deregulation of PKC by antigen challenge. The mechanism by which this occurs is not known, but may reflect a recently described novel G-protein receptor coupled (GPCR)-PKC-regulated switch that enhances receptor signalling, and prevents receptor internalization with consequent loss of responsiveness . Treatment with feG re-established sensitivity to rottlerin, and corrected the supposedly deregulated PKC function, although the mechanism of action is unknown.
An up-regulation of CD49d expression on circulating neutrophils occurs with ischemia-reperfusion injury , in septic patients , and as shown herein with allergic reactions (Figure 5). This abnormal up-regulation of a β1-integrin on circulating neutrophils leads to inappropriate neutrophil homing and recruitment [56–58], and activation of NADPH oxidase [59, 60]. Thus, expression of β1-integrin on circulating neutrophils could cause inappropriate inflammatory responses not only at the leukocyte-endothelial cell interface but also at an extravascular interface [9, 59], possibly through a mechanism involving frustrated phagocytosis and the leakage of the dismutated product of intracellular superoxide, hydrogen peroxide, from intracellular compartments. Concurrent with a decreased expression of CD49d by feG treatment of OA-challenged animals (Figure 5) the intracellular oxidative burst was correspondingly decreased (Figure 3) with a consequent reduction in the severity of allergic reactions. These observations may explain why antibodies to and small molecule antagonists against CD49d are effective in blocking asthmatic reactions in rats and sheep [61, 62].
The mechanism by which feG, administered 18 h after antigen, decreases circulating neutrophil accumulation, intracellular oxidative activity and CD49d expression remains undefined. However, previous studies suggest that feG and related peptides probably exert their anti-allergic actions on early cellular events as they reduce rapidly initiated anaphylactic events such as hypotension, intestinal motility and vascular permeability [10, 20]. A mode of action for feG independent of mast cells may predominant as the peptides do not modify antigen-evoked mast cell degranulation , whereas this peptide effectively reduces neutrophil adhesion and leukocyte migration both in vivo and in vitro [6, 17]. Since neither binding nor cellular uptake of [3H]feG has been observed with rat leukocytes or neutrophilic transformed HL60 cells (unpublished), we are currently determining if feG may act as a high affinity, low avidity allosteric regulator of integrins and associated co-stimulatory molecules , in a manner similar to a regulation of CD11a/CD18 affinity for counter ligands by a conformational switch in the I domain of this integrin . Since engagement of integrins contributes to increases in vascular permeability and superoxide production [64, 65], this mechanism of action may account for the observed properties of feG.