Our studies demonstrate that LPS-induced IL-6 production was significantly increased in PMNs harvested from β-arrestin 2 (-/-) mice compared to (+/+) mice. Thus, β-arrestin 2 is a negative regulator of pro-inflammatory mediator production in PMNs. β-arrestin 2 deficiency had no effect on LPS binding/uptake to PMNs or TLR4 expression on the surface of PMNs. However, we found that PMN chemotaxis was greatly augmented in β-arrestin 2-deficient mice, suggesting that it may play a significant role in the mediation of PMN recruitment and activity at the site of inflammation. Expression of adhesion receptors CD18 and particularly CD62L were also found to be increased in β-arrestin 2-deficient mice, suggesting a role for the β-arrestin 2 in expression of these receptors at the cell surface. In our study with β-arrestin 2 we demonstrated a marked increase in pulmonary MPO activity relative to WT mice. These findings suggest that β-arrestin 2 negatively regulates PMN tissue infiltration during sepsis.
The measured basal +LPS stimulated production of TNFα and IL-6 in oyster glycogen recruited PMNs from β-arrestin 2-deficient mice suggest a predominant anti-inflammatory function of β-arrestin 2 in PMNs. Our recent studies showed that both β-arrestin 1 and 2 negatively regulate NFκB activation [20
]. In HEK293 cells rendered LPS-responsive by stable transfections with CD14 and TLR4, we demonstrated by siRNA depletion of β-arrestin 1 and 2 augmented NFκB activation in response to LPS [20
]. On the other hand, over-expression of WT β-arrestins 1 and 2 in these cells suppressed LPS-induced NFκB activation [20
]. These findings agree with studies that β-arrestin 2 directly interacts with, IκBα thus preventing the phosphorylation and degradation of IκBα [21
]. Recent studies have demonstrated that β-arrestins 1 and 2 directly interact with TRAF6 following TLR or IL-6 activation [29
]. The complexes of β-arrestins and TRAF6 prevented its autouitination and activation of NFκB [29
]. These studies further support an inhibitory role for β-arrestins in the regulation of LPS signaling. Since cytokine production was also increased in oyster glycogen recruited PMNs, it is probable that β-arrestin 2 may negatively regulate other inflammatory stimuli through similar mechanisms.
In addition to studying the effect of β-arrestin 2 on the production of pro-inflammatory cytokines, we demonstrated an increase in chemotaxis of PMNs induced by oyster glycogen in the peritoneal cavity of β-arrestin 2 deficient mice as compared to (+/+) mice. These findings are in accordance with other studies implicating β-arrestins as negative regulators of chemotaxis. The interactions between β-arrestins and Gi protein-coupled receptors involved in regulation of PMN chemotaxis, specifically in neutrophils, are pronounced in the CXC subfamily of receptors, including CXCR1 and CXCR2 [29
]. Exposure to increased concentrations of chemokines, e.g. IL-8 can cause neutrophils to become unresponsive to further stimulation by other inflammatory cytokines; therefore, desensitization and internalization of CXC receptors is necessary for proper neutrophil functioning during inflammation. Barlic et al. showed that CXCR1 internalization is decreased in HEK293 cells with low β-arrestin 2 expression, and that such internalization is increased with additional expression of β-arrestin 1 or 2 [30
]. Su et al. showed that neutrophil recruitment with the binding of the chemokine CXCR1 to the CXCR2 receptor was increased in β-arrestin 2 deficient mice, and that although increased neutrophil activity in the form of calcium mobilization and superoxide anion production were increased in KO mice, receptor internalization was markedly decreased [32
]. These two studies suggest that β-arrestin 2 may be negatively regulate chemotaxic activity of neutrophils by mediating chemokine receptor internalization and ultimately terminating chemokine signaling. However, such an interpretation may be an over simplification of the response.
One concept of chemotaxis is that desensitization and recycling of chemotactic receptors are essential for maintaining cellular polarity that promotes chemotaxis [29
]. This concept is, in part, based upon in vitro studies where β-arrestin 2 deficiency actually suppresses chemotaxis [29
]. Thus, β-arrestin regulation of PMN recruitment in vivo likely reflects responses to other signals at the site of inflammation that are not present in transwell filter assays. Also, in contrast to CRCR1 and CXCR2, β-arrestin 2 appears to positively regulate CXCR4 in vivo lymphocyte chemotaxis [33
]. Therefore, the effect of β-arrestins in regulation of chemotaxis in vivo depends upon the chemotactic receptor activation.
We also observed an increase in CD18 and CD62L (L-selectin) expression in the KO mice as compared to WT, in the recruited PMNs. This suggests that β-arrestin 2 is involved in the inhibition of signaling by these two integrins in PMNs. Mulligan et al. [25
] examined the role of adhesion molecule expression in recruitment of neutrophils and found that blocking the selectins reduced the accumulation of neutrophils in the peritoneal cavity after oyster glycogen-induced peritonitis. A similar trend was seen after the use of blocking antibodies for CD11a, CD11b, and CD18 [32
], indicating that these adhesion molecules are involved in the recruitment of neutrophils and the subsequent release of cytokines. β-arrestin may thus reduce the relative expression of these adhesion molecules and thus reduce PMNs to endothelial surfaces.
The CLP model is accepted as a clinically relevant sepsis model. CLP-induced pulmonary MPO activity was significantly increased in β-arresin 2 KO compared to WT mice demonstrating that β-arrestin 2 negatively regulates pulmonary leukosequestration. In WT mice, CLP-induced small but statistically significant increase of pulmonary MPO activity suggesting that the strain of mice has low response to sepsis induced inflammation. However, the β-arrestin 2 (-/-) mice with same background exhibited a more severe response suggesting that β-arrestin 2 negatively regulates tissue PMN infiltration in sepsis. Whether this is a result of altered PMN or endothelial adhesion receptor expression and/or altered chemokine production is currently under investigation. However this finding highlights the translational significance of β-arrestin 2 in polymicrobial sepsis.
Our studies demonstrate an effect of β-arrestin 2 on PMN inflammatory responses as reflected by altered cytokine production, PMN recruitment, adhesion molecule expression, and pulmonary leukosequtration in response to CLP. The extent that such responses may affect other pathogenesic events of sepsis remains to be determined.