Decay accelerating factor (CD55) protects neuronal cells from chemical hypoxia-induced injury

Background: Activated complement system is known to mediate neuroinflammation and neurodegeneration following exposure to hypoxic-ischemic insults. Therefore, inhibition of the complement activation cascade may represent a potential therapeutic strategy for the management of ischemic brain injury. Decay-accelerating factor (DAF, also known as CD55) inhibits complement activation by suppressing the function of C3/C5 convertases, thereby limiting local generation or deposition of C3a/C5a and membrane attack complex (MAC or C5b-9) production. The present study investigates the ability of DAF to protect primary cultured neuronal cells subjected to sodium cyanide (NaCN)-induced hypoxia from degeneration and apoptosis. Methods: Cultured primary cortical neurons from embryonic Sprague-Dawley rats were assigned one of four groups: control, DAF treatment alone, hypoxic, or hypoxic treated with DAF. Hypoxic cultures were exposed to NaCN for 1 hour, rinsed, followed by 24 hour exposure to 200 ng/ml of recombinant human DAF in normal medium. Human DAF was used in the present study and it has been shown to effectively regulate complement activation in rats. Neuronal cell function, morphology and viability were investigated by measuring plateau depolarization potential, counting the number dendritic spines, and observing TUNEL and MTT assays. Complement C3, C3a, C3a receptor (R) production, C3a-C3aR interaction and MAC formation were assessed along with the generation of activated caspase-9, activated caspase-3, and activated Src. Results: When compared to controls, hypoxic cells had fewer dendritic spines, reduced plateau depolarization accompanied by increased apoptotic activity and accumulation of MAC, as well as up-regulation of C3, C3a and C3aR, enhancement of C3a-C3aR engagement, and elevated caspase and Src activity. Treatment of hypoxic cells with 200 ng/ml of recombinant human DAF resulted in attenuation of neuronal apoptosis and exerted significant protection against neuronal dendritic spine loss and plateau depolarization reduction. Furthermore, treatment with DAF resulted in decreased accumulation of C3a, MAC, C3a-C3aR interaction, caspase-9, activated caspase-3, and pTyr416-Src (activated Src) tyrosine kinase. Conclusion: DAF was found to reduce neuronal cell death and apoptosis in NaCN induced hypoxia. This effect is attributed to the ability of DAF to limit complement activation and inhibit the activity of Src and caspases 9 and 3. This study supports the inhibiting of complement as a neuroprotective strategy against CNS ischemia/reperfusion injury

The Role of Platelet Factor 4 in Local and Remote Tissue Damage in a Mouse Model of Mesenteric Ischemia/Reperfusion Injury

The robust inflammatory response that occurs during ischemia reperfusion (IR) injury recruits factors from both the innate and adaptive immune systems. However the contribution of platelets and their products such as Platelet Factor 4 (PF4; CXCL4), during the pathogenesis of IR injury has not been thoroughly investigated. We show that a deficiency in PF4 protects mice from local and remote tissue damage after 30 minutes of mesenteric ischemia and 3 hours of reperfusion in PF4-/- mice compared to control B6 mice. This protection was independent from Ig or complement deposition in the tissues. However, neutrophil and monocyte infiltration were decreased in the lungs of PF4-/- mice compared with B6 control mice. Platelet-depleted B6 mice transfused with platelets from PF4-/- mice displayed reduced tissue damage compared with controls. In contrast, transfusion of B6 platelets into platelet depleted PF4-/- mice reconstituted damage in both intestine and lung tissues. We also show that PF4 may modulate the release of IgA. Interestingly, we show that PF4 expression on intestinal epithelial cells is increased after IR at both the mRNA and protein levels. In conclusion, these findings demonstrate that may PF4 represent an important mediator of local and remote tissue damage

Platelet-Associated CD40/CD154 Mediates Remote Tissue Damage after Mesenteric Ischemia/Reperfusion Injury

Several innate and adaptive immune cell types participate in ischemia/reperfusion induced tissue injury. Amongst them, platelets have received little attention as contributors in the process of tissue damage after ischemia reperfusion (I/R) injury. It is currently unknown whether platelets participate through the immunologically important molecules including, CD40 and when activated, CD154 (CD40L), in the pathogenesis of I/R injury. We hypothesized that constitutive expression of CD40 and activation-induced expression of CD154 on platelets mediate local mesenteric and remote lung tissue damage after I/R injury. Wild type (WT; C57BL/6J), CD40 and CD154 deficient mice underwent mesenteric ischemia for 30 minutes followed by reperfusion for 3 hours. WT mice subjected to mesenteric I/R injury displayed both local intestinal and remote lung damage. In contrast, there was significantly less intestinal damage and no remote lung injury in CD40 and CD154 deficient mice when compared to WT mice. Platelet-depleted WT mice transfused with platelets from CD40 or CD154 deficient mice failed to reconstitute remote lung damage. In contrast, when CD40 or CD154 deficient mice were transfused with WT platelets lung tissue damage was re-established. Together, these findings suggest that multiple mechanisms are involved in local and remote tissue injury and also identify platelet-expressed CD40 and/or CD154 as mediators of remote tissue damage

C4d Deposits on the Surface of Red Blood Cells in Trauma Patients and Interferes with their Function

Objective Complement system is activated in patients with trauma. Although complement activation is presumed to contribute to organ damage and constitutional symptoms, little is known about the involved mechanisms. Because complement components may deposit on red blood cells (RBC), we asked whether complement deposits on the surface of RBC in trauma and whether such deposition alters RBC function. Design A prospective experimental study Setting Research laboratory Subjects Blood samples collected from 42 trauma patients and 21 healthy donors Intervention None Measurements and Main Results RBC and sera were collected from trauma patients and control donors. RBC from trauma patients (n=40) were found to display significantly higher amounts of C4d on their surface by flow cytometry compared to normal RBC (n=17) (P<0.01). Increased amounts of iC3b were found in trauma sera (n=27) (vs. 12 controls, P<0.01) by ELISA. Incubation of RBC from universal donors (O,Rh-) with trauma sera (n=10) promoted C4d deposition on their surface (vs. 6 controls, P<0.05). Complement-decorated RBC (n=6) displayed limited their deformability (vs. 6 controls, P<0.05) in 2-dimensional microchannel arrays. Incubation of RBC with trauma sera (n=10) promoted the phosphorylation of band 3, a cytoskeletal protein important for the function of the RBC membrane (vs. 8 controls, P<0.05), and also accelerated calcium influx (n=9) and enhanced nitric oxide production (n=12) (vs. 4 and 8 controls respectively, P<0.05) in flow cytometry. Conclusions Our study found the presence of extensive complement activation in trauma patients and presents new evidence in support of the hypothesis that complement activation products deposit on the surface of RBC. Such deposition could limit RBC deformability and promote the production of nitric oxide. Our findings suggest that RBC in trauma patients malfunction, which may explain organ damage and constitutional symptoms that is not accounted for otherwise by previously known pathophysiologic mechanisms

C3a Enhances the Formation of Intestinal Organoids through C3aR1

C3a is important in the regulation of the immune response as well as in the development of organ inflammation and injury. Furthermore, C3a contributes to liver regeneration but its role in intestinal stem cell function has not been studied. We hypothesized that C3a is important for intestinal repair and regeneration. Intestinal organoid formation, a measure of stem cell capacity, was significantly limited in C3-deficient and C3a receptor (C3aR) 1-deficient mice while C3a promoted the growth of organoids from normal mice by supporting Wnt-signaling but not from C3aR1-deficient mice. Similarly, the presence of C3a in media enhanced the expression of the intestinal stem cell marker leucine-rich repeat G-protein-coupled receptor 5 (Lgr5) and of the cell proliferation marker Ki67 in organoids formed from C3-deficient but not from C3aR1-deficient mice. Using Lgr5.egfp mice we showed significant expression of C3 in Lgr5+ intestinal stem cells whereas C3aR1 was expressed on the surface of various intestinal cells. C3 and C3aR1 expression was induced in intestinal crypts in response to ischemia/reperfusion injury. Finally, C3aR1-deficient mice displayed ischemia/reperfusion injury comparable to control mice. These data suggest that C3a through interaction with C3aR1 enhances stem cell expansion and organoid formation and as such may have a role in intestinal regeneration

Transfusion of either <i>CD40^−/−</i> and <i>CD154^−/−</i> platelets protects platelet depleted B6 mice from remote lung injury but not from local intestinal injury.

<p>(A–L) Hematoxylin and eosin stained sections of mouse small intestine and lung from B6 and platelet depleted B6 mice transfused with either <i>CD40^−/−</i> or <i>CD154^−/−</i> platelets after 30 minutes of ischemia and 3 hours reperfusion. Images are representative of 3–4 mice per group in two experiments. All images shown are 200×magnification. (M)Intestinal injury score and (N) Lung injury score (mean ± SD). ns: not significant *p≤0.05, **p≤0.01, and ***p≤0.001 for I/R compared to sham controls.</p

Remote lung injury is re-established in <i>CD154^−/−</i> and <i>CD40^−/−</i> mice transfused with B6 platelets.

<p>(A–D) Hematoxylin and eosin, (E–H) platelets and (I–L) C3 complement factor stained sections of mouse lung from <i>CD154^−/−</i> and <i>CD40^−/−</i> mice transfused with B6 platelets after 30 minutes of ischemia and 3 hours reperfusion. Images are representative of 3–4 mice per group in two experiments. All images shown are 200×magnification. (M)Injury score (mean ± SD) in intestine and lung. ns: not significant *p≤0.05, **p≤0.01, and ***p≤0.001 for I/R compared to sham controls. Small arrows: indicate areas of positive staining.</p

Intestinal and lung injury is reduced after mesenteric ischemia/reperfusion in <i>CD40^−/−</i> and <i>CD154^−/−</i> mice.

<p>Hematoxylin and eosin stained sections of mouse small intestine after 30 minutes of ischemia and 3 hours reperfusion. Images are representative of 3–4 mice per group in two experiments. (A–F) Images of intestinal villi from sham and I/R. (G–L) Images of lung from sham and I/R, platelet-deficient sham and I/R. All images shown are 200×magnification. (M, N) Injury score (mean ± SD) in intestine and lung. *p≤0.05, **p≤0.01, and ***p≤0.001 for I/R compared to sham controls.</p

Tissue damage in PF4-/- mice is not associated with immunoglobulin (Ig) deposition.

<p>Tissue sections of lung and intestine from B6 and PF4-/- mice after 30 minutes of mesenteric ischemia and 3 hrs of reperfusion were stained for IgM (A-L, red) and IgA (M-X, red) and counterstained with hematoxylin (blue). Images are representative of 3–4 mice per group. Red: Positive Staining.</p