Functional roles for C5a and C5aR but not C5L2 in the pathogenesis of human and experimental cerebral malaria.

The host immune response plays an important role in the onset and progression of cerebral malaria (CM). The complement system is an essential component of the innate immune response to malaria, and its activation generates the anaphylatoxin C5a. To test the hypothesis that C5a signaling contributes to the pathogenesis of CM, we investigated a causal role for the C5a receptors C5aR and C5L2 in a mouse model of experimental CM (ECM) induced by Plasmodium berghei ANKA infection, and using a case-control design, we examined levels of C5a in plasma samples from Ugandan children presenting with CM or uncomplicated malaria (UM). In the ECM model, C5aR(-/-) mice displayed significantly improved survival compared to their wild-type (WT) counterparts (P = 0.004), whereas C5L2(-/-) mice showed no difference in survival from WT mice. Improved survival in C5aR(-/-) mice was associated with reduced levels of the proinflammatory cytokines tumor necrosis factor (TNF) and gamma interferon (IFN-γ) and the chemokine, monocyte chemoattractant protein 1 (MCP-1) (CCL2). Furthermore, endothelial integrity was enhanced, as demonstrated by increased levels of angiopoietin-1, decreased levels of angiopoietin-2 and soluble ICAM-1, and decreased Evans blue extravasation into brain parenchyma. In the case-control study, the median levels of C5a at presentation were significantly higher in children with CM versus those in children with UM (43.7 versus 22.4 ng/ml; P < 0.001). These findings demonstrate that C5a is dysregulated in human CM and contributes to the pathogenesis of ECM via C5aR-dependent inflammation and endothelial dysfunction.

Complement C3 and C5 deficiency affects fracture healing.

There is increasing evidence that complement may play a role in bone development. Our previous studies demonstrated that the key complement receptor C5aR was strongly expressed in the fracture callus not only by immune cells but also by bone cells and chondroblasts, indicating a function in bone repair. To further elucidate the role of complement in bone healing, this study investigated fracture healing in mice in the absence of the key complement molecules C3 and C5. C3(-/-) and C5(-/-) as well as the corresponding wildtype mice received a standardized femur osteotomy, which was stabilized using an external fixator. Fracture healing was investigated after 7 and 21 days using histological, micro-computed tomography and biomechanical measurements. In the early phase of fracture healing, reduced callus area (C3(-/-): -25%, p=0.02; C5(-/-): -20% p=0.052) and newly formed bone (C3(-/-): -38%, p=0.01; C5(-/-): -52%, p=0.009) was found in both C3- and C5-deficient mice. After 21 days, healing was successful in the absence of C3, whereas in C5-deficient mice fracture repair was significantly reduced, which was confirmed by a reduced bending stiffness (-45%; p=0.029) and a smaller callus volume (-17%; p=0.039). We further demonstrated that C5a was activated in C3(-/-) mice, suggesting cleavage via extrinsic pathways. Our results suggest that the activation of the terminal complement cascade in particular may be crucial for successful fracture healing.

LPS-induced murine abortions require C5 but not C3, and are prevented by upregulating expression of the CD200 tolerance signaling molecule.

PROBLEM: Lipopolysaccharide (LPS) acts via tlr4 to promote Th1 cytokine secretion and abortions. LPS is an essential co-factor in spontaneous abortion in the CBA x DBA/2 model and in stress-triggered abortions. In the CBA x DBA/2 model, C3a, C5a, and fgl2 prothrombinase participate in triggering inflammation that terminates embryo viability. As fgl2 prothrombinase (via thrombin) can generate C5a, it was predicted that LPS-driven abortions (which require fgl2) would be independent of C3. CD200Fc can prevent abortions in the CBA x DBA/2 model, but an action through Fc could not be excluded. METHOD OF STUDY: C3(-/-) and C5(-/-) knock-out mice on a B6 background were syngeneically mated and Salmonella enteritidis LPS was administered i.p. on day 6.5 or pregnancy along with 2 mg progesterone in sesame oil s.c. The total number of implants and the number of resorbing embryos were counted on day 13.5 of pregnancy. CD200-rtTA double transgenic homozygous males (B6 background) mated with B6(+/+) females were similarly treated. To up-regulate CD200 expression in embryonic trophoblasts, doxycycline was added to the drinking water from the time of mating. RESULTS: The LPS boosted the abortion rate from 15.5% (control) to 42.0% in C3(-/-) mice (chi(2) = 9.28, P < 0.005). In C5(-/-) mice, there was no increase in abortion rate with LPS compared to progesterone-treated controls (22.8%versus 26.3%, P = NS). LPS-treated transgenic mice given LPS + progesterone had a 42.5% abortion rate, but when the mice were given doxycycline to induce expression of CD200 by the embryo, the abortion rate was only 8.3% (chi(2) = 14.40, P < 0.005, Fisher's exact test P = 0.00007). CONCLUSION: C5, but not C3, appears necessary for LPS-driven abortions. Up-regulation of CD200 can prevent LPS-driven abortions, possibly by altering dendritic cells to promote Treg cell development or by a direct suppressive action on macrophages and mast cells that also express CD200 receptors.

A role for tumour necrosis factor-alpha, complement C5 and interleukin-6 in the initiation and development of the mycobacterial cord factor trehalose 6,6'-dimycolate induced granulomatous response.

Trehalose 6,6'-dimycolate (TDM) is a glycolipid component of the mycobacterial cell wall that causes immune responses in mice similar to Mycobacterium tuberculosis (MTB) infection, including granuloma formation with production of proinflammatory cytokines. The precise roles of tumour necrosis factor (TNF)-alpha, complement C5 and interleukin (IL)-6 in the molecular events that lead to the initiation and maintenance of the granulomatous response to TDM have not been fully elucidated. Macrophage proinflammatory responses from wild-type and complement-deficient mice after infection with MTB were assessed, and compared to responses from organisms in which surface TDM had been removed. Removal of TDM abolished proinflammatory responses, markedly so in the complement-deficient macrophages. Mice deficient in TNF-alpha, C5a and IL-6, along with wild-type C57BL/6 controls, were intravenously injected with TDM in a water-in-oil emulsion, and analysed for histological response and cytokine production in lungs. Wild-type C57BL/6 mice formed granulomas with increased production of IL-1beta, IL-6, TNF-alpha, macrophage inflammatory protein-1alpha (MIP-1alpha), IL-12p40, interferon-gamma (IFN-gamma), and IL-10 protein and mRNA. TNF-alpha-deficient mice failed to produce a histological response to TDM, with no increases in cytokine production following TDM administration. While C5a-deficient mice exhibited inflammation, they did not form structured granulomas and initially had decreased production of proinflammatory mediators. IL-6-deficient mice initiated granuloma formation, but failed to maintain the granulomas through day 7 and demonstrated decreased early production of proinflammatory mediators in comparison to wild-type mice. These data suggest that TNF-alpha is critical for initiation of the granulomatous response, C5a is necessary for formation of cohesive granulomas, and IL-6 plays a key role in the granuloma maintenance response to mycobacterial TDM.

B-1 B cell IgM antibody initiates T cell elicitation of contact sensitivity.

Although B-1 B cells have received considerable attention, their actual role in the normal functioning of the immune system is unclear. The hypothesized role of B-1 cell IgM in natural protective immunity is just being established. We have uncovered a separate and novel role for B-1 cell IgM in initiating the elicitation of acquired T cell-dependent contact sensitivity (CS), the prototype of in vivo T cell immunity, early after immunization (within 4 days). The recent recognition of a similarly unanticipated role of B cells in a variety of T cell responses, may indicate that B-1 cell IgM has a broader role in immunity than thought previously. We showed that 24 hr CS responses, and rises in local IFN-gamma levels at 24 hrs later after antigen (Ag) challenge the ears, were absent in pan B cell and antibody deficient mice. The mechanism of B cell involvement in CS-initiation is via local C5a generation early (1-2 hrs) after antigen (Ag) challenge of the ears, in 4 day contact sensitized mice. C5a activates local mast cells to release serotonin (5-HT) and TNF alpha to induce endothelial ICAM-1 and VCAM-1, leading to T cell recruitment. We hypothesized that C5a was generated via complement activation due to antibodies forming local AgAb complexes, and that B-1 cell IgM was involved because isotype switching of B-2 cells to produce C-activating IgG isotypes, could not occur as early as day 4. Indeed, B-1 cell deficient CBA/N-xid mice lacked C5a in 2 hr ear extracts, and had impaired CS ear swelling and elaboration of IFN-gamma at 24 hrs. Importantly, adoptive transfer of purified normal peritoneal B-1 cells, or just i.v. injection of Ag-specific IgM monoclonal antibodies in sensitized xid, restored deficient early C5a and late 24 hr ear swelling. These results suggest that early after Ag challenge, specific B-1 cell IgM, produced at distant sites by prior sensitization, forms AgAb complexes that trigger elaboration of C5a, to activate mast cell release of vasoactive TNF alpha and 5-HT to initiate CS, leading to T cell recruitment. We postulate that antibody of various isotypes possibly may lead to local vascular activation to aid in T cell recruitment in a variety of T cell responses, but that very early after immunization, Ag-specific IgM produced by B-1 cells, preferentially serves this important function.

Differing patterns of P-selectin expression in lung injury.

Using two models of acute lung inflammatory injury in rats (intrapulmonary deposition of immunoglobulin G immune complexes and systemic activation of complement after infusion of purified cobra venom factor), we have analyzed the requirements and patterns for upregulation of lung vascular P-selectin. In the immune complex model, upregulation of P-selectin was defined by Northern and Western blot analysis of lung homogenates, by immunostaining of lung tissue, and by vascular fixation of 125I-labeled anti-P-selectin. P-selectin protein was detected by 1 hour (long before detection of mRNA) and expression was sustained for the next 7 hours, in striking contrast to the pattern of P-selectin expression in the cobra venom factor model, in which upregulation was very transient (within the 1st hour). In the immune complex model, injury and neutrophil accumulation were P-selectin dependent. Upregulation of P-selectin was dependent on an intact complement system, and the presence of blood neutrophils was susceptible to the antioxidant dimethyl sulfoxide and required C5a but not tumor necrosis factor alpha. In contrast, in the cobra venom factor model, upregulation of P-selectin, which is C5a dependent, was also dimethyl sulfoxide sensitive but neutrophil independent. Different mechanisms that may explain why upregulation of lung vascular P-selectin is either transient or sustained are discussed.