Interferons (IFNs) are cytokines that possess potent anti-viral and immunoregulatory activities. In contrast, their potential role(s) in anti-bacterial defense and neutrophil activation mechanisms is less well explored. By comparing gene expression patterns between immature and mature human neutrophils, we obtained evidence that intracellular proteases and other anti-bacterial proteins are produced at earlier stages of maturation, whereas the genes for receptors and signaling molecules required for the release of these effector molecules are preferentially induced during terminal differentiation. For instance, mature neutrophils strongly expressed genes that increase their responses to type I and type II IFNs. Interestingly, granulocyte/macrophage colony-stimulating factor was identified as a repressor of IFN signaling <em>components</em> and consequently of IFN-responsive genes. Both IFN-alpha and IFN-gamma induced strong tyrosine phosphorylation of STAT1 in mature but not in immature neutrophils. Functional in vitro studies suggested that IFNs act as priming factors on mature neutrophils, allowing the formation of extracellular traps upon subsequent stimulation with <em>complement</em> factor <em>5a</em> (C<em>5a</em>). In contrast, both IFN-alpha and IFN-gamma had only little capacity to prime immature cells in this system. Moreover, both IFNs did not have significant anti-proliferative effects on immature neutrophils. These data contribute to our understanding regarding changes of gene expression during neutrophil differentiation and IFN-mediated anti-bacterial defense mechanisms.

BACKGROUND

Age related macular degeneration (AMD) is the leading cause of irreversible blindness in elderly populations worldwide. Inflammation, among many factors, has been suggested to play an important role in AMD pathogenesis. Recent studies have demonstrated a strong genetic association between AMD and <em>complement</em> factor H (CFH), the down-regulatory factor of <em>complement</em> activation. Elevated levels of <em>complement</em> activating molecules including <em>complement</em> <em>component</em> <em>5a</em> (C<em>5a</em>) have been found in the serum of AMD patients. Our aim is to study whether C<em>5a</em> can impact human T cells and its implication in AMD.

METHODS

Human peripheral blood mononuclear cells (PBMCs) were isolated from the blood of exudative form of AMD patients using a Ficoll gradient centrifugation protocol. Intracellular staining and enzyme-linked immunosorbent assays were used to measure protein expression. Apoptotic cells were detected by staining of cells with the annexin-V and TUNEL technology and analyzed by a FACS Caliber flow cytometer. SNP genotyping was analyzed by TaqMan genotyping assay using the Real-time PCR system 7500.

RESULTS

We show that C<em>5a</em> promotes interleukin (IL)-22 and IL-17 expression by human CD4+ T cells. This effect is dependent on B7, IL-1β and IL-6 expression from monocytes. We have also found that C<em>5a</em> could protect human CD4+ cells from undergoing apoptosis. Importantly, consistent with a role of C<em>5a</em> in promoting IL-22 and IL-17 expression, significant elevation in IL-22 and IL-17 levels was found in AMD patients as compared to non-AMD controls.

CONCLUSIONS

Our results support the notion that C<em>5a</em> may be one of the factors contributing to the elevated serum IL-22 and IL-17 levels in AMD patients. The possible involvement of IL-22 and IL-17 in the inflammation that contributes to AMD may herald a new approach to treat AMD.

The 74 amino acid glycoprotein, <em>complement</em> <em>component</em> <em>5a</em> (C<em>5a</em>), is a potent pro-inflammatory mediator cleaved enzymatically from its precursor, C5, upon activation of the <em>complement</em> cascade. C<em>5a</em> is quickly metabolised by carboxypeptidases, forming the less potent C<em>5a</em>desArg. Acting via a classical G protein-coupled receptor, CD88, C<em>5a</em> and C<em>5a</em>desArg exert a number of effects essential to the innate immune response, while their actions at the more recently discovered non-G protein-coupled receptor, C5L2 (or GPR77), remain unclear. The widespread expression of C<em>5a</em> receptors throughout the body allows C<em>5a</em> to elicit a broad range of effects. Thus, C<em>5a</em> has been found to be a significant pathogenic driver in a number of immuno-inflammatory diseases, making C<em>5a</em> inhibition an attractive therapeutic strategy.

Leukocyte migration into tissues is characteristic of inflammation. It is usually measured in vitro as the average displacement of populations of cells towards a chemokine gradient, not acknowledging other patterns of cell migration. Here, we designed and validated a microfluidic migration platform to simultaneously analyse four qualitative migration patterns: chemoattraction, -repulsion, -kinesis and -inhibition, using single-cell quantitative metrics of direction, speed, persistence and fraction of cells responding. We find that established chemokines, <em>complement</em> <em>component</em> <em>5a</em> and IL-8 induce chemoattraction and repulsion in equal proportions, resulting in the dispersal of cells. These migration signatures are characterized by high persistence and speed and are independent of the chemokine dose or receptor expression. Furthermore, we find that twice as many T lymphocytes migrate away than towards stromal cell-derived factor 1 and their directional migration patterns are not persistent. Overall, our platform helps discover migratory signature responses and uncovers an avenue for precise characterization of leukocyte migration and therapeutic modulators.

Endotoxic fever is regulated by endogenous factors that provide pro- and anti-pyretic signals at different points along the febrigenic pathway, from the periphery to the brain. Current evidence indicates that the febrile response to invading Gram-negative bacteria and their products is initiated upon their arrival in the liver via the circulation and their uptake by Kupffer cells (Kc). These pathogens activate the <em>complement</em> cascade on contact, hence generating <em>complement</em> <em>component</em> <em>5a</em>. It, in turn, very rapidly stimulates Kc to release prostaglandin (PG)E2. Pyrogenic cytokines (TNF-alpha, etc.) are produced later and are no longer considered to be the immediate triggers of fever. The Kc-generated PGE2 either (1) may be transported by the bloodstream to the ventromedial preoptic-anterior hypothalamus (POA, the locus of the temperature-regulating center), presumptively diffusing into it and acting on thermoregulatory neurons; PGE2 is thus taken to be the final, central fever mediator. Or (2) it may activate hepatic vagal afferents projecting to the medulla oblongata, thence to the POA via the ventral noradrenergic bundle. Norepinephrine consequently secreted stimulates alpha1-adrenoceptors on thermoregulatory neurons, rapidly evoking an initial rise in core temperature (Tc) not associated with any change in POA PGE2; this neural, PGE2-independent signaling pathway is quicker than the blood-borne route. Elevated POA PGE2 and a secondary Tc rise occur later, consequent to alpha2 stimulation. Endogenous counter-regulatory factors are also elaborated peripherally and centrally at different points during the course of the febrile response; they are, therefore, anti-pyretic. These multiple interacting pathways are the subject of this review.

<em>Complement</em> fragment <em>5a</em> (C<em>5a</em>)-C<em>5a</em> receptor (C<em>5a</em>R) signaling plays an essential role in neutrophil innate immunity. Blockade of either the ligand or the receptor improves survival rates in experimental sepsis. In the current study, sepsis was induced in rats by cecal ligation/puncture. Early in sepsis C<em>5a</em>R content on neutrophils significantly dropped, reached the nadir at 24 h after onset of sepsis, and progressively elevated thereafter. Western-blot, RT-PCR, and confocal microscopy analyses revealed that the loss and re-expression of C<em>5a</em>R during sepsis might be due, at least in part, to the receptor internalization and reconstitution. The reduction and reconstitution of C<em>5a</em>R correlate with the loss and restoration of innate immune functions of blood neutrophils (chemotaxis and reactive oxygen species production), respectively. Quantitative measurements of C<em>5a</em>R on blood neutrophils are highly predictive of survival or death during sepsis. These data suggest that neutrophil C<em>5a</em>R content represents an essential <em>component</em> of an efficient defense system in sepsis and may serve as a prognostic marker for the outcome.

Increased microvascular dilatation and permeability is observed during allograft rejection. Because vascular integrity is an important indicator of transplant health, we have sought to limit injury to blood vessels by blocking complement activation. Although complement component 3 (C3) inhibition is known to be vasculoprotective in transplantation studies, we recently demonstrated the paradoxical finding that, early in rejection, C3(-/-) transplant recipients actually exhibit worse microvascular injury than controls. In the genetic absence of C3, thrombin-mediated complement component 5 (C5) convertase activity leads to the generation of C5a (anaphylatoxin), a promoter of vasodilatation and permeability. In the current study, we demonstrated that microvessel thrombin deposition is significantly increased in C3(-/-) recipients during acute rejection. Thrombin colocalization with microvessels is closely associated with remarkably elevated plasma levels of C5a, vasodilatation, and increased vascular permeability. Administration of NOX-D19, a specific C5a inhibitor, to C3(-/-) recipients of airway transplants significantly improved tissue oxygenation, limited microvascular leakiness, and prevented airway ischemia, even in the absence of conventional T-cell-directed immunosuppression. As C3 inhibitors enter the clinics, the simultaneous targeting of this thrombin-mediated complement activation pathway and/or C5a itself may confer significant clinical benefit.

The <em>complement</em> system is a key <em>component</em> of innate host defence that, under normal conditions, is responsible for the opsonization and destruction of potential pathogens. However, inappropriate or excessive activation of <em>complement</em> can have a detrimental effect on the host and has been implicated in the pathophysiology of numerous disease states. Recently, there has been increasing evidence for a role of the <em>complement</em> system and, in particular, the potent pro-inflammatory anaphylatoxin <em>complement</em> <em>component</em> <em>5a</em> (C<em>5a</em>) in both normal and complicated pregnancy. The following review describes the results of in vitro, animal, and human clinical studies investigating the role of the <em>complement</em> system in healthy pregnancy, recurrent miscarriage, preterm birth, and preeclampsia.

OBJECTIVE

Stroke is a leading cause of morbidity and mortality in the United States. Recent animal studies have implicated the complement system in cerebral ischemia/reperfusion injury and suggest that complement inhibition may improve stroke outcomes. To assess the applicability of these findings to humans, we evaluated the characteristics and time course of human complement activation after stroke.

METHODS

We compared peripheral blood levels of complement factor 3a (C3a), 5a (C5a), and sC5b-9 drawn from 15 patients on poststroke Days 1, 2, 3, 7, 14, 21, and 28 to age-, race/ethnicity-, and sex-matched controls from the same population. Statistical analysis was performed using unpaired Mann-Whitney nonparametric tests with Bonferroni correction. All data is presented as the mean +/- standard deviation.

RESULTS

Mean C3a concentrations showed significant early elevations in stroke patients relative to matched controls (controls: 1080 +/- 189 ng/ml; Day 1: 1609 +/- 422 ng/ml, P = 0.0008; Day 3: 1520 +/- 317 ng/ml, P = 0.0005; Day 7: 1526 +/- 386 ng/ml, P = 0.001). C3a was also significantly elevated on Day 28 (1448 +/- 386 ng/ml, P = 0.004). Before poststroke Day 7, mean C5a levels did not differ significantly from controls. However, beginning on Day 7 and continuing through Day 14, there were significant elevations in C5a (controls: 3.33 +/- 2.1 ng/ml; day 7: 6.86 +/- 3.5 ng/ml, P = 0.005; Day 14: 7.65 +/- 4.6 ng/ml, P = 0.004). Mean sC5b-9 concentrations showed early depressions that reached significance on Days 1 and 2 (controls: 275.6 +/- 107 ng/ml; Day 1: 167.0 +/- 108 ng/ml, P = 0.006; Day 2: 156.3 +/- 80.0 ng/ml, P = 0.005) and did not differ significantly from controls at any other time point.

CONCLUSIONS

C3a is acutely elevated after human ischemic stroke, C5a shows delayed elevations 7 to 14 days after cerebral ischemia, and sC5b-9 is acutely depressed after stroke. Together, these data confirm complement activation after stroke and suggest that this activation is a heterogeneous process, with varying responses for different components.

Salmonella enterica serovar Typhi (S. Typhi) causes typhoid fever, a disseminated infection, while the closely related pathogen S. enterica serovar Typhimurium (S. Typhimurium) is associated with a localized gastroenteritis in humans. Here we investigated whether both pathogens differ in the chemotactic response they induce in neutrophils using a single-cell experimental approach. Surprisingly, neutrophils extended chemotactic pseudopodia toward Escherichia coli and S. Typhimurium, but not toward S. Typhi. Bacterial-guided chemotaxis was dependent on the presence of <em>complement</em> <em>component</em> <em>5a</em> (C<em>5a</em>) and C<em>5a</em> receptor (C<em>5a</em>R). Deletion of S. Typhi capsule biosynthesis genes markedly enhanced the chemotactic response of neutrophils in vitro. Furthermore, deletion of capsule biosynthesis genes heightened the association of S. Typhi with neutrophils in vivo through a C<em>5a</em>R-dependent mechanism. Collectively, these data suggest that expression of the virulence-associated (Vi) capsular polysaccharide of S. Typhi obstructs bacterial-guided neutrophil chemotaxis.

The epithelial <em>complement</em> inhibitory proteins (CIPs) cluster of differentiation 46 and 55 (CD46 and CD55) regulate circulating immune complex-mediated <em>complement</em> activation in idiopathic pulmonary fibrosis (IPF). Our previous studies demonstrated that IL-17A mediates epithelial injury via transforming growth factor β1 (TGF-β1) and down-regulates CIPs. In the current study, we examined the mechanistic role of TGF-β1 in <em>complement</em> activation-mediated airway epithelial injury in IPF pathogenesis. We observed lower epithelial CIP expression in IPF lungs compared to normal lungs, associated with elevated levels of <em>complement</em> <em>component</em> 3a and <em>5a</em> (C3a and C<em>5a</em>), locally and systemically. In normal primary human small airway epithelial cells (SAECs) treated with TGF-β1 (10 ng/ml), C3a, or C<em>5a</em> (100 nM), we observed loss of CIPs and increased poly(ADP-ribose) polymerase (PARP) activation [also observed with RNA interference (RNAi) of CD46/CD55]. TGF-β1-mediated loss of CIPs and Snail induction [SNAI1; a transcriptional repressor of E-cadherin (E-CAD)] was blocked by inhibiting mitogen-activated protein kinase (p38MAPK; SB203580) and RNAi silencing of SNAI1. C3a- and C<em>5a</em>-mediated loss of CIPs was also blocked by p38MAPK inhibition. While C3a upregulated TGFb transcripts, both C3a and C<em>5a</em> down-regulated SMAD7 (negative regulator of TGF-β), and whereas TGF-β1 induced C3a/C<em>5a</em> receptor (C3aR/C<em>5a</em>R) expression, pharmacologic C3aR/C<em>5a</em>R inhibition protected against C3a-/C<em>5a</em>-mediated loss of CIPs. Taken together, our results suggest that epithelial injury in IPF can be collectively amplified as a result of TGF-β1-induced loss of CIPs leading to <em>complement</em> activation that down-regulates CIPs and induces TGF-β1 expression

Clear-cell renal cell carcinoma (ccRCC) possesses an unmet medical need, particularly at the metastatic stage, when surgery is ineffective. <em>Complement</em> is a key factor in tissue inflammation, favoring cancer progression through the production of <em>complement</em> <em>component</em> <em>5a</em> (C<em>5a</em>). However, the activation pathways that generate C<em>5a</em> in tumors remain obscure. By data mining, we identified ccRCC as a cancer type expressing concomitantly high expression of the <em>components</em> that are part of the classical <em>complement</em> pathway. To understand how the <em>complement</em> cascade is activated in ccRCC and impacts patients' clinical outcome, primary tumors from three patient cohorts (<i>n</i> = 106, 154, and 43), ccRCC cell lines, and tumor models in <em>complement</em>-deficient mice were used. High densities of cells producing classical <em>complement</em> pathway <em>components</em> C1q and C4 and the presence of C4 activation fragment deposits in primary tumors correlated with poor prognosis. The <i>in situ</i> orchestrated production of C1q by tumor-associated macrophages (TAM) and C1r, C1s, C4, and C3 by tumor cells associated with IgG deposits, led to C1 complex assembly, and <em>complement</em> activation. Accordingly, mice deficient in C1q, C4, or C3 displayed decreased tumor growth. However, the ccRCC tumors infiltrated with high densities of C1q-producing TAMs exhibited an immunosuppressed microenvironment, characterized by high expression of immune checkpoints (i.e., PD-1, Lag-3, PD-L1, and PD-L2). Our data have identified the classical <em>complement</em> pathway as a key inflammatory mechanism activated by the cooperation between tumor cells and TAMs, favoring cancer progression, and highlight potential therapeutic targets to restore an efficient immune reaction to cancer.

Despite numerous reports, the role of tumor necrosis factor (TNF) in polymorphonuclear leukocyte (PMN) function remains controversial. We found TNF to be a potent, pertussis toxin-independent stimulator of PMN adhesion (ED50 2.6 pM). TNF-stimulated PMN under adherent conditions released up to 65% of their transcobalamine content (ED50 3.9 pM) and increased their burst activity 10-fold (ED50 3.2 pM) as measured by the hexose monophosphate shunt, whereas PMN held in suspension hardly degranulated at all and only little burst activity was demonstrable. However, preincubation of PMN with TNF in suspension led to a decrease in cellular adhesiveness, degranulation, and burst activity in response to a secondary stimulus of TNF under adherent conditions, although cells remained fully responsive toward phorbol myristate acetate. A concomitant dose-dependent decline of TNF receptor numbers that correlated well with the inhibition of PMN function (r = 0.91) suggests receptor down-regulation as the mechanism of functional PMN deactivation. Remarkably, preincubation with other PMN stimuli such as N-formyl-methionyl-leucyl-phenylalanine, platelet-activating factor, leukotriene B4, <em>complement</em> <em>component</em> fragment <em>5a</em> (C<em>5a</em>)/C<em>5a</em> (desarginated), and endotoxin also led to a reduction of TNF-specific PMN responses (cross-deactivation) from 35% (LTB4) to 90% (endotoxin), corresponding with the down-regulation of TNF receptors. Deactivation and receptor down-regulation are independent of pertussis toxin-sensitive G proteins and protein kinase C but seemed to depend on changes in calcium metabolism. Granulocyte hyporesponsiveness towards TNF in sepsis (with elevated blood levels of endotoxin and TNF) might be a mechanism of self-protection or, to the contrary, might impair a possibly central mode of host defense.

Actinobacillus pleuropneumoniae is resistant to <em>complement</em>-mediated killing, even in the presence of specific Ab. Our studies focused on identifying the mechanism(s) responsible for this resistance. Encapsulated A. pleuropneumoniae was susceptible to killing in precolostral calf serum (PCS) but not in normal serum as a <em>complement</em> source in the presence of anti-capsular polysaccharide (CP) IgG. In contrast, two capsule-deficient mutants were sensitive to killing in normal serum and one was sensitive to killing in PCS alone. Electron microscopy demonstrated that A. pleuropneumoniae serotype <em>5a</em> synthesized a thick, adherent CP that bound anti-CP Ab distant from the outer membrane. The CP of A. pleuropneumoniae did not prevent <em>complement</em> activation or the attachment of C3 to the cell surface. However, the CP did limit the amount of C9, a <em>component</em> of the membrane attack complex, that bound to A. pleuropneumoniae in PCS. A second mechanism of serum resistance was a result of an LPS-specific Ab present in the IgG fractions of normal swine serum, swine anti-K17 serum, and guinea pig anti-K17 LPS that blocked anti-CP IgG <em>complement</em>-mediated killing of A. pleuropneumoniae. Incubation of swine anti-K17 IgG with purified K17 LPS depleted Abs specific for K17 LPS but not for K17 proteins and removed all blocking activity. Immune swine serum containing this blocking Ab reduced the deposition of C9 on A. pleuropneumoniae in the presence of anti-CP IgG and also directed the deposition of C9 to sites on the bacteria in which the bound C9 was easily eluted. Thus, CP and anti-LPS Ab may act synergistically or at different stages of infection to limit the ability of <em>complement</em> to eliminate A. pleuropneumoniae.

S19 ribosomal protein is a <em>component</em> of the protein-producing machinery, ribosome. When recombinant S19 proteins were cross-linked intermolecularly by plasma transglutaminase (coagulation factor XIIIa), this homodimer newly exhibited monocyte chemotactic activity. This effect was specific to monocytes. The S19 protein homodimer shared the immunoreactivity with the <em>complement</em>-derived chemotactic factor, <em>component</em> <em>5a</em> (C<em>5a</em>). Monocytes pretreated with an anti-C<em>5a</em> receptor antibody or with a synthetic C<em>5a</em> receptor antagonist responded poorly in chemotaxis to this homodimer. These data indicate that the S19 protein homodimer possesses a 3-dimensional structure similar to C<em>5a</em> in terms of the immunologic epitope and the receptor ligand, although homology between their primary structures is only 4%. In contrast, the S19 protein homodimer did not attract polymorphonuclear leukocytes. In addition, the homodimer inhibited a chemotactic response of polymorphonuclear leukocytes to C<em>5a</em> in vitro and in vivo as a receptor antagonist. Furthermore, the S19 protein homodimer competitively inhibited the binding of radiolabeled C<em>5a</em> to polymorphonuclear leukocytes. The S19 protein homodimer with these opposite effects in the leukocyte chemotaxis seems to induce the monocyte/macrophage predominant infiltration in chronic inflammation.

Studies in fibroblasts, neurons, and platelets have demonstrated the integration of signals from different G protein-coupled receptors (GPCRs) in raising intracellular free Ca(2+). To study signal integration in macrophages, we screened RAW264.7 cells and bone marrow-derived macrophages (BMDM) for their Ca(2+) response to GPCR ligands. We found a synergistic response to <em>complement</em> <em>component</em> <em>5a</em> (C<em>5a</em>) in combination with uridine 5'-diphosphate (UDP), platelet activating factor (PAF), or lysophosphatidic acid (LPA). The C<em>5a</em> response was Galpha(i)-dependent, whereas the UDP, PAF, and LPA responses were Galpha(q)-dependent. Synergy between C<em>5a</em> and UDP, mediated by the C<em>5a</em> and P2Y6 receptors, required dual receptor occupancy, and affected the initial release of Ca(2+) from intracellular stores as well as sustained Ca(2+) levels. C<em>5a</em> and UDP synergized in generating inositol 1,4,5-trisphosphate, suggesting synergy in activating phospholipase C (PLC) beta. Macrophages expressed transcripts for three PLCbeta isoforms (PLCbeta2, PLCbeta3, and PLCbeta4), but GPCR ligands selectively used these isoforms in Ca(2+) signaling. C<em>5a</em> predominantly used PLCbeta3, whereas UDP used PLCbeta3 but also PLCbeta4. Neither ligand required PLCbeta2. Synergy between C<em>5a</em> and UDP likewise depended primarily on PLCbeta3. Importantly, the Ca(2+) signaling deficiency observed in PLCbeta3-deficient BMDM was reversed by re-constitution with PLCbeta3. Neither phosphatidylinositol (PI) 3-kinase nor protein kinase C was required for synergy. In contrast to Ca(2+), PI 3-kinase activation by C<em>5a</em> was inhibited by UDP, as was macropinocytosis, which depends on PI 3-kinase. PLCbeta3 may thus provide a selective target for inhibiting Ca(2+) responses to mediators of inflammation, including C<em>5a</em>, UDP, PAF, and LPA.

Sepsis, a widely prevalent disease with increasing morbidity and mortality, is thought to result from uncontrolled inflammatory responses to microbial infection and/or <em>components</em>. However, failure of several experimental anti-inflammatory therapies has necessitated re-evaluation of the paradigm underlying the pathogenesis of this complex disorder. Apoptotic cell death forms a second dominant feature of septic shock in patients and animal models. Anti-apoptotic strategies may protect animals from septic death. However, simultaneous occurrence of apoptosis and inflammation is necessary for septic death. At the cellular level, apoptosis plays a central role in the development of the lymphoid system and regulation of immune responses. Immune activation renders cells refractory to apoptosis while apoptosis of activated lymphocytes is an important immunoregulatory mechanism. Factors such as <em>complement</em> factor <em>5a</em>, caspase-1 and mitogen-activated protein kinase, which participate in apoptosis as well as pro-inflammatory pathways, may be responsible for simultaneous activation of apoptosis and inflammation in sepsis. Further identification of other similar biochemical events capable of co-activating inflammation and apoptosis may provide new targets for therapy of this hitherto untreatable disease.

Excessive activation of the complement system is detrimental in acute inflammatory disorders. In this study, we analyzed the role of complement-derived anaphylatoxins in the pathogenesis of experimental acute lung injury/acute respiratory distress syndrome (ALI/ARDS) in C57BL/6J mice. Intratracheal administration of recombinant mouse complement component (C5a) caused alveolar inflammation with abundant recruitment of Ly6-G(+)CD11b(+) leukocytes to the alveolar spaces and severe alveolar-capillary barrier dysfunction (C5a-ALI; EC(50[C5a]) = 20 ng/g body weight). Equimolar concentrations of C3a or desarginated C5a (C5a(desArg)) did not induce alveolar inflammation. The severity of C5a-ALI was aggravated in C5-deficient mice. Depletion of Ly6-G(+) cells and use of C5aR1(-/-) bone marrow chimeras suggested an essential role of C5aR1(+) hematopoietic cells in C5a-ALI. Blockade of PI3K/Akt and MEK1/2 kinase pathways completely abrogated lung injury. The mechanistic description is that C5a altered the alveolar cytokine milieu and caused significant release of CC-chemokines. Mice with genetic deficiency of CC-chemokine receptor (CCR) type 5, the common receptor of chemokine (C-C motif) ligand (CCL) 3, CCL4, and CCL5, displayed reduced lung damage. Moreover, treatment with a CCR5 antagonist, maraviroc, was protective against C5a-ALI. In summary, our results suggest that the detrimental effects of C5a in this model are partly mediated through CCR5 activation downstream of C5aR1, which may be evaluated for potential therapeutic exploitation in ALI/ARDS.

Human telomerase RNA (hTR), an important <em>component</em> of telomerase, is a possible target of telomerase-based cancer gene therapy. The present study was undertaken to assess the efficacy of antisense hTR therapy using newly developed 2-<em>5A</em> (5'-phosphorylated 2'-5'-linked oligoadenylate)-linked oligonucleotides against cervical cancer cells. ME180 and SiHa cells were treated with 2-<em>5A</em>-linked antisense hTR designed to <em>complement</em> the region of hTR between residues 76 and 94. The hTR expression, telomerase activity, cell viability, and apoptosis were then examined. The 2-<em>5A</em> anti-hTR effectively degraded hTR and inhibited telomerase activity. The 2-<em>5A</em> mutant anti-hTR and the anti-hTR without 2-<em>5A</em> were not capable of inhibiting telomerase activity. Inhibition of telomerase by 2-<em>5A</em> anti-hTR rapidly decreased cell viability only in telomerase-positive cells within 3-6 days after the treatment, when telomere length has not yet been shortened. This inhibition was associated with apoptosis, possibly through activation of caspase family members. These findings suggest that 2-<em>5A</em>-linked antisense-hTR therapy has a potent telomerase-inhibitory effect associated with a cytocidal effect from caspase-induced apoptosis, and may therefore be a potential tool in telomerase-based gene therapy against cervical cancers.

BACKGROUND

Cellular adhesion is crucial for eosinophil effector functions.

OBJECTIVE

We sought to elucidate the role of the actin cytoskeleton in cellular adhesion and superoxide anion generation by human eosinophils.

METHODS

Eosinophils were stimulated with platelet-activating factor (PAF) or <em>complement</em> <em>component</em> <em>5a</em> on human serum albumin-coated plates with or without an actin-polymerization inhibitor, cytochalasin B (CB), or cytochalasin D (CD). Superoxide anion generation was measured on the basis of reduction of absorbance associated with cytochrome c.2 Eosinophil adhesion was assessed on the basis of eosinophil protein X content in adherent cells. Transient stimulus-induced increase of intracellular calcium and translocation of protein kinase C (PKC) betaII, PKC delta, PKC zeta, and p47 phagocyte oxidase (a <em>component</em> of nicotinamide adenine dinucleotide phosphate oxidase) were also investigated.

RESULTS

CB, CD, or antibodies against CD18 (the beta2 chain of integrin, alphaMbeta2) inhibited stimulus-induced eosinophil superoxide anion generation. Stimulus-induced eosinophil adhesion was unaltered by CB, whereas it was significantly suppressed by CD or anti-CD18 antibodies. Transient PAF-induced intracellular calcium increase was also unaffected by CB or CD, but stimulus-induced eosinophil shape changes and translocation of PKCs and p47 phagocyte oxidase to the cell membrane region were completely inhibited by CB. PAF-induced eosinophil degranulation was inhibited by CB, CD, or anti-CD18 antibodies, whereas complement component 5-induced degranulation was not suppressed by CB.

CONCLUSIONS

By itself, beta2 integrin-dependent cellular adhesion is not sufficient for promoting eosinophil effector function. Adequate actin assembly is required for eosinophil adhesion and also for full superoxide anion generation in eosinophils.

Agonist binding to guanine nucleotide-binding protein (G protein)-coupled receptors in membranes of myeloid differentiated human leukemia (HL-60) cells is inhibited by guanine nucleotides, most potently by the GTP analog guanosine 5'-(gamma-thio)triphosphate (GTP gamma S). In order to study whether GTP gamma S formed locally from adenosine 5'-(gamma-thio)triphosphate (ATP gamma S) and GDP by nucleoside diphosphokinase has any advantage over exogenously added GTP gamma S in binding to and activating G proteins, regulation of <em>complement</em> <em>component</em> <em>5a</em> (C<em>5a</em>) binding to its receptors, as well as formation of GTP gamma S, was studied in membranes of HL-60 cells. GTP gamma S added to HL-60 membranes potently inhibited binding of 125I-C<em>5a</em> (IC50 about 3 nM), an effect not influenced by addition of either GDP or ATP gamma S. When HL-60 membranes were incubated with the combination of ATP gamma S and GDP, a marked potentiation (up to 300-fold) of the inhibition caused by either GDP or ATP gamma S alone was observed. By measuring nucleoside diphosphokinase-catalyzed formation of GTP gamma S and inhibition of 125I-C<em>5a</em> binding in the presence of GDP and ATP gamma S under identical assay conditions, it was found that formed GTP gamma S inhibited binding of 125I-C<em>5a</em> with an IC50 value of about 0.3 nM, thus being about 10-fold more potent than exogenously added GTP gamma S. These data suggest that the GTP gamma S-forming nucleoside diphosphokinase is closely associated with the C<em>5a</em> receptor-G protein complex and channels the formed GTP gamma S into the G protein.

Mesenchymal stem cells (MSCs) reside within tissues such as bone marrow, cord blood, and dental pulp and can differentiate into other mesenchymal cell types. Differentiated MSCs, called circulating fibrocytes, have been demonstrated in human lungs and migrate to injured lung tissue in experimental models. It is likely that MSCs migrate from the bone marrow to sites of injury by following increasing chemokine concentrations. In the present study, we show that primary mouse bone marrow mesenchymal stem cells (BM-MSCs) exhibit directed chemotaxis through transwell inserts toward increasing concentrations of the chemokines <em>complement</em> <em>component</em> <em>5a</em>, stromal cell-derived factor-1alpha, and monocyte chemotactic protein-1. Prior research has indicated that myristoylated alanine-rich C kinase substrate (MARCKS) protein is critically important for motility in macrophages, neutrophils, and fibroblasts, and here we investigated a possible role for MARCKS in BM-MSC directed chemotaxis. The presence of MARCKS in these cells as well as in human cord blood MSC was verified by Western blotting, and MARCKS was rapidly phosphorylated in these cells after exposure to chemokines. A synthetic peptide that inhibits MARCKS function attenuated, in a concentration-dependent manner, directed chemotaxis of BM-MSCs, while a missense control peptide had no effect. Our results illustrate, for the first time, that MARCKS protein plays an integral role in BM-MSC-directed chemotaxis in vitro.

Autoantibodies in sera from patients with autoimmune diseases have long been known and have become diagnostic tools. Analysis of their functional role again became popular with the availability of mice mutant for several genes of the <em>complement</em> and Fcgamma receptor (FcgammaR) systems. Evidence from different inflammatory models suggests that both systems are interconnected in a hierarchical way. The <em>complement</em> system mediators such as <em>complement</em> <em>component</em> <em>5a</em> (C<em>5a</em>) might be crucial in the communication between the <em>complement</em> system and FcgammaR-expressing cells. The split <em>complement</em> protein C<em>5a</em> is known to inactivate cells by its G-protein-coupled receptor and to be involved in the transcriptional regulation of FcgammaRs, thereby contributing to the complex regulation of autoimmune disease.

Pre-eclampsia is associated with increased levels of cholesterol and uric acid and an inflamed placenta expressing danger-sensing pattern recognition receptors (PRRs). Crystalline cholesterol and uric acid activate the PRR Nod-like receptor protein (NLRP)3 inflammasome to release interleukin (IL)-1β and result in vigorous inflammation. We aimed to characterize crystal-induced NLRP3 activation in placental inflammation and examine its role in pre-eclampsia. We confirmed that serum total cholesterol and uric acid were elevated in pre-eclamptic compared to healthy pregnancies and correlated positively to high sensitivity C-reactive protein (hsCRP) and the pre-eclampsia marker soluble fms-like tyrosine kinase-1 (sFlt-1). The NLRP3 inflammasome pathway <em>components</em> (NLRP3, caspase-1, IL-1β) and priming factors [<em>complement</em> <em>component</em> <em>5a</em> (C<em>5a</em>) and terminal <em>complement</em> complex (TCC)] were co-expressed by the syncytiotrophoblast layer which covers the placental surface and interacts with maternal blood. The expression of IL-1β and TCC was increased significantly and C<em>5a</em>-positive regions in the syncytiotrophoblast layer appeared more frequent in pre-eclamptic compared to normal pregnancies. In-vitro activation of placental explants and trophoblasts confirmed NLRP3 inflammasome pathway functionality by <em>complement</em>-primed crystal-induced release of IL-1β. This study confirms crystal-induced NLRP3 inflammasome activation located at the syncytiotrophoblast layer as a mechanism of placental inflammation and suggests contribution of enhanced NLRP3 activation to the harmful placental inflammation in pre-eclampsia.