Complement System Part II: Role in Immunity.

The complement system has been considered for a long time as a simple lytic cascade, aimed to kill bacteria infecting the host organism. Nowadays, this vision has changed and it is well accepted that complement is a complex innate immune surveillance system, playing a key role in host homeostasis, inflammation, and in the defense against pathogens. This review discusses recent advances in the understanding of the role of complement in physiology and pathology. It starts with a description of complement contribution to the normal physiology (homeostasis) of a healthy organism, including the silent clearance of apoptotic cells and maintenance of cell survival. In pathology, complement can be a friend or a foe. It acts as a friend in the defense against pathogens, by inducing opsonization and a direct killing by C5b-9 membrane attack complex and by triggering inflammatory responses with the anaphylatoxins C3a and C5a. Opsonization plays also a major role in the mounting of an adaptive immune response, involving antigen presenting cells, T-, and B-lymphocytes. Nevertheless, it can be also an enemy, when pathogens hijack complement regulators to protect themselves from the immune system. Inadequate complement activation becomes a disease cause, as in atypical hemolytic uremic syndrome, C3 glomerulopathies, and systemic lupus erythematosus. Age-related macular degeneration and cancer will be described as examples showing that complement contributes to a large variety of conditions, far exceeding the classical examples of diseases associated with complement deficiencies. Finally, we discuss complement as a therapeutic target.

Inhibition of the mTORC pathway in the antiphospholipid syndrome.

BACKGROUND: Although thrombosis is considered the cardinal feature of the antiphospholipid syndrome, chronic vascular lesions are common, particularly in patients with life-threatening complications. In patients who require transplantation, vascular lesions often recur. The molecular pathways involved in the vasculopathy of the antiphospholipid syndrome are unknown, and adequate therapies are lacking. METHODS: We used double immunostaining to evaluate pathway activation in the mammalian target of rapamycin complex (mTORC) and the nature of cell proliferation in the vessels of patients with primary or secondary antiphospholipid syndrome nephropathy. We also evaluated autopsy specimens from persons who had catastrophic antiphospholipid syndrome. The molecular pathways through which antiphospholipid antibodies modulate the mTORC pathway were evaluated in vitro, and potential pharmacologic inhibitors were also tested in vitro. Finally, we studied the effect of sirolimus in kidney-transplant recipients with the antiphospholipid syndrome. RESULTS: The vascular endothelium of proliferating intrarenal vessels from patients with antiphospholipid syndrome nephropathy showed indications of activation of the mTORC pathway. In cultured vascular endothelial cells, IgG antibodies from patients with the antiphospholipid syndrome stimulated mTORC through the phosphatidylinositol 3-kinase (PI3K)-AKT pathway. Patients with antiphospholipid syndrome nephropathy who required transplantation and were receiving sirolimus had no recurrence of vascular lesions and had decreased vascular proliferation on biopsy as compared with patients with antiphospholipid antibodies who were not receiving sirolimus. Among 10 patients treated with sirolimus, 7 (70%) had a functioning renal allograft 144 months after transplantation versus 3 of 27 untreated patients (11%). Activation of mTORC was also found in the vessels of autopsy specimens from patients with catastrophic antiphospholipid syndrome. CONCLUSIONS: Our results suggest that the mTORC pathway is involved in the vascular lesions associated with the antiphospholipid syndrome. (Funded by INSERM and others.).

Complement factor B mutations in atypical hemolytic uremic syndrome-disease-relevant or benign?

Atypical hemolytic uremic syndrome (aHUS) is a genetic ultrarare renal disease associated with overactivation of the alternative pathway of complement. Four gain-of-function mutations that form a hyperactive or deregulated C3 convertase have been identified in Factor B (FB) ligand binding sites. Here, we studied the functional consequences of 10 FB genetic changes recently identified from different aHUS cohorts. Using several tests for alternative C3 and C5 convertase formation and regulation, we identified two gain-of-function and potentially disease-relevant mutations that formed either an overactive convertase (M433I) or a convertase resistant to decay by FH (K298Q). One mutation (R178Q) produced a partially cleaved protein with no ligand binding or functional activity. Seven genetic changes led to near-normal or only slightly reduced ligand binding and functional activity compared with the most common polymorphism at position 7, R7. Notably, none of the algorithms used to predict the disease relevance of FB mutations agreed completely with the experimental data, suggesting that in silico approaches should be undertaken with caution. These data, combined with previously published results, suggest that 9 of 15 FB genetic changes identified in patients with aHUS are unrelated to disease pathogenesis. This study highlights that functional assessment of identified nucleotide changes in FB is mandatory to confirm disease association.

The interaction between factor H and VWF increases factor H cofactor activity and regulates VWF prothrombotic status.

Vascular endothelial cells (ECs) link hemostasis, thrombosis, and complement. ECs synthesize both the clotting initiator von Willebrand factor (VWF) and the complement regulator factor H (FH). VWF is stored in EC Weibel-Palade bodies (WPBs), but the intracellular location of FH is not well defined. We found that FH colocalizes with VWF in WPBs of human umbilical vein ECs. Moreover, FH bound to VWF with an apparent nanomolar affinity and the complex was present in normal plasma. The binding of VWF to FH enhanced FH cofactor activity toward factor I-mediated downregulation of complement activation. Besides, this interaction inhibited ADAMTS13-mediated proteolysis of VWF and promoted platelet aggregation. Here, we describe a novel interaction between complement and hemostasis. The simultaneous secretion of VWF and FH by activated ECs may promote adhesion of platelets to endothelial injury sites to assure wound healing, simultaneously dampening the proinflammatory effect of complement to limit bystander tissue damage.

Complement activation by heme as a secondary hit for atypical hemolytic uremic syndrome.

Atypical hemolytic uremic syndrome (aHUS) is characterized by genetic and acquired abnormalities of the complement system leading to alternative pathway (AP) overactivation and by glomerular endothelial damage, thrombosis, and mechanical hemolysis. Mutations per se are not sufficient to induce aHUS, and nonspecific primary triggers are required for disease manifestation. We investigated whether hemolysis-derived heme contributes to aHUS pathogenesis. We confirmed that heme activates complement AP in normal human serum, releasing C3a, C5a, and sC5b9. We demonstrated that heme-exposed endothelial cells also activate the AP, resulting in cell-bound C3 and C5b9. This was exacerbated in aHUS by genetic abnormalities associated with AP overactivation. Heme interacted with C3 close to the thioester bond, induced homophilic C3 complexes, and promoted formation of an overactive C3/C5 convertase. Heme induced decreased membrane cofactor protein (MCP) and decay-accelerating factor (DAF) expression on endothelial cells, giving Factor H (FH) a major role in complement regulation. Finally, heme promoted a rapid exocytosis of Weibel-Palade bodies, with membrane expression of P-selectin known to bind C3b and trigger the AP, and the release of the prothrombotic von Willebrand factor. These results strongly suggest that hemolysis-derived heme represents a common secondary hit amplifying endothelial damage and thrombosis in aHUS.

A prevalent C3 mutation in aHUS patients causes a direct C3 convertase gain of function.

Atypical hemolytic uremic syndrome (aHUS) is a rare renal thrombotic microangiopathy commonly associated with rare genetic variants in complement system genes, unique to each patient/family. Here, we report 14 sporadic aHUS patients carrying the same mutation, R139W, in the complement C3 gene. The clinical presentation was with a rapid progression to end-stage renal disease (6 of 14) and an unusually high frequency of cardiac (8 of 14) and/or neurologic (5 of 14) events. Although resting glomerular endothelial cells (GEnCs) remained unaffected by R139W-C3 sera, the incubation of those sera with GEnC preactivated with pro-inflammatory stimuli led to increased C3 deposition, C5a release, and procoagulant tissue-factor expression. This functional consequence of R139W-C3 resulted from the formation of a hyperactive C3 convertase. Mutant C3 showed an increased affinity for factor B and a reduced binding to membrane cofactor protein (MCP; CD46), but a normal regulation by factor H (FH). In addition, the frequency of at-risk FH and MCP haplotypes was significantly higher in the R139W-aHUS patients, compared with normal donors or to healthy carriers. These genetic background differences could explain the R139W-aHUS incomplete penetrance. These results demonstrate that this C3 mutation, especially when associated with an at-risk FH and/or MCP haplotypes, becomes pathogenic following an inflammatory endothelium-damaging event.

M-ficolin and leukosialin (CD43): new partners in neutrophil adhesion.

M-ficolin specificity for sialylated ligands prompted us to investigate its interactions with the main membrane sialoprotein of human neutrophils, CD43. rM-ficolin bound CD43 and prevented the access of anti-CD43 mAb. Moreover, rM-ficolin reacted exclusively with CD43 on Western blots of neutrophil lysate. We confirmed that M-ficolin is secreted by fMLP-activated neutrophils, and this endogenous M-ficolin also binds to CD43 and competes with anti-CD43 mAb. Anti-CD43 antibody cross-linking or fMLP resulted in M-ficolin and CD43 colocalization on polarized neutrophils. The binding of rM-ficolin to resting neutrophils induced cell polarization, adhesion, and homotypic aggregation as anti-CD43 mAb. The M-ficolin Y271F mutant, unable to bind sialic acid, neither reacted with neutrophils nor modulated their functions. Finally, rM-ficolin activated the lectin complement pathway on neutrophils. These results emphasize a new function of M-ficolin, different from ficolin pathogen recognition, i.e., a participation to neutrophil adhesion potentially important in early inflammation, as nanomolar agonist concentrations are sufficient to mobilize M-ficolin to the neutrophil surface. This multivalent lectin could then endow the antiadhesive CD43, essentially designed to prevent leukocyte aggregation in the blood flow, with new adhesive properties and explain, at least in part, dual-adhesive/antiadhesive roles of CD43 in neutrophil recruitment.

LPS-induced galectin-3 oligomerization results in enhancement of neutrophil activation.

Galectin-3 (Gal 3) is a glycan-binding protein that can be secreted by activated macrophages and mast cells at inflammation sites and plays an important role in inflammatory diseases caused by Bacteria and their products, such as lipopolysaccharides (LPS). Although it is well established that Gal 3 can interact with LPS, the pathophysiological importance of LPS/Gal 3 interactions is not fully understood. Data presented herein demonstrate for the first time that the interaction of Gal 3, either via its carbohydrate binding C-terminal domain or via its N-terminal part, with LPS from different bacterial strains, enhances the LPS-mediated neutrophil activation in vitro. Gal 3 allowed low LPS concentrations (1 µg/mL without serum, 1 ng/mL with serum) to upregulate CD11b expression and reactive oxygen species (ROS) generation on human neutrophils in vitro and drastically enhanced the binding efficiency of LPS to the neutrophil surface. These effects required LPS preincubation with Gal 3, before neutrophil stimulation and involved specific Gal 3/LPS interaction. A C-terminal Gal-3 fragment, which retains the lectin domain but lacks the N-terminal part, was still able to bind both to Escherichia coli LPS and to neutrophils, but had lost the ability to enhance neutrophil response to LPS. This result emphasizes the importance of an N-terminus-mediated Gal 3 oligomerization induced by its interaction with LPS. Finally we demonstrated that Balb/C mice were more susceptible to LPS-mediated shock when LPS was pretreated with Gal 3. Altogether, these results suggest that multimeric interactions between Gal 3 oligomers and LPS potentiate its pro-inflammatory effects on neutrophils.

Functional complement C1q abnormality leads to impaired immune complexes and apoptotic cell clearance.

C1q plays a key role in apoptotic cell and immune complex removal. Its absence contributes to the loss of tolerance toward self structures and development of autoimmunity. C1q deficiencies are extremely rare and are associated with complete lack of C1q or with secretion of surrogate C1q fragments. To our knowledge, we report the first case of a functional C1q abnormality, associated with the presence of a normal C1q molecule. Homozygous GlyB63Ser mutation was found in a patient suffering from lupus with neurologic manifestations and multiple infections. The GlyB63Ser C1q bound to Igs, pentraxins, LPSs, and apoptotic cells, similarly to C1q from healthy donors. However, the interaction of C1r(2)C1s(2) and C1 complex formation was abolished, preventing further complement activation and opsonization by C3. The mutation is located between LysB(61) and LysB(65) of C1q, suggested to form the C1r binding site. Our data infer that the binding of C1q to apoptotic cells in humans is insufficient to assure self-tolerance. The opsonization capacity of C4 and C3 fragments has to be intact to fight infections and to prevent autoimmunity.

Alternative complement pathway assessment in patients with atypical HUS.

The atypical Hemolytic Uremic Syndrome (aHUS) is a rare thrombotic microangiopathy leading to end stage renal disease in approximately 60% of patients. Over the last decade, a clear link has been demonstrated between this disease and defective complement regulation. The hallmark of the aHUS is the association with mutations in complement alternative pathway genes. Endothelial damage is related to complement dysregulation, but the exact mechanism is just starting to be elucidated. Screening for and characterization of mutations in the components of the C3 convertase (C3 and FB) or its regulators (FH, FI, MCP, and Thrombomodulin) or anti-FH antibodies has become an indispensable part of the disease's diagnostic. This review will initially summarize current knowledge on the understanding of complement activation and regulation, followed by a description on the genetic analysis as well as the methods used for complement protein quantification. Another part of this review will focus on the mechanisms of action of aHUS-associated mutations. We will emphasize on when and why some mutations lead to protein deficiency, while others result in - to dysfunctional but normally expressed proteins. Finally, we will discuss how the therapy of aHUS patients can be modified according to the functional consequences of each particular genetic defect.

Complement alternative pathway acts as a positive feedback amplification of neutrophil activation.

Complement alternative pathway plays an important, but not clearly understood, role in neutrophil-mediated diseases. We here show that neutrophils themselves activate complement when stimulated by cytokines or coagulation-derived factors. In whole blood, tumor necrosis factor/formyl-methionyl-leucyl-phenylalanine or phorbol myristate acetate resulted in C3 fragments binding on neutrophils and monocytes, but not on T cells. Neutrophils, stimulated by tumor necrosis factor, triggered the alternative pathway on their surface in normal and C2-depleted, but not in factor B-depleted serum and on incubation with purified C3, factors B and D. This occurred independently of neutrophil proteases, oxidants, or apoptosis. Neutrophil-secreted properdin was detected on the cell surface and could focus "in situ" the alternative pathway activation. Importantly, complement, in turn, led to further activation of neutrophils, with enhanced CD11b expression and oxidative burst. Complement-induced neutrophil activation involved mostly C5a and possibly C5b-9 complexes, detected on tumor necrosis factor- and serum-activated neutrophils. In conclusion, neutrophil stimulation by cytokines results in an unusual activation of autologous complement by healthy cells. This triggers a new amplification loop in physiologic innate immunity: Neutrophils activate the alternative complement pathway and release C5 fragments, which further amplify neutrophil proinflammatory responses. This mechanism, possibly required for effective host defense, may be relevant to complement involvement in neutrophil-mediated diseases.

Human neutrophil integrin alpha9beta1: up-regulation by cell activation and synergy with beta2 integrins during adhesion to endothelium under flow.

Neutrophil beta1 integrin expression and contribution to cell adhesion were revisited in this study. alpha9beta1 and alpha5beta1 appeared here as the main beta1 integrins expressed on the membrane of resting platelet-depleted neutrophils-alpha6beta1 representing <15% and alpha2beta1 undetectable. Neutrophil activation slightly enhanced alpha5 expression, did not change alpha6, but resulted in a two- to threefold increase of alpha9beta1, which then became the major beta1 integrin of the neutrophil membrane. alpha9beta1 was the only beta1 integrin to be up-regulated after transendothelial migration across TNF-alpha-activated HUVECs. As alpha9beta1 binds VCAM-1, we analyzed its participation to neutrophil adhesion to TNF-alpha-activated endothelial cells. Blocking anti-alpha9 mAb had little effect on neutrophil static adhesion, contrasting with the strong inhibition by anti-beta2 mAb. Under flow conditions, the anti-alpha9 mAb had no effect by itself on neutrophil adhesion to activated HUVECs but enhanced the blocking effect of anti-beta2 antibodies significantly and further enhanced the velocity of beta2-blocked rolling neutrophils. In conclusion, we describe here for the first time a nearly exclusive up-regulation of alpha9beta1 expression among all beta1 integrins during neutrophil activation and transendothelial migration and a possibly important synergy between alpha9beta1 and beta2 integrins in stabilizing neutrophil adhesion to endothelium under flow conditions.

Hyperfunctional C3 convertase leads to complement deposition on endothelial cells and contributes to atypical hemolytic uremic syndrome.

Complement is a major innate immune defense against pathogens, tightly regulated to prevent host tissue damage. Atypical hemolytic uremic syndrome (aHUS) is characterized by endothelial damage leading to renal failure and is highly associated with abnormal alternative pathway regulation. We characterized the functional consequences of 2 aHUS-associated mutations (D(254)G and K(325)N) in factor B, a key participant in the alternative C3 convertase. Mutant proteins formed high-affinity C3-binding site, leading to a hyperfunctional C3 convertase, resistant to decay by factor H. This led to enhanced complement deposition on the surface of alternative pathway activator cells. In contrast to native factor B, the 2 mutants bound to inactivated C3 and induced formation of functional C3-convertase on iC3b-coated surface. We demonstrated for the first time that factor B mutations lead to enhanced C3-fragment deposition on quiescent and adherent human glomerular cells (GEnCs) and human umbilical vein endothelial cells (HUVECs), together with the formation of sC5b-9 complexes. These results could explain the occurrence of the disease, since excessive complement deposition on endothelial cells is a central event in the pathogenesis of aHUS. Therefore, risk factors for aHUS are not only mutations leading to loss of regulation, but also mutations, resulting in hyperactive C3 convertase.

The macrophage-derived neutrophil chemotactic factor, MNCF: a lectin with TNF-alpha-like activities on neutrophils.

The macrophage-derived neutrophil chemotactic factor (MNCF) is an alpha-galactoside-binding lectin, known to induce dexamethasone-insensitive neutrophil recruitment. We further characterized MNCF effects on neutrophils and showed that it shares with TNF-alpha the ability to delay apoptosis and to trigger degranulation. MNCF and TNF-alpha effects show similar kinetics and involve Src kinases and MAPKinases dependent pathways. They were, however, clearly distinguished, since the soluble TNF-receptor etanercept prevented TNF but not MNCF effects, while melibiose disaccharide inhibited MNCF but not TNF effects. Absorption of MNCF on detoxi-gel did not alter its properties, precluding an LPS contamination effect. By contrast, galectin-3 required LPS to activate neutrophils. Specific antibodies allowed to further demonstrate that MNCF and galectin-3 are two distinct molecules. Finally, MNCF- and IL-8-induced neutrophil activation differed by their kinetic and sensitivity to pertussis toxin. In conclusion, MNCF is a distinct neutrophil agonist, with pro-inflammatory activities involving its carbohydrate recognition domain.

The cleavage of neutrophil leukosialin (CD43) by cathepsin G releases its extracellular domain and triggers its intramembrane proteolysis by presenilin/gamma-secretase.

The highly negatively charged membrane sialoglycoprotein leukosialin, CD43, is shed during neutrophil activation. This is generally thought to enhance cell adhesion. We here describe two novel consequences of this shedding, during neutrophil activation by phorbol esters or by chemoattractants after TNF-alpha priming. CD43 proteolysis was investigated by Western blotting, using a polyclonal antibody to CD43 intracellular domain. Our data emphasize the importance of a juxtamembranous cleavage of about 50% of membrane CD43 molecules by cathepsin G. Indeed, it is inhibited by alpha1-antichymotrypsin and cathepsin G inhibitor I and is reproduced by exogenous purified cathepsin G. The resulting membrane-anchored C-terminal fragment, CD43-CTF, becomes susceptible to presenilin/gamma-secretase, which releases CD43 intracytoplasmic domain: preincubation with three different gamma-secretase inhibitors, before PMN treatment by agonists or by purified cathepsin G, results in the accumulation of CD43-CTF. Because CD43 binds E-selectin, we also investigated the effect of the soluble extracellular domain CD43s, released by cathepsin G juxtamembranous cleavage, on neutrophil adhesion to endothelial cells. A recombinant CD43s-Fc fusion protein inhibited neutrophil E selectindependent adhesion to endothelial cells under flow conditions, while it had no effect on neutrophil static adhesion. We thus propose that, in addition to its potential pro-adhesive role, CD43 proteolysis results in: (i) the release, by cathepsin G, of CD43 extracellular domain, able to inhibit the adhesion of flowing neutrophils on endothelial cells and thus to participate to the natural control of inflammation; (ii) the release and/or the clearance, by presenilin/gamma-secretase, of CD43 intracellular domain, thereby regulating CD43-mediated signaling.

Increased membrane expression of proteinase 3 during neutrophil adhesion in the presence of anti proteinase 3 antibodies.

We investigated membrane proteinase 3 (mPR3) expression during TNF-alpha-induced adhesion of neutrophils in the presence of anti-PR3 antibodies, a situation occurring during anti-neutrophil cytoplasmic autoantibodies (ANCA)-associated vasculitis. Three increasing levels of mPR3 expression were observed on the mPR3(+) neutrophil subset after stepwise cell activation. TNF-alpha activation without adhesion, TNF-alpha-induced adhesion, and adhesion in the presence of anti-PR3 mAb or human anti-PR3 ANCA resulted, respectively, in a two-, seven-, and 24-fold increase of mPR3 levels. In plasma, anti-PR3 antibodies poorly recognized suspended neutrophils, whereas they bound to mPR3 on adherent cells. mPR3 upregulation was also triggered by IL-8, formyl-methionyl-leucyl-phenylalanine (fMLP), and neutrophil adhesion to activated human umbilical vein endothelial cells. It involved beta2 integrins and Fcgamma receptor, because it was prevented by anti-CD18 antibodies and was not observed with anti-PR3 F(ab')(2). Furthermore, it was specific to anti-PR3 mAb, and no mPR3 upregulation was observed with anti-myeloperoxidase or anti-HLA-ABC mAb. Newly expressed mPR3 molecules, after TNF-induced adhesion, were mobilized from secretory vesicles (CD35(+)) and secondary granules (CD11b(+)). The adhesion- and antibody-dependent upregulations of mPR3 expression occurred with little azurophilic granule degranulation, no sign of apoptosis, and no further CD177 upregulation. In conclusion, this study describes an amplifying loop in polymorphonuclear neutrophil activation process, whereby ANCA are involved in the membrane expression of their own antigen during cell adhesion. This could explain the restriction of ANCA-associated vasculitis to small vessels, the main site of neutrophil adhesion.

Distinct signaling pathways are involved in leukosialin (CD43) down-regulation, membrane blebbing, and phospholipid scrambling during neutrophil apoptosis.

Although leukosialin (CD43) membrane expression decreases during neutrophil apoptosis, the CD43 molecule, unexpectedly, is neither proteolyzed nor internalized. We thus wondered whether it could be shed on bleb-derived membrane vesicles. Membrane blebbing is a transient event, hardly appreciated during the asynchronous, spontaneous apoptosis of neutrophils. Cell pre-synchronization at 15 degrees C made it possible to observe numerous blebbing neutrophils for a short 1-h period at 37 degrees C. CD43 down-regulation co-occurred with the blebbing stage and phosphatidylserine externalization, shortly after mitochondria depolarization and before nuclear condensation. Blebs detaching from the cell body were observed by time lapse fluorescence microscopy, and the release of bleb-derived vesicles was followed by flow cytometry. Phosphatidylserine externalization required caspases and protein kinase C (PKC) but not the myosin light chain kinase (MLCK). By contrast, bleb formation and release was caspase- and PKC-independent but required an active MLCK, whereas CD43 down-regulation involved caspases but neither PKC nor MLCK. Furthermore, CD43 appeared mostly excluded from membrane blebs by electron microscopy. Thus, CD43 down-regulation does not result from the release of bleb-derived vesicles. Ultracentrifugation of apoptotic cell supernatants made it possible to recover <1 microM microparticles, which contained the entire CD43 molecule. These microparticles expressed neutrophil membrane markers such as CD11b, CD66b, and CD63, together with CD43. In conclusion, we show that the three early membrane events of apoptosis, namely blebbing, phosphatidylserine externalization, and CD43 down-regulation, result from different signaling pathways and can occur independently from one another. CD43 down-regulation results from the shedding of microparticles released during apoptosis but unrelated to the blebbing.

TNF-induced beta2 integrin activation involves Src kinases and a redox-regulated activation of p38 MAPK.

We previously demonstrated that the TNF-alpha-induced inside-out signaling leading to beta(2) integrin activation is redox regulated. To identify kinases involved in this pathway, the effects of kinase inhibitors on the expression of beta(2) integrin activation neoepitope (clone 24) were investigated. We show that both p38 MAPK (inhibited by SB203580) and Src kinases (inhibited by PP2) are involved in beta(2) integrin activation by TNF and oxidants in human neutrophils. Src kinases appeared constitutively active in resting neutrophils and not further activated by TNF or oxidants in nonadherent conditions. However, PP2 blocked both TNF-induced expression of the 24 epitope and cell adhesion promoted by the integrin activating anti-CD18 KIM185 mAb, showing that both the inside-out and the outside-in signaling involve Src kinases. p38 MAPK was activated by TNF and oxidants in nonadherent conditions i.e., with 10 mM EDTA. This activation in EDTA resulted in CD11b, CD35 and CD66 up-regulation and in an oxidative response, all blocked by SB203580 and PP2. p38 MAPK was not activated upon direct integrin activation by KIM185 mAb. Thus, p38 activation allows the study to distinguish the initial transduction pathway leading to beta(2) integrin activation from the signaling resulting from integrin engagement. Finally, p38 MAPK activation by TNF was blocked by diphenylene iodonium, an inhibitor of flavoprotein oxidoreductase, and by the free radical scavenger N-acetylcystein. Taken together, these results demonstrate, for the first time, that constitutively activated Src tyrosine kinases and a redox-regulated activation of p38 MAPK are involved in TNF inside-out signaling leading to beta(2) integrin activation.

Long-term reversal of established autoimmunity upon transient blockade of the LFA-1/intercellular adhesion molecule-1 pathway.

Transgenic models and administration of mAbs directed against the LFA-1/intercellular adhesion molecule 1 (ICAM-1) pathway have shown that these costimulatory molecules play a key role in generating effector cells mediating inflammatory responses. In this report, durable remission of recent diabetes in nonobese diabetic (NOD) mice was induced by transient expression of an immunoadhesin gene encoding the soluble form of ICAM-1 (sICAM-1/Ig). A single i.v. injection of an adenovirus vector encoding the immunoadhesin gene led to 70% diabetes remission as opposed to 0% in mice injected with a control adenovirus vector. Despite the rapid decline of sICAM-1/Ig serum levels, diabetes remission remained stable in 50% of NOD mice for >6 mo. sICAM-1/Ig expression also led to long-term protection against diabetes in prediabetic NOD mice. sICAM-1/Ig in vitro induced an agonistic effect of T cell activation in a TCR-transgenic model, increasing T cell proliferation and IL-2 secretion. Importantly, protected mice were not immunosuppressed because they rejected skin allografts normally and developed immunity against the adenovirus vector. Rather, sICAM-1/Ig induced active tolerance, as assessed by the persistence of diabetogenic T cells in protected mice and the reversal of protection by immunosuppression with cyclophosphamide.