Alan Ezekowitz

Arthritis Critically Dependent on Innate Immune System Players

K/BxN T cell receptor transgenic mice are a model of inflammatory arthritis, similar to rheumatoid arthritis. Disease in these animals is focused specifically on the joints but stems from autoreactivity to a ubiquitously expressed antigen, glucose-6-phosphate isomerase (GPI). T and B cells are both required for disease initiation, but anti-GPI immunoglobulins (Igs), alone, can induce arthritis in lymphocyte-deficient recipients. Here, we show that the arthritogenic Igs act through both Fc receptors (in particular, FcgammaRIII) and the complement network (C5a). Surprisingly, the alternative pathway of complement activation is critical, while classical pathway components are entirely dispensable. We suggest that autoimmune disease, even one that is organ specific, can occur when mobilization of an adaptive immune response results in runaway activation of the innate response.

Mannan-binding lectin recognizes structures on ischaemic reperfused mouse kidneys and is implicated in tissue injury

Organ damage as a consequence of ischaemia and reperfusion (I/R) is a major clinical problem in an acute renal failure and transplantation. Ligands on surfaces of endothelial cells that are exposed due to the ischaemia may be recognized by pattern recognition molecules such as mannan‐binding lectin (MBL), inducing complement activation. We examined the contribution of the MBL complement pathway in a bilateral renal I/R model (45 min of ischaemia followed by 24 h of reperfusion), using transgenic mice deficient in MBL‐A and MBL‐C [MBL double knockout (MBL DKO)] and in wildtype (WT) mice. Kidney damages, which were evaluated by levels of blood urea nitrogen (BUN) and creatinine, showed that MBL DKO mice were significantly protected compared with WT mice. MBL DKO mice, reconstituted with recombinant human MBL, showed a dose‐dependent severity of kidney injury increasing to a comparable level to WT mice. Acute tubular necrosis was evident in WT mice but not in MBL DKO mice after I/R, confirming renal damages in WT mice. MBL ligands in kidneys were observed to be present after I/R but not in sham‐operated mice. C3a (desArg) levels in MBL DKO mice were decreased after I/R compared with that in WT mice, indicating less complement activation that was correlated with less C3 deposition in the kidneys of MBL DKO mice. Our data implicate a role of MBL in I/R‐induced kidney injury.

Characterization and Quantification of Mouse Mannan‐Binding Lectins (MBL‐A and MBL‐C) and Study of Acute Phase Responses

Rat monoclonal antibodies (MoAbs) against mouse mannan‐binding lectin (MBL)‐A and MBL‐C were generated and assays for MBL‐A and MBL‐C were constructed. This allowed for the quantitative analysis of both proteins for the first time. Previously only MBL‐A has been quantified using less standardized methods. In a mouse serum pool the concentrations were now determined at 7.5 µg MBL‐A and 45 µg MBL‐C per ml. On gel permeation chromatography of mouse serum, MBL‐A eluted corresponding to a Mr of 850 kDa whereas the majority of MBL‐C eluted corresponding to a Mr of 950 kDa. On sucrose density gradient centrifugation the sedimentation velocities of MBL‐A and MBL‐C were estimated at 7.3 S and 10.8 S, respectively. The MBL‐A and MBL‐C levels in 10 laboratory mice strains were compared and found to vary between 4 µg/ml to 12 µg/ml, and 16 µg/ml to 118 µg/ml, respectively. After the induction of acute phase responses by intraperitoneal injection of either casein or lipopolysaccharide (LPS), MBL‐A was found to increase approximately two‐fold, with a maximum after 32 h, while MBL‐C did not increase significantly. In comparison, serum amyloid A component (SAA) peaked at 15 h with an approximate 100‐fold increase.

Mannan-binding lectin modulates the response to HSV-2 infection

Viruses have developed numerous strategies to escape recognition by the immune system. However, some viruses such as herpes simplex virus‐2 (HSV‐2) are recognized by initiators of the complement system, e.g. mannan‐binding lectin (MBL). To study the effects of MBL deficiency during viral infection we have chosen a model of generalized HSV‐2 infection. We infected MBL‐A and MBL‐C double knock‐out mice (DKO) with HSV‐2 via the intraperitoneal (i.p.) route. DKO mice cleared HSV‐2 from the liver less efficiently than the comparable wild‐type animals. The impairment to effectively neutralize HSV‐2 correlated with compromised liver function as measured by increased plasma levels of alanine‐amino transferase. No differences in the viral burden were found in other organs such as spleen or brain. Thus, MBL‐mediated protection was limited to the effects of preservation of liver homeostasis. Reconstitution with recombinant human MBL before and during the HSV‐2 infection dramatically lowered the viral titres in the liver. Taken together, the data show that MBL modulates the response to HSV‐2 in mice by affecting neutralization of the virus. To analyse if MBL plays a role in establishment and progression of human HSV‐2 infection we analysed MBL levels in the serum samples from asymptomatic (virus‐exposed people who have never displayed symptoms of HSV‐2 infection) and symptomatic HSV‐2 patients (people with recurrent HSV‐2 infections). We found that the frequency of the MBL deficiency (<100 ng/ml) was higher in the symptomatic group and significantly different from that in the asymptomatic group (P = 0·0369). This suggests that lack of MBL‐mediated complement activation increases susceptibility to viral infection.

The Differing Roles of the Classical and Mannose-Binding Lectin Complement Pathways in the Events following Skeletal Muscle Ischemia-Reperfusion

Complement is an important mediator of the injuries observed after skeletal muscle ischemia and subsequent reperfusion. Although the classical pathway had been assumed to be the major pathway of activation leading to injury, the mannose-binding lectin (MBL) pathway might also play a contributing role. In this study, we found that MBL-deficient mice had significant protection after skeletal muscle reperfusion injury compared with wild-type, classical pathway-specific C1q-deficient mice, or MBL-deficient mice reconstituted with recombinant human MBL. MBL-deficient mice, however, were not protected from permeability edema or secondary lung injury after ischemia-reperfusion. These data indicate that blockade of the classical pathway alone (C1q) is protective against permeability edema and remote pulmonary injury but not protective against histologic muscle injury. In contrast, blocking the MBL pathway alone protects against histological injury but is not protective against permeability edema or lung injury. Thus, the activation of both pathways is likely responsible for the full spectrum of injuries observed after skeletal muscle reperfusion injury.

Reports of suicidality in clinical trials of montelukast.

BACKGROUND In recent years, a number of drugs and drug classes have come under scrutiny by the US Food and Drug Administration regarding suicidality (including suicidal behavior and ideation). OBJECTIVE We sought to perform 2 reviews (requested by the US Food and Drug Administration) of the number of events possibly related to suicidality reported in Merck clinical trials of montelukast. METHODS Method 1 was a descriptive review of clinical adverse experiences (AEs) from 116 studies (double-blind and open-label, adult and pediatric, and single- and multiple-dose studies) completed as of March 2008. Summaries were constructed from investigator-reported AE terms possibly related to suicidality (completed suicide, suicide attempt, and suicidal ideation) or self-injurious behavior. Method 2 used a retrospective adjudication of investigator-reported AEs and other events listed in the study database described as possibly suicidality-related adverse events (PSRAEs) in a prespecified set of 41 double-blind, placebo-controlled trials completed as of April 2008. RESULTS No completed suicides were reported in any study. For the descriptive review, 20,131 adults and children received montelukast, 9,287 received placebo, and 8,346 received active control; AEs possibly related to suicidality were rare and were similar between the montelukast and placebo or active-control groups. For the adjudicated review across 22,433 patients, there were 730 adjudicated events. In 9,929 patients taking montelukast, 1 PSRAE was identified (classified as suicidal ideation); none were identified in 7,780 and 4,724 patients taking placebo and active control, respectively. CONCLUSIONS Assessed by using 2 complementary methods, there were no reports of completed suicide, and reports of PSRAEs were rare in patients receiving montelukast and similar to those seen in control subjects.

Analysis of behavior-related adverse experiences in clinical trials of montelukast

BACKGROUND Frequencies of behavior-related adverse experiences (BRAEs) in controlled clinical studies of leukotriene modifier drugs have not been summarized. OBJECTIVE We sought to compare the frequency of BRAEs in patients receiving montelukast or placebo in a retrospective analysis of Merck clinical trial data. METHODS An adverse experience database was constructed to include all double-blind, placebo-controlled trials of montelukast meeting prespecified criteria. BRAEs (described using the Medical Dictionary for Regulatory Activities controlled vocabulary dictionary) were prespecified to include any term in the Psychiatric Disorders System Organ Class, selected terms related to general disorders, and terms related to akathisia. Frequencies of BRAEs (overall, leading to study discontinuation, and/or serious) were summarized. Analyses estimated the odds ratios (ORs) for montelukast versus placebo based on the frequency of patients with BRAEs in each study. RESULTS In total 35 adult and 11 pediatric placebo-controlled trials were included; 11,673 patients received montelukast, 8,827 received placebo, and 4,724 received active control. The frequency of patients with 1 or more BRAEs was 2.73% and 2.27% in the montelukast and placebo groups, respectively; the OR for montelukast versus placebo was 1.12 (95% CI, 0.93-1.36). The frequency of patients with a BRAE leading to study discontinuation was 0.07% and 0.11% in the montelukast and placebo groups, respectively (OR, 0.52; 95% CI, 0.17-1.51). The frequency of patients with a BRAE considered serious was 0.03% in both treatment groups. CONCLUSION Reports of BRAEs were infrequent in clinical trials of montelukast. Those leading to study discontinuation or considered serious were rare. Frequencies were similar regardless of treatment group.

Mannose binding lectin gene deficiency increases susceptibility to traumatic brain injury in mice

Mannose binding lectin (MBL) initiates complement activation and exacerbates tissue damage after systemic ischemia/reperfusion. We tested the hypothesis that MBL activates complement and worsens outcome using two levels of controlled cortical impact (CCI) in mice. After moderate CCI (0.6 mm depth), MBL immunostaining was detected on injured endothelial cells of wild-type (WT) mice and C3d was detected in MBL KO (deficient in MBL A/C) and WT mice, suggesting that MBL is dispensable for terminal complement activation after CCI. Brain neutrophils, edema, blood-brain barrier permeability, gross histopathology, and motor dysfunction were similar in injured MBL KO and WT mice. In mice subjected to mild CCI (0.2 mm), MBL KO mice had almost two-fold increased acute CA3 cell degeneration at 6 h (P<0.01 versus WT). Naive MBL KO mice had decreased brain volume but performed similar to WT mice in two distinct Morris water maze (MWM) paradigms. However, injured MBL KO mice had impaired performance in cued platform trials (P<0.05 versus WT), suggesting a transient nonspatial learning deficit in injured MBL KO mice. The data suggest that MBL deficiency increases susceptibility to CCI through C3-independent mechanisms and that MBL-deficient patients may be at increased risk of poor outcome after traumatic brain injury.

Lectin-Dependent Enhancement of Ebola Virus Infection via Soluble and Transmembrane C-type Lectin Receptors

Mannose-binding lectin (MBL) is a key soluble effector of the innate immune system that recognizes pathogen-specific surface glycans. Surprisingly, low-producing MBL genetic variants that may predispose children and immunocompromised individuals to infectious diseases are more common than would be expected in human populations. Since certain immune defense molecules, such as immunoglobulins, can be exploited by invasive pathogens, we hypothesized that MBL might also enhance infections in some circumstances. Consequently, the low and intermediate MBL levels commonly found in human populations might be the result of balancing selection. Using model infection systems with pseudotyped and authentic glycosylated viruses, we demonstrated that MBL indeed enhances infection of Ebola, Hendra, Nipah and West Nile viruses in low complement conditions. Mechanistic studies with Ebola virus (EBOV) glycoprotein pseudotyped lentiviruses confirmed that MBL binds to N-linked glycan epitopes on viral surfaces in a specific manner via the MBL carbohydrate recognition domain, which is necessary for enhanced infection. MBL mediates lipid-raft-dependent macropinocytosis of EBOV via a pathway that appears to require less actin or early endosomal processing compared with the filovirus canonical endocytic pathway. Using a validated RNA interference screen, we identified C1QBP (gC1qR) as a candidate surface receptor that mediates MBL-dependent enhancement of EBOV infection. We also identified dectin-2 (CLEC6A) as a potentially novel candidate attachment factor for EBOV. Our findings support the concept of an innate immune haplotype that represents critical interactions between MBL and complement component C4 genes and that may modify susceptibility or resistance to certain glycosylated pathogens. Therefore, higher levels of native or exogenous MBL could be deleterious in the setting of relative hypocomplementemia which can occur genetically or because of immunodepletion during active infections. Our findings confirm our hypothesis that the pressure of infectious diseases may have contributed in part to evolutionary selection of MBL mutant haplotypes.

Mannan‐binding lectin deficiency modulates the humoral immune response dependent on the genetic environment

Mannan‐binding lectin (MBL) is a plasma protein implicated in innate immune defence against a broad range of microorganisms, including viruses. It is also thought that MBL plays a role in the recruitment of the specific clonal immune response. This was studied by injecting soluble hepatitis B surface antigen (HBsAg) intravenously into mice deficient in both MBL‐A and MBL‐C (MBL DKO mice). The MBL DKO animals on mixed genetic background (SV129EvSv × C57BL/6) produced higher antibody titres than the wild‐type littermates. After primary challenge with the antigen the immunoglobulin M anti‐HBsAg antibody titres were threefold higher in the MBL DKO mice than in the wild‐type mice. Following the boost, the immunoglobulin G anti‐HBsAg antibody titres were 10‐fold higher in the MBL DKO mice, suggesting that MBL plays a role in a negative feedback regulation of adaptive immunity. However, the modulating effect of MBL was dependent on the genetic environment. The MBL DKO mice backcrossed on a C57BL/6 background showed the opposite response with the MBL DKO mice now producing fewer antibodies than the wild‐type animals, whereas MBL deficiency in mice with the SV129EvSv background did not show any effect in antibody production. These findings indicate that the modifying effect of MBL on the humoral immune response is influenced by the genetic environment.