Lisbeth Jensen

Control of the classical and the MBL pathway of complement activation

The activation of complement via the mannan-binding lectin (MBL) pathway is initiated by the MBL complex consisting of the carbohydrate binding molecule, MBL, two associated serine proteases, MASP-1 and MASP-2, and a third protein, MAp19. In the present report we used an assay of complement activation specifically reflecting the physiological activity of the MBL complex to identify biological and synthetic inhibitors. Inhibitor activity towards the MBL complex was compared to the inhibition of the classical pathway C1 complex and to a complex of MBL and recombinant MASP-2. A number of synthetic inhibitors were found to differ in their activities towards complement activation via the MBL pathway and the classical pathway. C1 inhibitor inhibited both pathways whereas alpha2-macroglobulin (alpha2M) inhibited neither. C1 inhibitor and alpha2M were found to be associated with the MBL complex. Upon incubation at 37 degrees C in physiological buffer, the associated inhibitors as well as MASP-1, MASP-2, and MAp19 dissociated from MBL, whereas only little dissociation of the complex occurred in buffer with high ionic strength (1 M NaCl). The difference in sensitivity to various inhibitors and the influence of high ionic strength on the complexes indicate that the activation and control of the MBL pathway differ from that of the classical pathway. MBL deficiency is linked to various clinical manifestations such as recurrent infections, severe diarrhoea, and recurrent miscarriage. On the other hand, impaired control of complement activation may lead to severe and often chronically disabling diseases. The results in the present report suggests the possibility of specifically inhibiting of the MBL pathway of complement activation.

Mannan-Binding Lectin-Associated Serine Protease (MASP)-1 Is Crucial for Lectin Pathway Activation in Human Serum, whereas neither MASP-1 nor MASP-3 Is Required for Alternative Pathway Function

The lectin pathway of complement is an important component of innate immunity. Its activation has been thought to occur via recognition of pathogens by mannan-binding lectin (MBL) or ficolins in complex with MBL-associated serine protease (MASP)-2, followed by MASP-2 autoactivation and cleavage of C4 and C2 generating the C3 convertase. MASP-1 and MASP-3 are related proteases found in similar complexes. MASP-1 has been shown to aid MASP-2 convertase generation by auxiliary C2 cleavage. In mice, MASP-1 and MASP-3 have been reported to be central also to alternative pathway function through activation of profactor D and factor B. In this study, we present functional studies based on a patient harboring a nonsense mutation in the common part of the MASP1 gene and hence deficient in both MASP-1 and MASP-3. Surprisingly, we find that the alternative pathway in this patient functions normally, and is unaffected by reconstitution with MASP-1 and MASP-3. Conversely, we find that the patient has a nonfunctional lectin pathway, which can be restored by MASP-1, implying that this component is crucial for complement activation. We show that, although MASP-2 is able to autoactivate under artificial conditions, MASP-1 dramatically increases lectin pathway activity at physiological conditions through direct activation of MASP-2. We further demonstrate that MASP-1 and MASP-2 can associate in the same MBL complex, and that such cocomplexes are found in serum, providing a scenario for transactivation of MASP-2. Hence, in functional terms, it appears that MASP-1 and MASP-2 act in a manner analogous to that of C1r and C1s of the classical pathway.

Assays for the functional activity of the mannan-binding lectin pathway of complement activation.

Mannan-binding lectin (MBL) activates complement independently of the adaptive, clonal immune system and thus presents an innate anti-microbial defence mechanism. Events in the MBL pathway of complement activation involve the binding of MBL to patterns of carbohydrate structures presented by the surface of micro-organisms. For the activation of complement to occur MBL must be associated with serine proteases (MBL-associated serine proteases, MASPs) in an MBL/MASP complex. When bound to micro-organisms, the MBL complex mediates the activation of C4 and C2, generating the C3 convertase, C4bC2b. The C4/C2 cleaving activity of the MBL complex is shared with the C1 complex of the classical pathway of complement activation. Different assays that allow for determination of the activity of the MBL complex in serum samples have been developed and are discussed in this report. We present data from one such assay (MBL/MASP activity test), which we have found useful for the routine evaluation of clinical samples. In this assay any influence of the classical pathway has been eliminated by using a hypertonic buffer, which inhibits the binding of C1q to immuncomplexes and disrupt the C1 complex, while leaving the function of the MBL complex intact. In parallel we determine the MBL concentration in the sample. As predicted a very high correlation is observed between the results of the two assays.

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.

Characteristics and Biological Variations of M-Ficolin, a Pattern Recognition Molecule, in Plasma

The three human ficolins, H-ficolin, L-ficolin and M-ficolin, are pattern recognition molecules of the innate immune system. All three ficolins can activate the lectin pathway of the complement system after binding to pathogens. H- and L-ficolin are serum proteins with an average concentration of 18 and 3 µg/ml, respectively. M-ficolin has been described as a membrane-associated pattern recognition receptor of monocytes, being also present in granulocytes; recently, minuscule amounts of M-ficolin have been found in serum, too. No assay specific for M-ficolin has yet been described and biological variations are unknown. We have raised specific monoclonal anti-human M-ficolin antibodies and have developed a quantitative assay for M-ficolin. M-ficolin elutes as a large, 900-kDa protein upon gel permeation chromatography of serum. Analysis of M-ficolin levels in serum samples of 350 blood donors reveals a mean concentration of 1.07 µg/ml, ranging from 0.28 to 4.05 µg/ml. Analyses of consecutive acute phase serum samples from major surgery patients indicated a complex response. Ontogeny was investigated through cord blood samples from healthy full-term babies, which showed adult levels, with sequential samples showing no increase from birth to 1 year of age. We suggest that M-ficolin should also be considered as a humoral pattern recognition molecule.

Biological variations of MASP-3 and MAp44, two splice products of the MASP1 gene involved in regulation of the complement system

The lectin pathway of complement is part of the innate immune system. The complement-activating pattern-recognition molecules (for which we suggest the abbreviation CAPREMs) mannan-binding lectin (MBL) and the three ficolins (H-, L- and M-ficolin) circulate in complexes with MBL-associated serine proteases (MASP-1, -2 and -3) and two additional proteins (MAp19 and MAp44, also termed sMAP and MAP-1, respectively). When MBL or ficolins recognize a microorganism or altered self components, activation of the MASPs ensues, leading to the activation of the complement system. MASP-1, MASP-3 and MAp44 are all three encoded by the MASP1 gene. MASP-1 and -3 share five domains (constituting the so-called A-chain), but have unique protease domains (B-chains). MAp44 shares the first four domains with MASP-1 and MASP-3, followed by 17 unique C-terminal amino acid residues. Thus, assays for the protease domain of MASP-3 and for the 17 C-terminal amino acids of MAp44 are required to measure these proteins specifically and here we present such assays for MASP-3 and MAp44. MASP-3 was captured with a monoclonal antibody (5F5) reacting with a common domain of the three proteins (CCP1) and the assay was developed with a monoclonal antibody (38.12.3) specific for the C-terminal part of the MASP-3 protease domain. MAp44 was captured with a monoclonal antibody (2D5) reacting with the C-terminus of MAp44 followed by assay development with a monoclonal anti-CCP1 antibody (4H2). Using Superose 6 gel permeation chromatography of serum, MASP-3 and MAp44 were found in complexes, which eluted in positions corresponding to 600-800 kDa and 500-700 kDa, respectively. The level of MASP-3 in donor sera (N=200) was log-normally distributed with a median value of 5.0 μg/ml (range: 1.8-10.6 μg/ml), and the corresponding value for MAp44, also log-normally distributed, was 1.7 μg/ml (range: 0.8-3.2 μg/ml). For MASP-3, the inter-assay coefficients of variation of low, intermediate and high level internal controls were 4.9%, 6.9% and 3.9% (N=12). For MAp44, the corresponding inter-assay CVs were 7.6%, 6.2%, and 7.0% (N=12). MASP-3 levels were low at birth and reached adult levels within the first 6 months, whereas MAp44 levels fell slightly during the first 6 months. Concomitant with the acute phase response in patients undergoing major surgery, levels of both proteins fell slightly over 1-2 days, but whereas MASP-3 recovered to baseline values over another 2 days, MAp44 only reached baseline values at around day 30. Thus, neither of the two proteins behaves as a classical acute phase protein.

Investigations on collectin liver 1.

Background: The collectin CL-L1 is a pattern recognition molecule of the innate immune system. Results: Several biological parameters are established and furnish a basis for continued investigations. Conclusion: CL-L1 has specificity similar to other collectins, is present as large oligomers in blood from birth, and is associated with MASPs. Significance: A number of novel data supporting a biological role for CL-L1 are presented. Collectins are pattern recognition molecules of the innate immune system showing binding to carbohydrate structures on microorganisms in a calcium-dependent manner. Recently, three novel collectins, collectin liver 1 (CL-L1), collectin kidney 1 (CL-K1 and CL-11), and collectin placenta 1 (CL-P1), were discovered. The roles of these three collectins remain largely unknown. Here, we present a time-resolved immunofluorometric assay for quantification of CL-L1. The concentration of CL-L1 in donor plasma (n = 210) was distributed log-normally with a median value of 3.0 μg/ml (range 1.5–5.5 μg/ml). We observed on average 30% higher concentrations of CL-L1 in plasma as compared with serum. Size analysis by gel-permeation chromatography showed CL-L1 in serum to elute as large 700–800-kDa complexes and smaller 200–300-kDa complexes. CL-L1 showed specific binding to mannose-TSK beads in a Ca2+-dependent manner. This binding could be inhibited by mannose and glucose, but not galactose, indicating that CL-L1 binds via its carbohydrate-recognition domain and has ligand specificity similar to that of mannan-binding lectin. Western blot analysis of CL-L1 showed the presence of several oligomeric forms in serum. Ontogeny studies showed CL-L1 to be present at birth at near adult levels. CL-L1 levels exhibit low variation in healthy adults over a 1-year period. During acute-phase responses, the CL-L1 levels display only minor variations. In serum, CL-L1 was found in complexes with mannan-binding lectin-associated serine proteases, suggesting a role in the lectin pathway of complement activation. The presented data establish a basis for future studies on the biological role of CL-L1.

Complement activation by ligand-driven juxtaposition of discrete pattern recognition complexes

Significance A salient feature of the immune system is its ability to discriminate self from nonself. We define the molecular mechanism governing activation of an ancient and central component: the lectin pathway of complement. The basis is the association of two proteases in distinct complexes with at least five pattern recognition molecules. Clustering of these complexes on ligand surfaces allows cross-activation of the proteases, which subsequently activate downstream factors to initiate a proteolytic cascade. This is conceptually similar to signaling by cellular receptors and could be viewed as cellular signaling turned inside out. Different pattern recognition complexes “talk to each other” to coordinate immune activation, which may impart differential activation based on recognition of simple vs. complex ligand patterns. Defining mechanisms governing translation of molecular binding events into immune activation is central to understanding immune function. In the lectin pathway of complement, the pattern recognition molecules (PRMs) mannan-binding lectin (MBL) and ficolins complexed with the MBL-associated serine proteases (MASP)-1 and MASP-2 cleave C4 and C2 to generate C3 convertase. MASP-1 was recently found to be the exclusive activator of MASP-2 under physiological conditions, yet the predominant oligomeric forms of MBL carry only a single MASP homodimer. This prompted us to investigate whether activation of MASP-2 by MASP-1 occurs through PRM-driven juxtaposition on ligand surfaces. We demonstrate that intercomplex activation occurs between discrete PRM/MASP complexes. PRM ligand binding does not directly escort the transition of MASP from zymogen to active enzyme in the PRM/MASP complex; rather, clustering of PRM/MASP complexes directly causes activation. Our results support a clustering-based mechanism of activation, fundamentally different from the conformational model suggested for the classical pathway of complement.

Co-Complexes of MASP-1 and MASP-2 Associated with the Soluble Pattern-Recognition Molecules Drive Lectin Pathway Activation in a Manner Inhibitable by MAp44

The lectin pathway of complement is an integral component of innate immunity. It is activated upon binding of mannan-binding lectin (MBL) or ficolins (H-, L-, and M-ficolin) to suitable ligand patterns on microorganisms. MBL and ficolins are polydisperse homo-oligomeric molecules, found in complexes with MBL-associated serine proteases (MASP-1, -2, and -3) and MBL-associated proteins (MAp19 and MAp44). This scenario is far more complex than the well-defined activation complex of the classical pathway, C1qC1r2C1s2, and the composition of the activating complexes of the lectin pathway is ill defined. We and other investigators recently demonstrated that both MASP-1 and MASP-2 are crucial to lectin pathway activation. MASP-1 transactivates MASP-2 and, although MASP-1 also cleaves C2, MASP-2 cleaves both C4 and C2, allowing formation of the C3 convertase, C4bC2a. Juxtaposition of MASP-1 and MASP-2 during activation must be required for transactivation. We previously presented a possible scenario, which parallels that of the classical pathway, in which MASP-1 and MASP-2 are found together in the same MBL or ficolin complex. In this study, we demonstrate that, although MASPs do not directly form heterodimers, the addition of MBL or ficolins allows the formation of MASP-1–MASP-2 co-complexes. We find that such co-complexes have a functional role in activating complement and are present in serum at varying levels, impacting on the degree of complement activation. This raises the novel possibility that MAp44 may inhibit complement, not simply by brute force displacement of MASP-2 from MBL or ficolins, but by disruption of co-complexes, hence impairing transactivation. We present support for this contention.

Further observations on the speed of death in hanging

Abstract:  Given that most fatal hangings are suicidal and occur in locations that have been selected to conceal this activity (thus maximizing the chances of a lethal outcome), there has been very little corroboration of the speed with which unconsciousness and death may occur. A 35‐year‐old male is reported who committed suicide by hanging immediately after talking to his spouse. Police investigations confirmed her reliability as a witness indicating that lethal anoxia in this case had occurred within a very short time (most likely in less than 1 min) of suspension. The speed with which death may result from hanging not only gives an insight into fatal pathophysiological mechanisms, but also provides useful information for situations where a lethal outcome is to be avoided, or is not intended. For example, individuals at risk of suicide who are being monitored in institutional facilities need to be constantly under direct visual surveillance as significant hypoxia can be rapidly induced, parents and caregivers with infants and children in potentially unsafe sleeping environments need to realize how swiftly death or irreversible anoxic brain damage may occur from neck compression, and those who engage in recreational asphyxia should be informed just how quickly a fatal outcome may ensue.