Søren E. Degn

Capture of influenza by medullary dendritic cells via SIGN-R1 is essential for humoral immunity in draining lymph nodes

A major pathway for B cell acquisition of lymph-borne particulate antigens relies on antigen capture by subcapsular sinus macrophages of the lymph node. Here we tested whether this mechanism is also important for humoral immunity to inactivated influenza virus. By multiple approaches, including multiphoton intravital imaging, we found that antigen capture by sinus-lining macrophages was important for limiting the systemic spread of virus but not for the generation of influenza-specific humoral immunity. Instead, we found that dendritic cells residing in the lymph node medulla use the lectin receptor SIGN-R1 to capture lymph-borne influenza virus and promote humoral immunity. Thus, our results have important implications for the generation of durable humoral immunity to viral pathogens through vaccination.

MAp44, a Human Protein Associated with Pattern Recognition Molecules of the Complement System and Regulating the Lectin Pathway of Complement Activation

Essential effector functions of innate immunity are mediated by complement activation initiated by soluble pattern recognition molecules: mannan-binding lectin (MBL) and the ficolins. We present a novel, phylogenetically conserved protein, MAp44, which is found in human serum at 1.4 μg/ml in Ca2+-dependent complexes with the soluble pattern recognition molecules. The affinity for MBL is in the nanomolar range (KD = 0.6 nM) as determined by surface plasmon resonance. The first eight exons of the gene for MAp44 encode four domains shared with MBL-associated serine protease (MASP)-1 and MASP-3 (CUB1-EGF-CUB2-CCP1), and a ninth exon encodes C-terminal 17 aa unique to MAp44. mRNA profiling in human tissues shows high expression in the heart. MAp44 competes with MASP-2 for binding to MBL and ficolins, resulting in inhibition of complement activation. Our results add a novel mechanism to those known to control the innate immune system.

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.

Trafficking of B Cell Antigen in Lymph Nodes

The clonal selection theory first proposed by Macfarlane Burnet is a cornerstone of immunology (1). At the time, it revolutionized the thinking of immunologists because it provided a simple explanation for lymphocyte specificity, immunological memory, and elimination of self-reactive clones (2). The experimental demonstration by Nossal & Lederberg (3) that B lymphocytes bear receptors for a single antigen raised the central question of where B lymphocytes encounter antigen. This question has remained mostly unanswered until recently. Advances in techniques such as multiphoton intravital microscopy (4, 5) have provided new insights into the trafficking of B cells and their antigen. In this review, we summarize these advances in the context of our current view of B cell circulation and activation.

Complement activation, regulation, and molecular basis for complement-related diseases.

The complement system is an essential element of the innate immune response that becomes activated upon recognition of molecular patterns associated with microorganisms, abnormal host cells, and modified molecules in the extracellular environment. The resulting proteolytic cascade tags the complement activator for elimination and elicits a pro‐inflammatory response leading to recruitment and activation of immune cells from both the innate and adaptive branches of the immune system. Through these activities, complement functions in the first line of defense against pathogens but also contributes significantly to the maintenance of homeostasis and prevention of autoimmunity. Activation of complement and the subsequent biological responses occur primarily in the extracellular environment. However, recent studies have demonstrated autocrine signaling by complement activation in intracellular vesicles, while the presence of a cytoplasmic receptor serves to detect complement‐opsonized intracellular pathogens. Furthermore, breakthroughs in both functional and structural studies now make it possible to describe many of the intricate molecular mechanisms underlying complement activation and the subsequent downstream events, as well as its cross talk with, for example, signaling pathways, the coagulation system, and adaptive immunity. We present an integrated and updated view of complement based on structural and functional data and describe the new roles attributed to complement. Finally, we discuss how the structural and mechanistic understanding of the complement system rationalizes the genetic defects conferring uncontrolled activation or other undesirable effects of complement.

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.

Mannan-binding lectin (MBL)-associated serine protease-1 (MASP-1), a serine protease associated with humoral pattern-recognition molecules: normal and acute-phase levels in serum and stoichiometry of lectin pathway components.

The pattern‐recognition molecules mannan‐binding lectin (MBL) and the three ficolins circulate in blood in complexes with MBL‐associated serine proteases (MASPs). When MBL or ficolin recognizes a microorganism, activation of the MASPs occurs leading to activation of the complement system, an important component of the innate immune system. Three proteins are produced from the MASP1 gene: MASP‐1 and MASP‐3 and MAp44. We present an assay specific for MASP‐1, which is based on inhibition of the binding of anti‐MASP‐1‐specific antibody to MASP‐1 domains coated onto microtitre wells. MASP‐1 was found in serum in large complexes eluting in a position corresponding to ∼600 kDa after gel permeation chromatography in calcium‐containing buffer and as monomers of ∼75 kDa in dissociating buffer. The concentration of MASP‐1 in donor sera (n = 105) was distributed log‐normally with a median value of 11 µg/ml (range 4–30 µg/ml). Serum and citrate plasma levels were similar, while the values in ethylenediamine tetraacetic acid plasma were slightly lower and in heparin plasma were 1·5 times higher than in serum. MASP‐1 was present at adult level at 1 year of age, while it was 60% at birth. In normal healthy individuals the level of MASP‐1 was stable throughout a 2‐month period. After induction of an acute‐phase reaction by operation we found an initial short decrease, concomitant with an increase in C‐reactive protein levels, followed by an increase, doubling the MASP‐1 concentration after 2 days. The present data prepare the ground for studies on the associations of MASP‐1 levels with disease.

Polymorphisms in Mannan-Binding Lectin (MBL)-Associated Serine Protease 2 Affect Stability, Binding to MBL, and Enzymatic Activity

Mannan-binding lectin-associated serine protease 2 (MASP-2) is an enzyme of the innate immune system. MASP-2 forms complexes with the pattern recognition molecules mannan-binding lectin (MBL), H-ficolin, L-ficolin, or M-ficolin, and is activated when one of these proteins recognizes microorganisms and subsequently cleaves complement factors C4 and C2, thus initiating the activation of the complement system. Missense polymorphisms of MASP-2 exist in different ethnic populations. To further characterize the nature of these, we have produced and characterized rMASP-2s representing the following naturally occurring polymorphisms: R99Q, D120G, P126L, H155R, 156_159dupCHNH (CHNHdup), V377A, and R439H. Only very low levels of CHNHdup were secreted from the cells, whereas quantities similar to wild-type MASP-2 were found intracellularly, indicating that this mutation results in a misfolded protein. We found that D120G and CHNHdup could not associate with MBL, whereas R99Q, P126L, H155R, V377A, R439H, and wild-type MASP-2 bound equally well to MBL. Accordingly, when D120G and CHNHdup were mixed with MBL, no activation of complement factor C4 was observed, whereas R99Q, P126L, and V377A cleaved C4 with an activity comparable to wild-type MASP-2 and H155R slightly better. In contrast, the R439H variant was deficient in this process despite its normal binding to MBL. This variant was also not able to autoactivate in the presence of MBL and mannan. We find the R439H variant is common in Sub-Saharan Africans with a gene frequency of 10%. Our results indicate that individuals with different types of MASP-2 defects may be identified through genotyping.

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.

MAp19, the alternative splice product of the MASP2 gene

Abstract The lectin pathway of complement is a central part of innate immunity, but as a powerful inducer of inflammation it needs to be tightly controlled. The MASP2 gene encodes two proteins, MASP-2 and MAp19. MASP-2 is the serine protease responsible for lectin pathway activation. The smaller alternative splice product, MAp19, lacks a catalytic domain but retains two of three domains involved in association with the pattern-recognition molecules (PRMs): mannan-binding lectin (MBL), H-ficolin, L-ficolin and M-ficolin. MAp19 reportedly acts as a competitive inhibitor of MASP-2-mediated complement activation. In light of a ten times lower affinity of MAp19, versus MASP-2, for association with the PRMs, much higher serum concentrations of MAp19 than MASP-2 would be required for MAp19 to exert such an inhibitory activity. Just four amino acid residues distinguish MAp19 from MASP-2, and these are conserved between man, mouse and rat. Nonetheless we generated monoclonal rat anti-MAp19 antibodies and established a quantitative assay. We found the concentration of MAp19 in serum to be 217ng/ml, i.e., 11nM, comparable to the 7nM of MASP-2. In serum all MASP-2, but only a minor fraction of MAp19, was associated with PRMs. In contrast to previous reports we found that MAp19 could not compete with MASP-2 for binding to MBL, nor could it inhibit MASP-2-mediated complement activation. Immunohistochemical analyses combined with qRT-PCR revealed that both MAp19 and MASP-2 were mainly expressed in hepatocytes. High levels of MAp19 were found in urine, where MASP-2 was absent.