Mannose-binding lectin (MBL) substitution: recovery of opsonic function in vivo lags behind MBL serum levels.

Abstract

Mannose-binding lectin (MBL) deficiency is often associated with an increased risk of infection or worse prognosis in immunocompromised patients. MBL substitution in these patients might diminish these risks. We therefore performed an open, uncontrolled safety and pharmacokinetic MBL-substitution study in 12 pediatric oncology patients with chemotherapy-induced neutropenia. Twice weekly MBL infusions with plasma-derived MBL yielded MBL trough levels >1.0 microg/ml. We tested whether MBL substitution in vivo increased MBL-dependent complement activation and opsonophagocytosis of zymosan in vitro. Upon MBL substitution, opsonophagocytosis by control neutrophils increased significantly (p < 0.001) but remained suboptimal, although repeated MBL infusions resulted in improvement over time. The MBL-dependent MBL-associated serine protease (MASP)-mediated complement C3 and C4 activation also showed a suboptimal increase. To explain these results, complement activation was studied in detail. We found that in the presence of normal MASP-2 blood levels, MASP-2 activity (p < 0.0001) was reduced as well as the alternative pathway of complement activation (p < 0.05). This MBL-substitution study demonstrates that plasma-derived MBL infusions increase MBL/MASP-mediated C3 and C4 activation and opsonophagocytosis, but that higher circulating levels of plasma-derived MBL are required to achieve MBL-mediated complement activation comparable to healthy controls. Other patient cohorts should be considered to demonstrate clinical efficacy in phase II/III MBL-substitution studies, because we found a suboptimal recovery of (in vitro) biological activity upon MBL substitution in our neutropenic pediatric oncology cohort.

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TitleThe role of mannose-binding lectin in vitro and in vivo
AuthorN. Brouwer
FacultyFaculty of Medicine
Year 2008
Pages192
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The Role of Mannose-Binding Lectin
in Vitro and in Vivo













Nannette Brouwer

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The research described in this thesis was performed at the Department of Blood Cell Research,
Sanquin Research, Amsterdam, the Netherlands and Landsteiner Laboratory, Academic Medical
Centre, University of Amsterdam, the Netherlands

The research project was supported by a grant of the Landsteiner Foundation of Blood
Transfusion Research (LSBR no. 0207).





















The role of mannose-binding lectin in vitro and in vivo.

ISBN : 978-90-9023095-5
Author: Nannette Brouwer
Cover: Wim Brouwer & Mark Glas
“MBL exists in oligomers that form tulip-like structures”
Layout: Nannette Brouwer
Print: PrintPartners Ipskamp, Enschede, the Netherlands


© 2008, Nannette Brouwer. All rights reserved. No part of this thesis may be reproduced or
transmitted in any form or by any means without permission of the author.

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The role of Mannose-Binding Lectin
in Vitro and in Vivo









ACADEMISCH PROEFSCHRIFT










ter verkrijging van de graad van doctor
aan de Universiteit van Amsterdam
op gezag van de Rector Magnificus
Prof. dr. D.C. van den Boom
ten overstaan van een door het college voor promoties
ingestelde commissie,
in het openbaar te verdedigen in de Agnietenkapel
op donderdag 26 juni 2008, te 10.00 uur



door



Nannette Brouwer

geboren te Amsterdam

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Promotiecommissie

Promotores:


Co-promotor: Dr. K.M. Dolman


Overige Leden: Prof. dr. L.A. Aarden
Prof. dr. T. van der Poll
Prof. dr. M.R. Daha
Prof. dr. J.C. Jensenius
Prof. dr. N.J. Klein
Dr. D. Hamann


Faculteit der Geneeskunde










For the publication of this thesis, financial support is greatly acknowledged from:
Academic Medical Centre, University of Amsterdam
Stichting Sanquin Research
J.E. Juriaanse Stichting
Statens Serum Institute, Copenhagen, Denmark
Stichting Afweerstoornissen
Hycult biotechnology BV
Glas-Klooster BV
Vlug&Zoon transport BV
Ten Dam Zonwering BV
Sanyo E&E Europe BV
Sanquin Reagents
Prof. dr. T.W. Kuijpers
Prof. dr. D. Roos

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Het werk loopt niet weg als je je kind de regenboog laat zien,
Maar de regenboog wacht niet tot jij klaar bent met je werk.




























Voor Hugo

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Table of Contents

Chapter 1 Introduction

Chapter 2 Mannan-binding lectin (MBL)-mediated opsonization is enhanced by the
alternative pathway amplification loop
Mol Immunol. 2006 May; 43(13): 2051-60

Chapter 3 Mannose-binding lectin (MBL) facilitates opsonophagocytosis of yeasts but
not of bacteria, despite MBL binding
JI. 2008 March; 180 (6): 4124-4133

Chapter 4 High prevalence of mannose-binding lectin (MBL) deficiency in premature
neonates
Clin Exp Immunol. 2006 Jul; 145(1): 5-12

Chapter 5 Low mannose-binding lectin (MBL) levels in neonates with pneumonia and
sepsis.
Clin Exp Immunol. 2007 Nov; 150(2): 255-62

Chapter 6 The role of mannose-binding lectin (MBL) in paediatric oncology patients
with febrile neutropenia
Eur J Cancer. 2006 May; 42(7): 909-16

Chapter 7 Mannose-binding lectin (MBL) as prognostic factor in pediatric oncology
patients
Manuscript in preparation

Chapter 8 Phase-II safety and pharmacokinetics study on Mannose-Binding Lectin
substitution in children with chemotherapy-induced neutropenia.
Submitted for publication

Chapter 9 Mannose-Binding Lectin (MBL) substitution: recovery of opsonic function
lags behind reconstitution of MBL serum levels.
Manuscript in preparation

Chapter 10 Summary and general discussion


Co-authors affiliations

Nederlandse samenvatting

Dankwoord / Acknowledgements

Curriculum vitae & List of publications


9

29



47



67



81



95



109



125



141



163


173

177

183

187

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Page 11

Introduction

CHAPTER

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10
Chapter 1
Immune systems

system. There are two main differences between the two immune systems. The adaptive immune
response is highly specific to an invading micro-organism and has a memory function, so that
immunity to infectious micro-organisms will be acquired over time. In contrast, the innate
immunity is unspecific with a relatively constant role over time, and does not have a memory. The
adaptive immune response can be divided in cellular immune response, which involves T
lymphocytes and a humoral immune response, which involves proteins in the body fluids. In the
cellular immune response T-cells get activated upon contact with an antigen and develop to
activated cytotoxic T-cells, which are capable of killing micro-organisms directly. In contrast, for
an optimal humoral response, the T-cells and B-cells cooperate together to generate antibodies
(immunoglobulins) to the invading micro-organisms. After antigen presentation by an antigen-
presenting cell (APC), B-cells develop to antibody-secreting plasma cells, with the help of T-
helper (Th) cells (Figure 1A). Secreted antibodies bind to antigens on the surface of invading
micro-organisms, which flags them for phagocytosis and destruction (1). The primary antibody
response takes about 7-10 days to fully develop. In this first period after an infection the innate
immune response is of great importance.
Human immunity can be divided in the innate immune system and the adaptive immune

particular in early childhood, during the interval between the loss of passively acquired maternal
antibodies and the acquisition of a mature immunologic repertoire of the adaptive immune system,
innate immunity needs to function optimally. But, as mentioned above, the innate immune
response also contributes to host defense during the first days after infection with new
microorganisms in later life, before the adaptive immune response has been adequately induced.
Therefore, the innate immune response has a critical role in the control of an infection during the
first days. All innate immune cells and proteins are present in the circulation prior to an infection
and will act immediately. Natural killer-cells, monocytes, macrophages and polymorphic
neutrophils are leukocytes that are innate immune cells, which able to bind to micro-organisms
and kill them after uptake of the bound micro-organisms by phagocytosis. The recognition and
phagocytosis of invading micro-organisms by phagocytes is enhanced by marking of the micro-
organisms with soluble immune proteins, already present in the circulation, like complement
proteins (Figure 1B).
The innate immune response is an unspecific response that forms the first line of defense. In

Innate immunity and the complement system
Complement is a central component of the innate immune system, comprising at least 35
soluble proteins that collaborate in a complex manner in the elimination of microorganisms and in
the removal of apoptotic cells, but complement also acts in enhancing and directing the adaptive

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11
Introduction
immune response (2-4). The numerous plasma proteins are present not only in the circulation but
also in body fluids and tissues and can interact with various cells and mediators of the immune
system (5). The activation of the complement cascade can be initiated via three pathways (Figure
2). The classical pathway of the complement system is activated after formation of an antibody–
antigen complex (immune complex). C1q triggers the activation process when it binds to
antibodies within these immune complexes. In this way, C1q acts to bridge the innate and adaptive
Figure 1: Immune response: opsonization and phagocytosis of bacteria. (A): Adaptive immune reaction.
Peptide fragments of antigens present on the bacterial surface are presented to T-helper (Th) cells by
antigen-presenting cells (APC). This stimulation of Th cells activates B cell (B) proliferation and
differentiation to antibody forming cells (AFC). The secreted antibodies recognize the antigens on the
surface of the bacteria and bind to its surface, upon formation of antibody-antigen complexes, the classical
pathway of complement is activated and C3b and iC3b will be deposited on the surface of the bacteria
(opsonization). (B): Innate immune reaction. Circulating complement proteins (MBL, C3b) can bind
directly to repeating structures on the bacterial surface. The complement proteins cover the surface of the
bacteria (opsonization). Both the adaptive and the innate immune response lead to complement proteins on
the surface of the opsonized bacteria that are recognized by Complement Receptors (CR) on the surface of
the phagocytic neutrophils, which leads to cell activation and phagocytosis of the bacteria.

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Chapter 1
immune systems (4). The lectin pathway of the complement system is activated after binding of
mannose-binding lectin (MBL, also known as mannan-binding lectin or mannose-binding protein)
or ficolins to repeating sugar structures on the surface of micro-organisms. Upon binding, the
MBL-associated serine proteases (MASP) activate the complement cascade (6). The alternative
pathway of the complement system is based on auto-activation after direct binding of C3b or
C3·H2O to the surface of micro-organisms. All three pathways lead to the cleavage of C3 as a
consequence of triggered enzyme cascades. Upon activation of the classical and the lectin pathway
of complement, both C4 and C2 are cleaved, thereby generating C4b2a complexes, with C3
convertase activity (7). The C3bBb complex is the C3 convertase of the alternative pathway. The
alternative pathway is of importance for enhancement of both the classical and the lectin pathway,
because via the amplification loop, C3b formation leads to more C3b deposition on the pathogenic
surface.
The complement system has three major effector functions. Opsonization, the process by
which a pathogen is covered with proteins that facilitate its ingestion and destruction by a
phagocyte, is one of the effector functions of the complement system. Immunoglobulin G (IgG),
C3b and iC3b, the covalently-bound cleavage fragments of C3, are the most important
complement opsonins in the defense against bacterial infection (8). Opsonins have binding sites
for attachment to the cell-membrane of phagocytes and an enzymatic site for cleavage of the next
component of the complement cascade. Bound B3b or iC3b reacts with C3b receptors present on
the membrane of phagocytes, known as complement receptors (CR). There are 4 complement
receptors, CR1-4. The CR1 and CR2 are mainly important for the humoral immune response. CR1
is the opsonin receptor on neutrophils, which activates the phagocytosis process after binding to a
covalently bound C3 fragment on a micro-organism. Besides functioning as receptor for C3b or
iC3b functions CR1 also as receptor for C1q and had been suggested to bind MBL (9). CR2
binding of a small C3 (C3d) fragment enhances B-cell activation and antibody secretion. Besides
opsonization, chemotaxis (by C5a) is an effector function of complement activation in which
phagocytic cells are activated and induced to move towards the site of inflammation. The last
effector function of complement activation is the terminal pathway of complement, C5b-C9, which
forms the so-called membrane attack complex (MAC) that causes cell lysis of the micro-organism
by forming a pore in the cell membrane (10). Most micro-organisms are cleared from the
circulation by phagocytes after opsonization of the micro-organisms. The only known bacterium
that it is efficiently lysed via MAC insertion into the bacterial wall is Neisseria meningitides,
which causes meningitis (11).
Although the three known ficolins, L-Ficolin (12), H-ficolin (13) and M-ficolin (14), also
act at the lectin pathway of complement activation, an established role for ficolin-mediated
opsonization has yet to be discovered. The ficolins will not be further discussed in this thesis,
which is focused on MBL-mediated complement activation, opsonization and MBL disease
association.

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13
Introduction
Structure and function of MBL

also from rat and human liver (16). MBL belongs to a family of proteins called collectins, with
both collagenous regions and lectin domains. Besides MBL, the surfactant proteins A and D (SP-
A, SP-D) are other known collectins (17). The MBL molecule is built from polypeptide chains (32
kDa), consisting of four regions: the carbohydrate recognition domain (CRD), an alpha helical
MBL has been isolated for the first time from rabbit liver in 1978 (15) and a few years later
Figure 2: The complement system. The complement cascade is composed of proteins that are activated
through cleavage by an upstream enzyme. The three pathways of complement activation are shown at the
top of the figure. From left to right: the classical, the lectin and the alternative pathway. The binding of the
C1 complex to antigen-antibody complexes initiates proteolytic cleavage of complement proteins of the
classical pathway. The binding of the MBL/MASP-2 complex to a pathogenic cell wall initiates the lectin
pathway of complement. The alternative pathway of complement is initiated by the spontaneous hydrolysis
of C3, leading to the formation of C3·H2O. Each pathway leads to the activation of the central complement
protein C3. The C3 convertase of the classical and the lectin pathway is C4b2a, while C3bBb forms the C3
convertase of the alternative pathway. Both C3 convertases cleave C3 to C3a and C3b. C3b on the surface
of a pathogen or dying cell acts as an opsonin. C3b further contributes to the formation of the C5
convertase, which initiates the formation of the membrane attack complex (C5b-9, MAC). The formation of
the MAC on the pathogen surface causes cell lysis. Once C3b is deposited on the surface of pathogens or
dying cells, it binds more factor B, and this binding amplifies the complement activation cascade. This
process is known as the amplification loop. The cleaved protein C5a induces chemotaxis and promotes
inflammation.