The Complement System In MS

The preliminary evidence for the involvement of Complement in promoting primary Humoral Responses dates back over a quarter of a century.

However, it is only in the course of the past decade or so that the detailed mechanisms underlying Complement's influence have been characterized in depth.

It is now clear that Complement serves as a regulator of several B-Cell functions, including specific AntiBody production, Antigen uptake, processing and presentation, and shaping of the B-Cell repertoire.

Of key importance, in this respect, is the role played by the B-Cell-signaling triad consisting of the B-Cell Receptor for Antigen (BCR).

A complex composed of the iC3b/C3d fragment-binding Complement type 2 receptor (CR2, CD21⁺) and its signaling element CD19⁺ and the IgG-binding Receptor FcγRIIb (CD32⁺).

The positive or negative outcome of signaling through this triad is determined by the context in which Antigen is seen, be it alone or in association with natural or induced AntiBodies and/or C3-Complement fragments.

The aim of this review is to describe the present status of our understanding of Complement's participation in Acquired Immunity and the regulation of AutoImmune Responses.

Normal human B-Lymphocytes are known to activate the Alternative Pathway (AP) of Complement, leading to C3-fragment deposition and Membrane Attack Complex (MAC) formation.

The process is mediated via Complement Receptor Type 2 (CR2, CD21), with Complement Receptor Type 1 (CR1, CD35) playing a subsidiary role.

In this study, we examine the relative contributions of CR1 and CR2 to the deposition of C3 fragments and MAC on B-Lymphocytes under circumstances where all Complement Pathways are operational.

C3-fragment deposition and MAC formation were assessed on human Peripheral B-Lymphocytes in the presence of 30% autologous Serum.

Blocking the CR2 Ligand-binding site with MonoClonal AntiBody (mAb) FE8 resulted in significant reduction (37.9+/-11.9%) in C3-fragment deposition, whereas, MAC formation was only marginally affected (12.1+/-22.2% reduction).

Blocking the CR1 binding-site resulted in significant reduction of both C3-fragment deposition (22.0+/-14.5%) and MAC formation (47.4+/-13.8%).

Both the lack of CR2 influence on MAC formation and the promotion of C3-fragment deposition by CR1 are in striking contrast to the situation where only the AP is operational.

The presence of Erythrocytes (E) bearing CR1, however, markedly reduced both C3-fragment deposition and MAC formation.

Our data suggest that C3-fragment deposition and MAC formation on B-Lymphocytes in vivo may involve both AP and Classical Pathway activation, with CR1 contributing significantly to the latter.

On the other hand, the presence of extrinsic CR1, on E, may serve to limit spontaneous MAC formation and thereby ensure cell survival in the circulation.

CD19⁺ is a CoReceptor that amplifies signaling initiated by Antigen cross-linking of the B-Cell Antigen Receptor (BCR). CD19⁺ can also signal independently of BCR CoLigation.

This study shows that B-Cell adaptor for PhosphoInositide 3-kinase (BCAP), previously characterized as a substrate of the Tyrosine Kinases upon BCR engagement, is Phosphorylated by cross-linking of CD19⁺.

Tyrosine Phosphorylation of BCAP, mediated by Lyn, provides binding site(s) for PhosphoInositide 3-Kinase (PI3K), thereby participating in Akt activation.

Thus, these results provide evidence that BCAP serves as an Adaptor molecule for CD19⁺ to activate the PI3K Pathway in B-Cells.

CD19⁺ is a B-Lymphocyte cell surface molecule that functions as a general response regulator or Rheostat.

Which defines intrinsic and B-Cell Antigen Receptor-induced signalling thresholds that are critical for Humoral Immunity and expansion of the Peripheral B-Cell pool.

In addition, B-Cell responses are influenced by signals transduced through a CD19⁺-CD21⁺ Cell Surface Receptor complex.

Where the binding of Complement C3d to CD21⁺ links Humoral Immune Responses with the Innate Immune System.

This review outlines recent BioChemical and genetic studies that characterize the signal transduction pathways utilized by this receptor complex to regulate B-Cell IntraCellular Calcium Responses.

B-Lymphocytes are critically regulated by signals transduced through the CD19⁺-CD21⁺ Cell Surface Receptor complex, where Complement C3d binding to CD21⁺ supplies an already characterized Ligand.

To determine the extent that CD19⁺ function is controlled by Complement activation, CD19⁺-deficient mice (that are HypoResponsive to transmembrane signals) and mice overexpressing CD19⁺ (that are HyperResponsive) were crossed with CD21⁺- and C3-deficient mice.

Cell surface CD19⁺ and CD21⁺ expression were significantly affected by the loss of CD21⁺ and C3 expression, respectively.

Mature B-Cells from CD21⁺-deficient littermates had approximately 36% higher cell surface CD19⁺ expression, whereas CD21⁺/35⁺ expression was increased by approximately 45% on B-Cells from C3-deficient mice.

Negative regulation of CD19⁺ and CD21⁺ expression by CD21⁺ and C3, respectively, may be functionally significant because small increases in cell surface CD19⁺ overexpression can predispose to AutoImmunity.

Otherwise, B-Cell development and function in CD19⁺-deficient and -overexpressing mice were not significantly affected by a simultaneous loss of CD21⁺ expression.

Although CD21⁺-deficient mice were found to express a Hypomorphic cell surface CD21⁺ protein at low levels that associated with mouse CD19⁺.

C3 deficiency did not significantly affect B-Cell development and function in CD19⁺-deficient or -overexpressing mice.

These results, and the severe phenotype exhibited by CD19⁺-deficient mice compared with CD21⁺- or C3-deficient mice, collectively demonstrate that CD19⁺ can regulate B-Cell signaling thresholds independent of CD21⁺ engagement and Complement activation.

The initiation of AntiBody responses to foreign Antigens requires that B-Cells receive and integrate a variety of signals through an array of Cell Surface Receptors including the B-Cell Antigen Receptor (BCR) as well as a number of essential CoReceptors.

Recent evidence indicates that Cholesterol-rich Plasma Membrane MicroDomains, referred to here as Lipid Rafts, serve as platforms for BCR signaling and trafficking in B-Cells.

The existence of rafts suggests a previously unappreciated level of organization at the B-Cell surface that may explain, at least in part, how BCR signaling is coordinated.

Here the current evidence that Lipid Rafts play a key role in B-Cell responses is reviewed.

Copyright 2001 Academic Press.

The CD19⁺/CD21⁺ complex is an essential B-Cell CoReceptor that functions synergistically to enhance signaling through the B-Cell Ag Receptor in response to T-Cell-dependent, Complement-tagged Ags.

In this study, we use a recombinant protein containing three tandemly arranged copies of C3d and the Ag hen egg Lysozyme.

Shown to be a highly effective Immunogen in vivo, to evaluate the role of the CD19⁺/CD21⁺ complex in Ag processing in B-Cells.

Evidence is provided that coengagement of the CD19⁺/CD21⁺ complex results in more rapid and efficient production of Antigenic Peptide/Class II complexes as compared with B-Cell Ag Receptor-mediated processing alone.

The CD19⁺/CD21⁺ complex does not itself target Complement-tagged Ags for processing, but rather appears to influence B-Cell Ag processing through its signaling function.

The ability of the CD19⁺/CD21⁺ complex to augment processing may be an important element of the mechanism by which the CD19⁺/CD21⁺ complex functions to promote B-Cell responses to T-Cell-dependent Complement-tagged Ags in vivo.

The CD19⁺/CD21⁺ complex functions to significantly enhance B-Cell Antigen Receptor (BCR) signaling in response to Complement-tagged Antigens.

Recent studies showed that following Antigen binding the BCR translocates into plasma membrane Lipid Rafts that serve as platforms for BCR signaling.

Here, we show that the binding of Complement-tagged Antigens stimulates the translocation of both the BCR and the CD19⁺/CD21⁺ complex into Lipid Rafts, resulting in prolonged residency in and signaling from the rafts, as compared to BCR cross-linking alone.

When coligated to the BCR, the CD19⁺/CD21⁺ complex retards the internalization and degradation of the BCR.

The colocalization and stabilization of the BCR and the CD19⁺/CD21⁺ complex in plasma membrane Lipid Rafts represents a novel mechanism by which a CoReceptor enhances BCR signaling.

It is becoming well accepted that Innate Immunity serves as a natural adjuvant in enhancing and directing the Adaptive Immune Response.

In this review, I have discussed how the Complement System, a major mediator of Innate Immunity, links the two systems.

The recent availability of knockout mice bearing selective deficiencies in the critical Complement proteins and receptors has allowed formal demonstration of the importance of Complement in enhancement of Humoral Immunity.

Characterization of the mice has also uncovered mechanisms for maintaining survival of activated B-Cells within the Lymphoid compartment.

For example, Co-Ligation of the CD21⁺/CD19⁺/Tapa-1 Receptor with the BCR not only reduces the threshold for B-Cell follicular survival but provides a unique signal for survival in the germinal centers.

In addition Complement Receptors are critical for localization of Antigen and C3d Ligand to FDCs for maintenance of long-term B-Cell memory.

A surprise that has come from analysis of the deficient mice is that Complement is also important in negative selection of B-Lymphocytes.

This observation provides new insight to a long-standing enigma that the major predisposing factor in Lupus is deficiency in Complement C1q or C4.

The seeming contradiction of dual role for Complement in both B-Cell activation and Tolerance is reconciled by the hypothesis that natural IgM provides a mechanism to selectively identify Self-Antigens.

That are highly conserved and cross-react with microbial ones such as DNA and nuclear proteins.

Thus, the importance of Complement in Tolerance to Self-Antigens is restricted to those Self-Antigens that are evolutionary conserved, and they are identified by natural AntiBody.

The future should hold further surprises as to the intricate interactions between the Complement system and Acquired Immunity.

The Membrane Protein Complex CD19⁺/CD21⁺ couples the Innate Immune recognition of Microbial Antigens by the Complement System to the activation of B-Cells.

CD21⁺ binds the C3d fragment of activated C3 that becomes covalently attached to targets of Complement activation, and CD19⁺ co-stimulates signaling through the Antigen receptor, membrane ImmunoGlobulin.

CD21⁺ is also expressed by Follicular Dendritic Cells and mediates the long-term retention of Antigen that is required for the maintenance of Memory B-Cells.

Understanding of the biology of this receptor complex has been enriched by analyzes of genetically modified mice; these analyzes have uncovered roles not only in positive responses to foreign Antigens.

But, also in the development of Tolerance to Self-Antigens. Studies of signal transduction have begun to determine the basis for the CoReceptor activities of CD19⁺.

The integration of Innate and Adaptive Immune recognition at this molecular site on the B-Cell guides the appropriate selection of Antigen by Adaptive Immunity and emphasizes the importance of this CoReceptor Complex.

Before Antigen-specific Immunity arises, the Complement System responds by activation through the Classical and/or Alternative Pathways leading to the covalent deposition of Complement fragments.

Three models, not mutually exclusive, have been proposed to explain how these Complement fragments interact with their Receptors, CD21⁺/CD35⁺, to enhance Humoral Immune Responses:

• CD21⁺/CD35⁺ retain and focus Antigens for optimal presentation
• CD21⁺/CD35⁺ on B-Cells serve as enhancing CoReceptors for B-Cell Antigen Receptor (BCR) signaling
• CD21⁺/CD35⁺ regulate B-Cell responses, by CD19⁺ aggregation

The CoReceptor model led us to predict that CD21⁺/CD35⁺ may lower the threshold of BCR affinity to diversify the repertoire of Humoral Immune Responses.

But surprisingly, CD21⁺/CD35⁺-deficient mice expressing a transgenic BCR with very low affinity (Kalpha approximately =1.2 x 10(5) M(-1)) for the (4-Hydroxy-3-Nitrophenyl) Acetyl Hapten generated significant AntiBody and germinal center responses to even low doses of Antigens in alum.

The magnitudes of these responses were much below those of normal controls but higher doses of Antigens substantially rectified these deficits.

Thus, while CD21⁺/CD35⁺ play important roles in Humoral Immunity, our observations provide little support to the hypothesis that CD21⁺/CD35⁺ promote Clonal diversity in Immune Responses by helping recruit low-affinity B-Cells.

Studies over the past three decades have clearly established a central role for Complement in the promotion of a Humoral Immune Response.

The primary function of Complement, in this regard, is to Opsonize Antigen or Immune Complexes for uptake by Complement Receptor Type 2 (CR2, CD21⁺) expressed on B-Cells, Follicular Dendritic Cells (FDC) and some T-Cells.

A variety of mechanisms appear to be involved in Complement-mediated promotion of the Humoral Response. These include:

1. Enhancement of Antigen (Ag) uptake and processing by both Ag-specific and non-specific B-Cells for presentation to specific T-Cells
2. The activation of a CD21⁺/CD19⁺ complex-mediated signalling pathway in B-Cells
   • which provides a stimulus synergistic to that induced by Antigen interaction with the B-Cell Receptor (BCR)
3. Promotion of the interaction between B-Cells and Dendritic Cells (FDC)
   • where C3d-bearing Immune Complexes participate in InterCellular bridging
4. Current studies suggest that CR2 may also play a role in the determination of B-Cell Tolerance towards Self-Antigens.

And, thereby hold the key to the previously observed correlation between deficiencies of the early Complement components and AutoImmune Disease.

Whereas Complement System was usually considered as a member of Innate defence, one of its components (C3) is now thought to facilitate Acquired Immunity.

This role is due first to its capacity to covalently bind to Antigens and secondly to the interactions of its ProteoLytic fragments with different receptors expressed on most cells involved in the Acquired Immune Response.

After activation in the Plasma, C3 is ProteoLysed in fragments which possess various biological activities, as a modification in cell activities occurred after binding to Cell Surface Receptors.

Injection of low amount of Antigen results in a modified Immune Response in C3 deficient animals with a decrease in the level of specific AntiBodies and an absence of IgM/IgG switch.

One of the fragments of C3 (C3d) and its Receptor (CR2) seem particularly important : knock out animals in C3 or CR2 have similar phenotypes.

Mice with a deficit in CR2 restricted to the B-Lymphocytes present a strong reduction in the number and the size of germinal centers. Moreover, expression of CR2 on Follicular Dendritic Cells is necessary for the generation of a strong Memory Response.

C3 is also involved in the control of the IntraCellular processing of the Antigen as the use of covalent complex (C3b-Antigen) instead of free Antigen increases the amount of stable MHC Class II molecules at the Antigen-Presenting Cells surface.

In summary, C3 fragments increase cell to cell interactions, induce IntraCellular signalling after binding to their Receptors and increases IntraCellular processing of Antigens.

A better knowledge of the different roles of C3 may be useful to modify the Immune Response and to promote the Immune Memory, a domain where C3 seems particularly important.

Complement Receptor 2 (CD21⁺, CR2) is a B-Cell Receptor for Complement degradation products bound to Ag or Immune Complexes. The role of CD21⁺ in mediating Ag presentation of soluble Immune Complexes by resting B-Cells was studied.

Complement-coated Immune Complexes were formed by the incubation of Influenza Virus with Serum from Immune donors.

These complexes bound to peripheral blood B-Cells in a Complement-dependent manner. The binding required CD21⁺ or, to a lesser extent, Complement Receptor 1 (CR1, CD35⁺).

B-Cells pulsed with Immune Complexes containing Complement elicited a response from a panel of influenza-specific T-Cell clones, while those pulsed with Immune Complexes formed in the absence of Complement did not.

The expression of the early activation marker CD69⁺ and the CoStimulatory Molecule CD86⁺ were not induced by CD21⁺ Ligation alone, suggesting that CD21⁺-mediated Ag presentation occurs independently of B-Cell activation.

Up-regulation of these markers required exposure to T-Cell factors elicited by the recognition of Ag derived from Complement-containing Immune Complexes.

These findings suggest that binding of Ag to CD21⁺ enables Ag-nonspecific B-Cells to participate in the activation of Ag-specific T-Cells in a process that occurs independently of well-characterized B-Cell activation events.