Phenotypes of complement knockouts

lmmunopharmaco y ELSEVIER

Immunopharmacology 49 (2000) 125-131 www.elsevier.com/locate/immpharm

Review

Phenotypes of complement knockouts V. Michael Holers Diuision

*

of Rheumatology, Box B-115, UniuersiQ of Colorado Health Sciences Center, 4200 East Ninth Auenue, Denuer. CO 80262, USA Accepted 2 1 March 2000

Abstract Although complete and partial complement deficiencies are well described in humans and several spontaneous animal models, many questions have remained regarding the exact role that these deficiency states play in the observed clinical manifestations. Likewise, many important mechanistic questions cannot be addressed using patients or spontaneously arising animal models of deficiency states. To provide additional insights and create readily manipulable experimental systems, over the last 5 years mice have been created by several groups in which specifically targeted insertional mutagenesis has resulted in complete deficiencies of complement activation proteins, receptors or regulatory proteins. Many surprising findings have already been made using mice derived from these strategies, and clinically relevant studies have begun to provide great insights into human deficiency states. This review includes an overview of these complement deficient mice and highlights some of the important findings that have resulted from their creation. A discussion of future experimental directions thought to be important by this author then follows and concludes the review. 0 2000 Elsevier Science B.V. All rights reserved. Keywords:

Deficiency

state; Receptor;

Regulatory

protein

1. Introduction Complete or partial deficiency states have been described in humans for almost all complement activation pathway components as well as several receptors and many of the regulatory proteins (reviewed in Figueroa and Densen, 1991). Strains of animals from various species have also been previously identified and studied that manifest complete or essentially complement deficiencies. These include mice with C5 deficiency, guinea pigs with C2 and C4 deficiencies, dogs with C3 deficiency, rats and rabbits with C6 deficiency, and pigs with factor H deficiency, among others. Significant acquired deficiencies of

* Tel.: +I-303-315-7592;

fax: +l-303-315-5540, E-mail address: [email protected] (V.M. Holers).

0162.3109/00/$ PII: SO162-3

complement

activation

components

are well

known

to occur during systemic activation states (Dalmasso, 1986), and as well markedly low levels of complement receptors 1 and 2 (CR1 and CR21 are found in patients with systemic lupus erythematosus (SLE) (Wilson et al., 19861, likely through an acquired disease-related mechanism (Walport and Lachmann, 1988). Analysis of these deficiency states has led to the understanding that the complement system is important not only in the protection from infection and immune complex disease manifestations but also from defects in tolerance as manifested by the development of autoantibodies (Atkinson, 1989). However, despite a wealth of information gleaned from a careful analysis of these individuals and animal models, substantial questions have remained regarding

- see front matter 0 2000 Elsevier Science B.V. All rights reserved. 109(00)00209-5

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the specific mechanisms that underlie the particular clinical manifestation seen and whether the observed phenotype is due to complement deficiency itself or is secondary to other linked genes or conditions such as coincident infection. To address these issues, over the last 5 years several investigators have created mice with specifically targeted mutations that have eliminated expression of complement activation and regulatory proteins as well as complement receptors. This article will summarize this effort. Because of space limitations, it will not be a comprehensive review, and the author apologizes in advance for any oversights. It is also expected that many new and exciting results relative to this area will be presented at the XVIII ICW in Snowbird, Utah and thus will be included in the abstract section of this edition.

2. Results and discussion Studies will be reviewed not by their precedence in publication but rather in the general order of the complement activation pathway itself, followed then by complement receptors and concluding with regulatory proteins. 2.1. Serum amyloid P (SAP) SAP is a serum protein and member of the pentraxin family of proteins one of whose major roles is to bind DNA and chromatin as well as the surface blebs of apoptotic cells (Gewurz et al., 1996). Recently, mice deficient in SAP have been created (Bickerstaff et al., 1999). These mice have been particularly instructive in that they spontaneously develop anti-nuclear antibodies and severe glomerulonephritis in the absence of another strong genetic predisposition to autoimmune disease. These results suggest that complement activation through SAP, or SAP receptor mediated binding and clearance of chromatic:DNA containing complexes, is important in diminishing the load of potentially immunogenic apoptotic material, thus protecting from the development of autoimmunity. 2.2. Clq C lq-deficient mice have been particularly instructive because they appear to be very representative of

49 (2000) 125-131

the human complement deficiency of Cl components that is statistically the most tightly linked to the development of autoimmunity and immune complex disease (Bowness et al., 1994). These mice develop spontaneously enhanced humoral autoimmunity and glomerulonephritis that demonstrates strain dependence (Botto et al., 1998). Apparent abnormalities of the clearance of apoptotic bodies in the kidneys are also found. These results have suggested that the Clq-mediated binding of apoptotic bodies previously described (Korb and Ahearn, 1997) is likely to be important in clearance of this potentially immunogenic material. A further analysis has demonstrated that the enhanced autoimmunity and glomerulonephritis is independent of components at or downstream of factor B and C2 of the alternative and classical pathways (Mitchell et al., 1999), an important finding because patients with later complement deficiencies also manifest SLE-like disease (Amett, 1992). These particular results also suggest that autoimmune disease associated with each complement deficiency state may be the result of unique rather than common mechanisms.

2.3. Factor B

Factor-B-deficient mice were particularly important to create because no complete factor B deficiency has been described in humans, leading to the interpretation that this state was likely to be lethal. Surprisingly, however, these mice were viable and, while manifesting absent alternative pathway activation and decreased classical pathway activation through the amplification loop, no abnormalities of the immune system were detected (Matsumoto et al., 1997). Further analysis, though, by backcrossing these mice onto the MRL/Zpr model of SLE revealed that the lack of factor B lessened the vasculitis, glomerular disease, C3 consumption and IgG3 RF levels typically found in this model without altering levels of other autoantibodies (Watanabe et al., 2000). Thus, factor B has an unexpected role in autoimmune disease manifestations, either through the amplification loop, as yet unknown factor B receptors or IgA-mediated activation of the altemative pathway by pathogenic autoantibodies.

V.M. Holers/ Immunopharmacology 49 (2000) 12.5-131

2.4. Factor B/ C2 Dual factor B- and C2-deficient mice were created during efforts to create a specific factor B deficiency (Taylor et al., 1998). Unlike factor-B-deficient mice described above (Matsumoto et al., 1997) though, targeting of the factor B gene in this instance resulted in the lack of expression of both proteins, presumably because of the tight linkage of the two genes. This is not an unusual finding using insertional mutagenesis techniques and results from the regional effects of foreign DNA. These mice have been used, though, to demonstrate abnormalities of immune complex processing of a similar nature as those found in humans. 2.5. c3 Two groups have created C3-deficient mice (Wessels et al., 1995; Circolo et al., 1999). These mice have been used to demonstrate an important role for C3 in the clearance of group B streptococcal infection (Wessels et al., 1995) and in the generation of a normal T-dependent humoral immune response (Fischer et al., 1996). These mice have also been used to determine the relative complement dependence of various in vivo phenotypes such as endotoxic shock (Fischer et al., 1997) immune complex induced glomerulonephritis (Quigg et al., 1998a) and herpes virus infections (Da Costa et al., 1999). One of the most novel and striking findings made using these mice was in bone marrow transfer experiments, where one can transfer C3-producing bone marrow cells into a C3-deficient mouse, where it was found that locally generated and not serum C3 is primarily important in the amplification of humoral immunity (Fischer et al., 1998a). Currently these mice are being backcrossed in many model systems and it is anticipated that a substantial number of new insights will be generated from these ongoing studies. 2.6. C4 C4-deficient mice have also been created (Wessels et al., 1995). Because of the presence of only a single clearly functional C4 gene in mice (whether SLP has complement activity is in dispute) rather than the two found in humans (C4A and C4B), these

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targeted mice exhibit no classical pathway activity. C4-deficient mice exhibit defective clearance of group B streptococcal bacteria (Wessels et al., 1995). One of the most clinically relevant studies with these mice has addressed the mechanism of the observed increase in autoimmune disease in complete or partial C4 and C4A deficiency in humans (Arnett, 1992). In this study C4-deficient mice bred onto the autoimmune prone B6/lpr strain were reported to manifest strikingly accelerated autoimmune disease that was associated with apparent defects in B cell central tolerance utilizing the anti-HEL transgenic model (Prodeus et al., 1999). 2.7. CR1 / CR2 As opposed to humans where CR1 and CR2 are the products of separate genes and are more widely expressed, mouse CR1 and CR2 are alternatively spliced products of a single gene designated Cr2 and are expressed almost exclusively on B cells and follicular dendritic cells (FDC) (Molina et al., 1990; Kinoshita et al., 1990; Kurtz et al., 1990). Two groups have created Cr2 - / - mice and both have demonstrated substantial defects in T-dependent immune responses in these mice (Ahearn et al., 1996; Molina et al., 1996). In one (Aheam et al., 1996) but not the other report (Molina et al., 1996) Bl cells were decreased in number. Both B cell and FDC CR2 and CR1 have been found to be required for normal humoral responses (Fang et al., 1998). Germinal center development is also impaired due likely due to diminished survival signals (Fischer et al., 1998b) and perhaps also to insufficient T-cell help arising because of diminished B7 costimulation by B cells (Kozono et al., 1998). The biochemical mechanism of the defective B-cell response is likely related to the association of CR2 with CD19 (Fearon and Carter, 1995: Tedder et al., 1994). Cr2 - / - mice have also been bred to B6/lpr and anti-HEL transgenie mice and demonstrated a similarly enhanced autoimmune phenotype as C4-deficient mice (Prodeus et al., 1999). Despite these initial results in early crosses, though, it should be noted that analysis of the phenotype of extensively backcrossed Cr2 /B6/lpr mice has demonstrated a much less robust autoimmune phenotype (V.M. Holers and H. Molina, XVII ICW abstract, and H. Molina, personal

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communication). This result suggests that modifying genes found in early Sv129 X B6 crosses may amplify the apparent effects of the lack of CR2 and CRl. Ongoing studies are working to resolve this issue, which is a well-described confounding effect of the use of gene targeting strategies in one strain that is then followed by breeding the targeted allele into another. This is an issue in all knockouts, including those made in the complement field. 2.8. C5a receptor (CSaR) CSaRs have been found to be much more widely expressed than previously believed and are found, for instance, on hepatocytes and help to regulate the acute phase response (Wetsel, 1995). CSaR-deficient mice have been created and show substantial defects in the clearance of Pseudomonas, an important pulmonary pathogen (Hopken et al., 1996). In addition, CSaR-deficient mice demonstrate attenuated intraperitoneal and intradermal reverse passive Arthus reactions that are accompanied by decreased inflammatory cell infiltrates and TNF-o expression (Hopken et al, 1997).

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described. Despite the sequence similarity to CRl, the multiple inhibitory activities and wide distribution of Crry in rodents as compared to the narrow distribution of DAF-TM and especially MCP in mice has led, though, to the hypothesis that Crry plays a similar functional role in rodents as DAF and MCP in humans. &y-deficient mice have been created and have demonstrated a profound loss of fetal viability that begins relatively early during gestation and is associated with C3 deposition and an inflammatory infiltrate (Xu et al., 2000). While normal numbers of Crry - / - embryos are found initially in utero, no live Crry - / - pups are found, and time course analysis demonstrates a progressive decline in numbers of Crry - / - embryos in utero. Importantly, when bred back to C3 - / - mice, normal numbers of live Crry - / - pups are born, thus confirming that it is complement activation at or beyond the step of C3 that is mediating this phenotype. These results have substantial importance and clearly support the critical in vivo role of endogenous membrane complement inhibitors, especially during fetal development where alloantibodies and immune responses to the semi-allogeneic fetus are routinely found.

2.9. DAF In mice two DAF genes designated Daf-tm and Daf-gpi are found instead of the one found in humans (Spicer et al., 1995). Each appears to mediate complement inhibition, but the tissue distribution of DAF-GPI is much broader than DAF-TM. Mice specifically deficient in DAF-GPI have been created (Sun et al., 1999). In the unmanipulated state, there is no obvious phenotype in these mice. However, they do manifest increased sensitivity to hemolysis when cells are treated with antibody and complement or are subjected to reactive lysis conditions in vitro. 2.10. Crry Crry is a widely distributed potent membrane inhibitor of complement activation of both the classical and alternative pathways and exhibits both decay-accelerating function and cofactor activity for C3 and C4 cleavage (Molina et al., 1992; Kim et al., 1995). No human Crry structural homologue with closer sequence similarity than CR1 has yet been

3. Future directions The generation and analysis of complement knockout mice will continue to increase in scientific and clinical importance. Clearly early studies have led to many surprises but have also continued to support a critical role for the complement system in many aspects of immunity, tolerance and inflammatory injury. Certainly additional complement genes have been or will be inactivated, and there will be particular interest in the generation and analysis of mice deficient in later MAC components and CD59. Mice deficient in factor D and factor I as well as the regulators MCP, Cl-INH, factor H and C4-binding protein will also be of interest given the many roles these proteins have been proposed to fill. This author is particularly interested in the analysis of the roles of complement in chronic inflammatory and autoimmune diseases, and given the wealth of relevant models available in mice, years of work lie ahead to decipher which components are critically important in these states. This task is especially

V&l. Holers /Immunophatmacology

important given the increasing availability of complement inhibitors, such as a humanized inhibitory anti-C5 monoclonal antibody, that are in Phase II clinical trials. It is also anticipated that many diseases now stated to be “T-cell dependent” will manifest a clear complement dependence. One such recent example has been provided by the observation that overexpression of soluble Crry in the brain blocks the development of peptide-induced experimental autoimmune encephalomyelitis (EAE) (Davoust et al., 1999). Additional studies are underway in models of SLE, Type I diabetes (insulin-dependent diabetes mellitus, or juvenile diabetes), autoimmune vasculopathies, and many others. This list does not even consider the use of deficient mice to decipher the role of specific complement components in the pathogenesis of acute disease states such as ischemia-reperfusion and thermal injury. In addition to these strategies, the use of tissues and cells from knockout mice in transplantation settings will allow one to discern whether local or systemic effects underlie the phenotypic effects that are noted. This strategy has already provided important insights into the role of local C3 production in humoral immune responses and B cell versus FDC expression of CR2 and CR1 . Appropriate crosses with mice overexpressing complement inhibitors such as soluble Crry (Quigg et al., 1998b) will also allow the determination of the relative effects of soluble versus membrane regulatory proteins. Another area of particular importance will be the comparison of results using complement-deficient mice to those using species compatible complement inhibitors such as Crry-Ig (Quigg et al. 1998~). The relative value of knockout mice in general has been questioned by some because development of the immune system in the absence of a specific protein can potentially result in the generation of confounding and unanticipated compensatory mechanisms and, additionally, does not exactly mimic the clinical state when using an inhibitor. This concern may be of importance in the complement system as there is emerging evidence of changes in specific serum complement components when levels of inhibitors are changed using transgenic means (V.M. Holers and H.J. Kang, unpublished). This issue is one that should be critically addressed in both murine studies and clinical trials in patients.

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Additional insights will be gained by “humanizing” knockout mice by expressing human proteins using transgenic or knockin technologies. In this setting, one can utilize these mice as preclinical models to test reagents that specifically block human but not mouse complement components or receptors. This strategy should work in many instances, and our initial experience with reconstitution of Cr2 - / mice with human CR2 which results in a functional reconstitution of humoral immunity provides strong support for this strategy (K. Marchbank and V.M. Holers, submitted for publication). Finally, as in other immune systems the generation and analysis of dual complement component knockouts will address whether additional effects can be discovered when potentially parallel functional pathways are blocked. In sum, the use of contemporary knockout technologies has helped to revolutionize and re-energize the field by showing substantial in vivo effects of eliminating complement expression in an experimental setting that is directly comparable to other important immunoregulatory systems. The future of these efforts in the complement field is bright, and we look forward to the many new insights that will be generated.

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