CHRISTOPHER C. GOODNOW AUTOIMMUNITY

Safe havens for self-reactive cells Transgenic and mutant mice are providing new insights

into the steps leading to the breakdown of immunological tolerance to self antigens and ultimately to autoimmune disease.

Our immune systems normally tolerate the unique con- stellation of antigens that are expresse d on our own tis- sues, and specifically avoid producing antibodies against them. As first noted by Paul Ehrlich at the turn of the century, one striking example of this is the absence of an- tibodies directed to our own red blood cells despite the frequent presence of antibodies to red cells from indi- viduals with differing blood types. Studies in recent years have revealed a good deal of how self-tolerance comes about, outlining the array of mechanisms designed either to kill off clones of self-reactive lymphocytes or to prevent them from actively participating in an immune response. Despite these layers of control, break down of self-toler- ance to one or more tissue antigens nevertheless occurs in a significant fraction of people at some point in their lives. Production of autoantibodies to self red blood cells, in the human disease autoimmune hemolytic anemia, was the first identified example of such an autoimmune dis- ease. New results with a mouse model of autoimmune hemolytic anemia [1,2] highlight some of the steps in B- cell development where self-tolerance can break down leading to autoimmune disease (Fig. 1).

The latest addition to the hemolytic anemia story has its roots in mouse genetics some thirty years ago, with

the identification of a mouse strain, New Zealand Black (NZB), which develops autoimmune hemolytic anemia between 6 and 12 months of age (mice normally live for 1-2 years, so this is roughly equivalent to a 30--50 year old person). The NZB propensity for autoimmune disease has since been extensively studied, but has nev- ertheless proved ditficult to dissect. Inheritance of the au- toimmune predisposition is multigenic, and because the disease takes a good deal of time to become apparent and presents to varying degrees, classical genetic linkage analysis has not yet been successful. Studies have demon- strated that cells of hemopoietic origin are sufficient to transfer autoimmune potential to normal mice and ab- normalities in hemopoietic cells, macrophages, T cells (which help to regulate antibody responses) and B cells (which produce antibodies) have all been described. It has not been possible, however, to resolve which cellu- lar abnormalities are a cause and which a consequence of the autoimmune syndrome.

To start to unravel the sequence of events leading to autoimmunity in NZB mice, Okamoto et al. [1] have started with the B cells that produce the erythrocyte- destroying autoantibodies themselves. Immortalized hy- bridoma clones of these B cells have previously been

Bone Marrow

Pre-B cells

Newly-formed ~ B cell

Dying cell

Germinal center B

cells 1

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3

Mature B cell I

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2

Periphery

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Fig. 1. Steps in the development of an autoantibody-secreting clone. 1, generation of autoreactive B-cell clones, which normally occurs by chance as a result of antibody gene rearrangements in the bone marrow or during hypermutation of antibody genes in germinal center reactions. 2, survival of self-reactive B-cell clones. 3, clonal expansion. 4, differentiation into antibody-secreting cells. The latter three steps are normally opposed by continuous binding of self antigens to antigen receptors on the B cell.

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isolated from anemic NZB mice [3], and genes encod- ing the heavy and light chains of one particularly de- structive autoantibody had been c loned from one cell line. The group then set out, armed with the autoan- tibody genes, to retrace the steps leading to the devel- opment of this particular 'forbidden clone' of antibody- secreting cells. As these steps are normally hidden in the long latent period and probably involve chance events affecting rare clones of antigen-specific B cells, Okamoto et al, reasoned that it would be easier to detect some of these steps if the frequency of B calls with the forbidden specificity was elevated. To do this, the group capitalized on previous work showing that functionally-rearranged immunoglobulin genes, when in- troduced into the germline of transgenic mice, pre- vent other immunoglobulin genes from rearranging in most developing B cells. As a result, the repertoire of B cells in immunoglobufin-gene transgenic mice con- sists predominantly of cells expressing the transgene-en- coded antibodies, making it straightforward to follow the development and fate of antigen-specific cells.

Following the transgenic mouse approach, Okamoto et a/. introduced the NZB anti-erythrocyte heavy chain and light chain genes separately into the germline of inbred mice, using a homogeneous strain backgrotmd lack- ing the NZB propensity for autoimmunity. Mice carry- ing the heavy chain and light chain transgenes were then cross-bred with one another to yield offspring carrying both immunoglobulin genes and therefore ca- pable of assembling the complete anti-erythrocyte au- toantibody. In the resulting animals, most of the self- reactive B cells developing in the bone marrow are exposed to self red blood cells and are triggered to die. This induced cell death reflects a tolerance mech- anism that normally censors B cells with antigen re- ceptors that bind to membrane-bound self antigens in the bone marrow microenvironment. The same pro- cess has previously been shown to censor B cells re- acting with other membrane-bound self antigens, such as histocompatibility antigens [4] and a membrane- bound form of lysozyme [5], in similar immunoglobulin transgenic mouse models.

In the case of the anti-erythrocyte transgenic mice, how- ever, elimination of developing B cells in the bone mar- row appears to be incomplete and a few percent of the self-reactive B cells escape from the bone marrow and reach the spleen and other lymphoid organs [1]. In the spleen, the escaping anti-erythrocyte B cells never- theless continue to be kept in check by another toler- ance mechanism - - referred to variously as 'clonal an- ergy' or 'functional silencing' - - which renders the B cells unable to mount an efficient antibody response. Silencing of self-reactive B cells also accounts for self- tolerance in other transgenic mouse models, in B cells reacting with a soluble form of lysozyme [6] and ap- parently in B cells that bind to single-stranded DNA [7]. The silencing mechanism acting on the anti-ery- throcyte B cells and on anti-soluble lysozyme B cells appears identical; in both cases the silenced B cells show greatly reduced IgM antigen-receptor expression

on their cell surface [1,6] and they make very little secreted antibody in response to the bacterial mito- gen lipopolysaccharide [1,8]. The important functional distinction between antigen-induced death of self-reactive B cells and functional silencing of self-reactive B cells is that the latter is potentially reversible. In transgenic mice expressing soluble lysozyme, for example, silenced B cells recover the capacity to mount an antibody response if they are removed from constant exposure to self anti- gen and triggered to proliferate through antigen receptor- independent pathways [8].

Taken together, the initial findings of the Honjo group show that the forbidden clone of anti-erythrocyte B cells is heavily censored by the same tolerance mecha- nisms that control other self-reactive B cells. However, in contrast to the findings with transgenic mice expressing antibody genes specific for ubiquitously expressed anti- gens, such as histocompatibility antigens [4], membrane- bound lysozyme [5], secreted lysozyme [6], or DNA [7], Okamoto et al. find that tolerance spontaneously breaks down at a young age in a large fraction of the anti-erythrocyte transgenic mice [1]. The cause of this has now been determined to a first approximation, and provides an important clue to the pathogenesis of the disease in NZB mice themselves. As described in a re- cent paper, Murakami et a l [2] have found that a criti- cal loophole, in the system of controls, is the presence of lymphoid microenvironments where anti-erythrocyte B cells are shielded from erythrocyte antigens. In the anti-erythrocyte transgenic mice, small numbers of self- reactive B cells manage to avoid B-cell elimination and colonize the peritoneal cavity, where they build up in considerable numbers by the time the mice are eight weeks old.

There are two important components to the build up of self-reactive B cells in the peritoneal cavity. Firstly, the peritoneal microenvironment shields the autoreac- tive cells from specific erythrocyte antigens, protecting them from counterbalancing mechanisms which would otherwise trigger their death. Murakami et al. demon- strate this spectacularly by deliberately introducing ery- throcytes into the peritoneal cavity of the transgenic mice and showing that this immediately triggers a wave of programmed cell death in the anti-erythrocyte B cells. The second factor lies in a poorly understood aspect of the peritoneal microenvironment. Previous work has es- tablished that clones of B cells with particular antibody variable (V) regions are preferentially expanded in the peritoneal and pleural cavities of the mouse [9]. Al- though much interest rests on whether these B cells constitute a distinct lymphoid lineage (the Lyl or B1- lineage), the most salient point is that clonal expan- sion of these B cells is clearly independent of helper T cells and may be independent of antigenic stimula- tion, in contrast to proliferation of conventional B cells in the germinal centers of spleen and lymph node. The anti-erythrocyte B cells are clearly capable of participat- ing in this proliferation reaction, and this presumably ac- counts for their accumulation. One speculative possibility is that the anti-erythrocyte B cells recognize carbohydrate

418 © 1992 Current Biology

epitopes on erythrocyte lipids [10], and are driven to pro- liferate in the peritoneum by crossreactivity with bacterial lipopolysaccharides.

The Murakami et al. [2] study also points to a third el- ement contributing to the escape of forbidden clones of antibody-secreting cells. Although all of the anti-ery- throcyte transgenic mice carry expanded populations of self-reactive B cells in the peritoneal cavity, this alone appears insufficient for the development of hemolytic anemia. In the animals where tolerance actually breaks down, an unknown additional event triggers some of the peritoneal B cells to differentiate into antibody-secreting cells. Acute exposure to higher concentrations of bac- terial mitogens such as lipopolysaccharide can be one such trigger, as the group has shown experimentally [1]. Again, shielding from autoantigens is likely to be an important contributing factor, as the autoantibody- secreting cells fail to develop in erythrocyte-exposed sites such as the spleen [1,2], and continued self-anti- gen exposure has been found to prevent differentiation of lipopolysaccharide-stimulated self-reactive B cells in other situations [8].

Does shielding of self-reactive peritoneal B cells explain the propensity of NZB mice to autoimmune disease, or indeed all autoimmunity? Obviously, as Murakami et al. are quick to point out, there are many events in the NZB mouse which will have been sidestepped in the antibody- transgenic animals. Moreover, entirely different routes may also lead to autoimmunity. What is most impor- tam, however, is that the Murakami et al. study high- lights four basic steps that are likely to be of general significance in autoimmunity, clone generation, survival, growth and differentiation (Fig. 1). In the anti-erythrocyte transgenic mice, the first of these steps is bypassed, as an- toantibody transgenes are already rearranged and appear also to have undergone hypermutation in germinal cen- ters [11]. In the NZB mouse and other natural examples of autoimmunity, by contrast, appearance of self-reactive B cells may depend on inheritance of particular antibody Vregion genes or on factors that promote germinal cen- ter hypermutation, such as chronic viral infection or the break down of tolerance in T cells.

Survival of self-reactive B-cell clones (step 2, Fig. 1) can be promoted by shielding from self antigens, as shown by Murakami et al., or by the concurrent presence of signals from the microenvironment, such as T cells or follicular dendritic cells. Genetic defects may also favour survival of inappropriate B-cell clones and promote autoimmu- nity, by disrupting the function of cell-death pathways, such as apoptosis stimulated by the Fas receptor ([12], discussed in last month's Current Biology [13] ), or by ac- tivating the function of pro-survival pathways, as results from inappropriate expression of the Bcl2 gene [ 14].

Clonal expansion (step 3) and differentiation (step 4) into antibody-secreting cells can, in theory, be favoured by any circumstance which shifts the balance between growth and differentiation signals from microenviron- ments, from bacteria or from helper T cells, and an- tagonistic signals transmitted by self antigens through the B-cell antigen receptor. Environmental factors such

as those operating in the peritoneum of the anti-erythro- cyte immunoglobulin-transgenic mice, or genetic events comparable to those underlying neoplasia, may both play a part in shifting the balance. As with neoplasia, the ap- proach of identifying and using important genes to create animal models where other genetic or environmental fac- tors can be visualized is likely to be a particularly fruitful one.

References 1. OKAMOTO M, MURAKAMI M, SHtMIZU A, OZAKI S, TSUBATA T,

KUMAGAI S-I, HONJO T: A transgenic model of au to immune hemolytic anemia. J Exp Med 1991, 175:71-79.

2. MURAKAMI M, TSUBATA T, OKAMOTO M, SH1MIZU A, KUMAGAI S, IMURA H, HONJO T: Ant igen-induced apoptotic dea th of Ly- 1 B cells responsible for au to immune disease in transgenic mice. Nature 1992, 357:77-80.

3. SHIBATA T, BERNE'{ T, REININGER L, CHICHEPORTICHE Y, OZAKI S, SHIRAI T, IzLrl S: Monoclonal anti-erythrocyte autoantibodies derived from NZB mice cause hemolytic anemia by two dis- t inct pathogenic mechanisms. Int Immuno11990, 2:1133-1141.

4. NEMAZEE D, Bt3P4a K: Clonal delet ion of B lymphocytes in a t ransgenic mouse bearing anti-MHC class I antibody genes. Nature 1989, 357:562~66.

5. HARTLEY SB, CROSBIE J, BRINK RA, KANTOR AB, BASTEN A, GOODNOW CC: Elimination from peripheral lymphoid t issues of self-reactive B lymphocytes recognizing membrane-bound antigens. Nature 1991, 353:765-769.

6. GOODNOW CC, CROSBIE J, ADELSTE1N S, LAVOIE TB, SMITH-GILL SJ, BmNK RA, PRITCHARO-BmSCOE H, WOTHERSPOON JS, LOBLAY RH, RAPHAEL K, TRENT RJ, BASTEN Az Altered immunoglobul in express ion and functional silencing of self-reactive B lym- phocytes in transgenic mice. Nature 1988, 334:6764582.

7. ERIKSON J, RADIC MZ, CAMPER SA~ HARDY RR, CARMACK C, WEIGERT M: Expression of anti-DNA immunoglobul in trans- genes in non-au to immune mice. Nature 1991, 349:331-334.

8. GOODNOW CC, BRINK RA, ADAMS E: Break down of self-toler- ance in anergic B lymphocytes. Nature 1991, 352:532-536.

9. HARDY RR: Variable gene usage, physiology and develop- m e n t of Ly-1 + (CD5 + ) B cells. Curr Opin Immunol 1992, 4:181-185.

10. NOGUCH1 M, IWAMOPa M, HmANO T, HASHIMOTO H, HIROSE S, SHIRAI T, NAGAI Y: Preferential reactivity of autoantibodies in mur ine lupus NZB mice to neuraminidase- treated monosialo- gangliosides on B cells of mouse spleen. Cell Immunol 1991, 135:184-195.

11. REININGER L, SHIBATA T, OZAKI S, SHIRAI T, JATON JC, IZUI S: Variable-region sequences of pathogenic ant i-mouse red blood cell autoantibodies f rom au to immune NZB mice. Eur J Immunol 1990, 20:771~77.

12. WATANABE-FUKUNAGA R, BRANNAN CI, COPELAND NG, JENKINS NA~ NAGATA S: Lymphoproliferation disorder in mice explained by defects in Fas antigen that mediates apoptosis. Nature 1992, 356:314-317.

13. KRAMMER PH, DEBATIN K-M: W h e n apoptosis fails. Curr Biol 1992, 2:383-385.

14. STRASSER A, WHITI1NGHAM S, VAUX DL, BATH ML, ADAMS JM, CORY S, HARRIS AW: Enforced Bcl2 express ion in B-lymphoid cells prolongs antibody responses and elicits au to immune disease. Proc Natl Acad Sci USA 1991, 88:8661-8665.

Christopher C. Goodnow, Howard Hughes Medical Insti- tute and Department of Microbiology and Immunology, Stanford University, Stanford, California 94305, USA.

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