Biological properties of interleukin 10

Journal o f Clinical Immunology, Vol. 12, No. 4, 1992

Special Article

Biological Properties of Interleukin 10

MAUREEN HOWARD,I'2 ANNE O'GARRA,1 HIROSHI ISHIDA,1 RENt~ DE WAAL MALEFYT, I and JAN DE VRIES 1

Accepted: March 10, 1992

INTRODUCTION

Intefleukin I0 (IL-10) was discovered in 1989 by Mosmann and colleagues as an activity produced by murine type 2 helper T cells which suppressed cytokine production by type 1 helper T cells (1). This activity, initially designated cytokine synthesis inhibitory factor (CSIF), was isolated by expression cloning by Moore and colleagues (2), who subsequently identified and isolated a human analogue of CSIF by cross hybridization (3), using as their source a human helper T-cell clone produced by Roncarolo et al. (M.-G. Roncarolo and H. Spits, unpublished). While a search of the available se- quence data banks indicated that CSIF represented a novel cytokine, this search also revealed a re- markable homology between CSIF and a previously uncharacterized open reading frame, termed BCRF~, in the Epstein-Barr virus (EBV) genome (2-4). After further analysis of CSIF genomic Struc- ture, Moore and colleagues proposed the intriguing possibility that at some stage, EBV may have captured this mammalian gene, presumably to con- fer some survival advantage on itself.

The availability of recombinant CSIF, and neutralizing monoclonal antibodies that recognized it specifically (5), rapidly led to the discovery that this new cytokine accounted for two other novel activ-

1DNAX Research Institute, 901 California Avenue, Palo Alto, California 94304.

2To whom correspondence should be addressed.

ities under investigation at DNAX: (i) B-TCGF, a B cell-derived growth costimulator for thymocytes (6, 7); and (ii) MCGFIII, a B cell-derived growth costimulator for mast cells that could be distinguished from IL-3 and IL-4, the first two cytokines known to exhibit mast-cell stimulating function (8, 9). The realization that CSIF was a pleiotropic cytokine exerting effects on a diversity of cell types led to its renaming as interleukin I0 and initiated Studies which demonstrated that a number of immunostimulatory and immunosuppressive properties could be attributed to this cytokine (6, 7, 9-16). In addition to a diversity of cell types that respond to IL-10, it is now apparent that interleukin 10 can be produced by B cells (8, IT) and monocyte/macrophages (12, 14) in addition to the TH0 and TH2 subsets (re- viewed in Ref. 18) of helper T cells. In this review, we attempt to summarize what is currently known of the biological properties of IL-10 and speculate on its clinical potential and usefulness.

IMMUNOSUPPRESSIVE PROPERTIES OF IL-10 IN VITRO (TABLE I)

As indicated above, IL-10 was originally identified as a product of murine T helper 2 (Th2) clones that inhibited the production of IL-2, IL-3, tumor necrosis factor beta (TNFI3), granulocyte-macrophage colony-stimulating factor (GM-CSF), and interferon gamma (IFNT) by Thl clones following antigen presenting cell (APC)-dependent stimulation with antigen or soluble anti-CD3 monoclonal antibodies (I). The inhibitory effects of IL-10 on Thl cytokine synthesis were especially pronounced

239 0271-9142/92/0700-0239506,50/0 © 1992 Plenum Publishing Corporation

240 HOWARD ET AL.

Table I. Immunosuppressive Properties of IL-10 in Vitro

• Inhibits monocyte/macrophage-dependent cytokine synthesis by PBMNC, NK cells, and Thl clones

• Inhibits monocyte/macrophage-dependent Th0, Thl, and Th2 ~ proliferation

• Inhibits cytokine synthesis by activated macrophages/monocytes

• Inhibits monocyte class II MHC expression • Inhibits macrophage production of reactive nitrogen oxides • Inhibits macrophage intracellular and extracellular killing of

parasites

aSO far shown only in human system.

on the production of IFN~/and IL-3, both of which are normally synthesized late following activation of these clones. While murine-IL-10 is species specific, human- and EBV-derived viral IL-10 (vlL-10) inhibit cytokine synthesis by both mouse Thl clones and human peripheral blood mononuclear cells (PBMNC) (3, 19). Human IL-10 inhibited strongly the production of IFN~/, GM-CSF, TNFtx, and TNFI3 by PBMNC following activation with phytohemagglutinin (PHA), anti-CD3 monoclonal antibodies, or IL-2. This inhibition of cytokine synthesis by murine Thl clones and human PBMNC has been shown to occur at both the protein and the mRNA level (1, 3).

The mechanism by which IL-I0 inhibits cytokine synthesis by helper T-cell clones or PBMNC has not been completely elucidated. Mosmann and colleagues initially showed that m-IL-10 does not act directly on murine Thl clones, since IFN~/synthesis was unaffected when the T-cell clones were stimulated by immobilized anti-CD3 monoclonal antibodies or concanavalin A (Con A) in the absence of accessory cells (1). It was subsequently shown by O'Garra and Fiorentino, using purified populations of splenic or peritoneal APC, that IL- 10 inhibited IFN~/synthesis by antigen-stimulated Thl clones only when purified macrophages were used as APC, and had no effect on Thl activation when antigen was presented by either conventional or Lyl + B cells (13). Additional studies to elucidate this inhibition of macrophage APC function showed that IL-10 affected the antigen presenting capacity of metabolically active macrophages only. Further- more, since macrophage-dependent Thl stimulation by superantigens was also inhibited by IL-10, it appeared that the mechanism of IL-10-mediated suppression was not only at the level of antigen uptake or processing (13). The direct effect of IL-10 on processing has not been addressed as yet. Ex- periments to date make it unlikely that IL-10 acts by

inhibiting the production of a soluble costimulatory activity or by inducing a suppressor activity by macrophages. Some effort has been focused on the possibility that IL-10 suppresses APC function of macrophages by down-regulation of class II major histocompatibility complex (MHC) expression on these cells. Indeed, Stein et al. (M. Stein and S. Gordon, personal communication) have demonstrated that IL-10 inhibits IFN~-induced class II expression on polyacrylamide bead-elicited peritoneal macrophages. It remains to be determined whether this effect is also observed on resident macrophages which are inhibited by IL-10 from stimulating TH1 clones. Regardless of this out- come, several observations indicate that IL-10- mediated suppression of macrophage APC function involves additional factors other than class I I MHC expression. First, IL-10 inhibited the antigen presenting capacity of an activated murine macrophage line without apparently decreasing its expression of class II antigens, although this could be due to slow turnover of class II (13). Second, IL-10 inhibited the IL-2-induced production of IFN~/by Thl cells (13), a mode of stimulation which is dependent on a Mac 1 + accessory cell but not on antigen or class II MHC molecules (20). Finally, increases in either APC or antigen dose did not affect IL-10 action (13), again implicating suppression of an activation pathway other than T-cell receptor [TCR]-MHC class II interactions.

In a concurrent investigation of the mechanism of IL-10-mediated immunosuppression, de Waal- Malefyt and colleagues have analyzed the ability of human IL-10 to inhibit the production of IFN~/by PHA- or IL-2-activated human PBMNC (3, 19). Consistent with the above murine experiments, the inhibition of cytokine production by PHA-activated peripheral blood T cells is indirectly mediated via inhibitory effects of IL-10 on the antigen presenting or accessory function of human monocytes (R. de Waal-Malefyt, in preparation). Similar results were obtained to explain IL-10-mediated inhibition of IFN-~ production by human natural killer (NK) cells stimulated with accessory cells plus IL-2 (19). In this system, IL-10 did not effect the small amounts of IFN~ produced by highly purified human NK cells stimulated with IL-2 in the absence of accessory cells. However, IL-10 caused profound suppression of the enhanced production of IFN~ and TNFet that occurs when purified monocytes are added to IL-2-stimulated NK cells. IL-10-mediated inhibition of NK cell derived-IFN-,! has also been

Journal o f Clinical Immunology, Vol. 12, No. 4, 1992

BIOLOGICAL PROPERTIES OF INTERLEUKIN 10 241

shown in a murine in vitro model by Bancroft and Webster (21).

In addition to its cytokine synthesis inhibitory activity, IL-10 inhibits accessory cell-dependent, antigen-specific proliferation of resting human T cells and human Th0-, Thl-, and Th2-"like" CD4 ÷ T-cell clones (11) and of mouse Thl clones (S. Macatonia and A. O'Garra, in preparation). IL-10 inhibited antigen-specific proliferation when monocytes, but not when EBV-lymphoblastoid cell line [LCL] were used as APC, indicating that IL-10 affected the antigen presenting capacity of monocytes but not transformed B cells (11). Inhibition of human T-cell proliferation in this system appears to be independent of antigen processing, since human IL-10 was equally as effective at inhibiting human T-cell proliferation induced by monocyte-presented peptide antigens which did not require processing. Consistent with the observation on CSIF activity in mouse systems, no evidence has been obtained in the human T-cell proliferation system forthe induction of a monocyte suppressor factor by IL-10 or the inhibition of a soluble activator. However, down-regulation of class II MHC expression appears to play a significant role in IL-10-mediated suppression of human monocyte APC function (I 1). Human IL-10 strongly down-regulated constitutive, IL-4, and IFN~/-induced expression of HLA DR/DP and HLA DQ antigens on human monocytes; furthermore, it rendered monocytes refractory to induction of class II MHC expression by IFN~/and IL-4. The down-regulation of class II MHC expression correlated with the inhibition of proliferation by the T-cell clones, suggesting that IL-10 prevented the activation of the T-cell clones rather than actively suppressing their proliferation (I1). This notion was confirmed by the observation that Ca 2÷ fluxes were strongly reduced in T-cell clones following their activation with monocytes that had been pretreated with antigen and IL-10 and that IL-10 prevented the induction of the IL-2 receptor ~x chain (TAC) in T-cell cultures stimulated by antigen and monocytes. Importantly, IL-10-induced inhibition of T-cell proliferation was only partially re- versed by the addition of high concentrations of IL-2, suggesting that the reduced proliferative responses may not simply reflect reduced IL-2 production.

Extending this concept of IL-10-mediated suppression of human T-cell proliferation, Roncarolo et al. have shown that IL-10 also inhibits the proliferation of class II MHC alloantigen-specific T-cell

clones when monocytes are used as APC (M.-G. Roncarolo, in preparation). Furthermore, IL-10 inhibits the proliferation and cytokine production generated in primary one-way mixed leukocyte cultures (MLC) (M. T. Bejarano, submitted for publication). Separation of responder and stimulator cells indicated that IL-10 strongly inhibited the proliferation when allogeneic monocytes were used as stimulators and purified T cells as responders. IL-10 did not inhibit the proliferation induced by allogeneic EBV-LCL. Although the total number of responder T cells was reduced in the cultures with IL-10, the ratio of CD4 + and CD8 ÷ T cells remained unaffected, indicating that IL-10 had no preferential inhibitory effects on CD4 + or CD8 + T cells. Thus, the effects of IL-10 on alloantigen-induced proliferative responses are comparable to those on T-cell proliferation induced by soluble antigens presented by monocytes. IL-10 also inhibited the generation of allospecific cytotoxic T cells in primary one-way MLC in these studies. However, it is not yet clear whether IL-10 inhibits directly the generation of cytotoxic CD8 + T cells or that it affects the activation of the CD4 + T helper cells which are required for the generation of the cytotoxic T cells. In murine systems, IL-10 did not affect proliferation in an MLC initiated by mouse dendritic cells or unseparated splenic APC, using unseparated or purified CD4 + cell responders (S. Macatonia and A. O'Garra, in preparation). In these studies, resident splenic macrophages could not initiate an MLC (S. Macatonia and A. O'Garra, in preparation), a finding which is consistent with previous analyses of murine MLC (22). These differences in mouse and human systems may be due to the different sources of responder and stimulator cells used throughout, e.g., peripheral blood in the human versus splenic or lymph node cells in the mouse. Thus, more studies are needed to predict whether IL-10 will play a role in the induction or maintenance of tolerance following allogeneic bone marrow or or- gan transplantations in vivo.

In addition to its effects on the accessory/antigen presenting functions of monocytes/macrophages, IL-10 also inhibits other monocyte/macrophage functions in both mouse and man. The addition of IL-10 to cultures of monocytes or peritoneal macrophages results in changes in morphology and adhesion (14; R. de Waal-Malefyt, in preparation). The explanation of these morphological changes is not yet known, however, several considerations have been eliminated, e.g., IL-10 does not alter the

Journal of Clinical Immunology, Vol. 12, No. 4, 1992

242 HOWARD ET AL.

expression of a variety of adhesion molecules in- cluding CD11 abc, CD18, CD54, CD58, VLA-4, and VLA-5 on cultured monocytes (I1). In addition to these dramatic morphological changes, IL-10 inhibits the production of numerous cytokines, e.g., IL-let, IL-6, IL-8, GM-CSF, G-CSF, and TNFot, by murine peritoneal macrophages, macrophage cell lines, and human monocytes stimulated with lipopolysaccharide (LPS), IFN~/, or LPS and IFN7 (I4, 12, 23, 24). The substantial suppression of IL-let, GM-CSF, and TNFet production, in particular, presumably reflects the relatively late production of these cytokines following monocyte activation. In vitro studies have shown that monocytes and macrophages can produce high levels of IL-10 following activation by a variety of stimulants, such as LPS (14, 12). Interestingly, in vitro activation of monocytes/macrophages in the presence of neutralizing anti-IL-10 monoclonal antibodies results in increased production of IL-la, IL-II3, IL-6, IL-8, GM-CSF, G-CSF, and TNFa, a strong induction of class II MHC expression by human monocytes (12), and enhanced IL-6 production by mouse peritoneal macrophages (14), suggesting that endogenously produced IL-10 normally suppresses cytokine production and class II MHC expression by these cells. These observations have now been extended by the recent finding that exogenous IL-10 inhibited IL-10 mRNA expression by LPS-activated monocytes, suggesting that production of this cytokine is itself regulated by a negative feedback mechanism (12). In addition to its effects on monokine production, IL-10 has also been shown to inhibit the production of reactive nitrogen oxides (NO) following IFN7 activation of thioglycollate-elicited peritoneal macrophages (23, 25, 26). The production of NO is involved in the IFN-f-mediated elimination of intracellular Toxoplasma gondii (25) and the extracellular killing of schistosomula of Schistosoma mansoni (26) by macrophages. Since IL-10 is present following host infection by a variety of parasites (27), it may provide a way in which intra- and extracellular parasites escape from IFNT-dependent cell- mediated immunity. IL-10 does not completely suppress all monocyte protein synthesis, since it has no effect on the constitutive expression of TGF[~ (12), and it up-regulates expression of the IL-1 receptor antagonist (IL-1 Ra), an important regulator of inflammatory reactions (de Waal-Malefyt, unpublished). Collectively, these in vitro data suggest that IL-10 may play an important role in regulating

Table H. Immunostimulatory Properties of IL-IO in Vitro

• Enhances B-cell class II MHC expression • Enhances B-cell viability • Enhances activated B-cell proliferation and Ig secretion • Growth costimulator for thymocytes • Enhances cytotoxic T-cell development • Growth costimulator for mast cells

inflammatory responses via inhibition of proinflammatory cytokines and stimulation of IL-1RA.

IMMUNOSTIMULATORY PROPERTIES OF IL-10 IN VITRO

While IL-10 is a potent immunosuppressant of macrophage function, it exerts a wide array of immunostimulatory effects on B cells (summarized in Table II). IL-10 up-regulates class II expression and in vitro viability of highly purified small dense B cells obtained from the spleens of unstimulated mice (10). Previous studies have indicated that IL-4 is also capable of mediating these same effects on resting B cells (28-3I); however, neutralizing monoctonal antibodies that specifically recognize each of these cytokines suggest that the two cytokines work independently in these bioassays (10). Interestingly, B cells from xid mice were incapable of up-regulating class II expression in response to IL-10 stimulation (10), despite the apparent exis- tence of functional IL-10 receptors on these cells and their ability to up-regulate class II expression in response to IL-4 stimulation (N. Go and M. How- ard, unpubiished). This currently unexplained observation raises the possibility that IL-10 in some way contributes to the immunodeficiency of xid mice, which are unresponsive to thymus-independent type II antigens in B-cell bioassays in vitro and susceptible to an array of microorganisms in vivo (32-36).

In addition to these effects on resting B cells, IL-10 augments proliferation of activated B cells and induces their differentiation into antibody secreting cells. This has been demonstrated most convincingly using human B cells stimulated with anti-CD40 antibodies extensively cross-linked on FcR/L cell transfectants (37). In the presence of IL-4, this mode of activating human B cells induces their vigorous and long-term proliferation, with little differentiation into Ig secreting cells (38). The addition of IL-10 to these cultures augments DNA synthesis and expansion of total B cells and induces their differentiation into antibody producing cells,



with subsequent expression of large amounts of IgM, IgG, and IgA antibodies (37). IgE can also be obtained in this system but has an absolute require- ment for IL-4. Similar results were obtained when human B lymphocytes were activated via their antigen receptors (37). More recently, these studies have been extended to demonstrate that IL-10 induces IgM secretion predominantly from surface IgD ÷ naive B cells stimulated with anti-CD40 antibodies and IgG secretion predominantly from surface IgD- "germinal center-like" B cells stimulated with anti-CD40 antibodies (39). In the latter studies, IL-I0 also appeared to be an important cofactor for driving naive B cells activated with anti-CIM0 antibodies and tumor growth factor beta (TGFI3) into IgA secretion (39).

In addition to its potent immunostimulatory effects on B lymphocytes, IL-10 is a stimulant of numerous other cell types (summarized in Table II). It is capable of augmenting the in vitro proliferative response of mouse thymocytes and T cells which have been activated with IL-2 and IL-4 (6, 7) and may, therefore, play a role in T-cell development. In single-cell cultures containing IL-2, IL-10 enhanced the frequency of murine cytotoxic T-cell precursors generated from Con A-activated fluores- cence-activated cell sorter (FACS)-purified CD8 + splenic T cells (16) and markedly expanded the clone size and lytic potential of these cells when assayed against appropriate target cells (16). The effect of IL-10 on other hemopoietic lineages is only starting to be investigated now. Rennick and colleagues have shown that IL-10 sustains the viability of several mouse mast-ceU lines in vitro and syner- gizes with IL-3 and IL-4 to stimulate proliferation of these cell lines (9, 8). Perhaps more significantly, IL-10 markedly enhanced the total number and average size of mast-cell colonies which developed after 14 days of culture in the presence of IL-3 and IL-4 from mesenteric lymph node cells of nema- tode-infected mice.

A C T I O N O F M U R I N E IL-10 I N VIVO

A major challenge to all investigators of cytokine function faced with a plethora of in vitro cytokine functions, such as those listed above for IL-10, is to ascertain whether these in vitro properties accu- rately reflect the physiological function of the cytokine in vivo. To address this in the case of IL-10, Ishida et al. (40) have undertaken a series of experiments in which IL-10 function in vivo has been

Table m . Changes in Anti-IL-10-Treated Mice

• Reduced serum IgM and IgA • Increased serum IgG2a and IgGEb • No peritoneal B cells • No in vivo antibody responses to two bacterial antigens • Increased IFN~/levels • Increased monokine levels

blocked by administering neutralizing anti-IL-10 antibodies to mice from birth until 8 weeks of age, with subsequent evaluation of the immune status of these animals. At 8 weeks of age, anti-IL-10-treated mice appear to be essentially healthy and show no alteration of numerous parameters: total body weight; gross histology of liver, lungs, intestines, spleens, and thymuses; hematocrits; total numbers of splenocytes, thymocytes, lymph node cells, and peritoneal wash cells; and proportions of B cells, T cells, and non-B/non-T cells in spleen, lymph nodes, and thymus. However, continuous neutralization of IL-10 in mice did produce a series of highly reproducible phenotypic changes (summarized in Table III), when these animals were compared to either untreated mice or mice treated in an analogous manner with an isotype control antibody. In terms of serum immunoglobulin levels, anti-IL- 10-treated mice were drastically depleted of circulating IgM and IgA antibodies and contained sub- stantially increased levels of IgGEa and IgG2b. The remaining isotypes (i.e., IgG1, IgG3, IgE) were either unchanged or slightly elevated. In addition, while anti-IL-10-treated mice contained normal numbers of B cells in their spleens, they contained few to no B cells in their peritoneal cavities, in contrast to normal mice or isotype control-treated mice, both of which generally yielded 1-5 x 106 B cells from this site. The peritoneal cavities of anti- IL-10-treated mice lacked both mature B cells expressing surface IgM and/or IgD and pre-B cells expressing B220. Anti-IL-10-treated mice were also found to be functionally devoid of peritoneal B cells, as demonstrated by the inability of their peritoneal wash cells to proliferate in response to LPS stimulation in vitro. The lack of peritoneal B cells in anti-IL-10-treated mice was compensated by an increase in peritoneal T cells and granulo- cytes, such that the total cellularity of the peritoneal cavity was unaltered. Importantly, the splenic B cells in antML-10-treated mice were unchanged in terms of total numbers, surface marker phenotype, and in vitro responsiveness to the B-cell mitogens LPS or anti-Ig antibodies (40).


244 HOWARD ET AL.

Several other immunological modifications were found to be characteristic of anti-IL-10-treated mice. While developing perfectly normal primary and secondary antibody responses to the T-dependent antigen TNP-KLH, anti-IL-10-treated mice were found to be deficient in antibody production to two bacterial antigens, phosphorylcholine and ct- 1,3-dextran. Furthermore, anti-IL-10-treated mice contained substantial levels of circulating IFN~/and frequently expressed IL-6 in their sera, two cytokines which were not found in the circulation of either normal mice or isotype control-treated mice. The increased IL-6 expression was probably representative of a broader monokine up-regulation, since anti-IL-I0-treated mice were found to be 50-fold more susceptible to death by endotoxin- induced shock (H. Ishida and M. Howard, unpublished data), an inflammatory reaction resulting from elevation of numerous monokines (41-44).

These changes in anti-IL-10-treated mice, as summarized in Table III, can be interpreted as follows. The anti-IL-10-induced increase in IFN~/ and IL-6 expression implied by the aberrant presence of these cytokines in the circulation of anti- IL-10 treated mice is consistent with the previously documented ability of IL-10 to suppress production of these cytokines in vitro. As outlined above, numerous in vitro studies have shown that IL-10 is capable of suppressing IFN~/production by helper T cells (I, 13) and NK cells (19, 21) via an indirect action on accessory cells. Similarly, IL-10 is a potent suppressant of monokine production in vitro (12, 14). The increase in serum IgG2a and IgG2b is presumably the result of IFN~/elevation in anti-IL- 10-treated mice, since IFN~/has been shown previously to up-regulate expression of these immunoglobulin isotypes in vitro (45, 46). The remaining changes summarized in Table III, i.e., reduction of serum IgM and IgA, depletion of peritoneal B cells, and elimination of antibacterial antibody responses, can be explained by the selective elimination of a subpopulation of B cells, designated CD5 or Ly-1 B cells, from anti-IL-10-treated mice. CD5 B cells (47-51) are highly prevalent in the fetal and neonatal immune systems of mice and man but represent only a minor B-cell subpopulation in adulthood of both species. They have been studied extensively in the murine system, where numerous intriguing properties of CD5 B cells have been deduced from a variety of animal cell-transfer experiments where irradiated mice or SCID mice are selectively recon- stituted with either CD5 B cells or conventional B

cells. Such experiments have shown that murine CD5 B cells generate most circulating IgM (52-54), and contain the precursors of IgA secreting cells (55) and of all antibody forming cells specific for the bacterial antigens phosphorylcholine and ct-l,3- dextran (53, 56). In normal mice, CD5 B cells are almost undetectable in primary and secondary lym- phoid tissues but become greatly enriched in the peritoneal and pleural cavities. Thus, the observed depletion of peritoneal B cells, circulating IgM and IgA, and in vivo antibody responses to phosphorylcholine and ot-l,3-dextran in anti-IL- 10-treated mice are highly suggestive of an elimination of CD5 B cells resulting from this treatment.

Several experiments were performed to explore further the mechanism of CD5 B-cell depletion in anti-IL-10-treated mice. We have eliminated the trivial explanation that anti-IL-10 antibodies are selectively cytotoxic for CD5 B cells by showing that injection of the same antibodies into the peritoneal cavities of normal adult mice had no effect on the subsequent numbers or phenotypes of B cells recovered from this site 1, 2, or 3 days later. As alternative possible mechanisms, we have consid- ered whether CD5 B-cell depletion was the result of a secondary cytokine perturbation occurring in anti- IL-10-treated mice or, indeed, whether IL-10 acted as an autocrine growth factor for CD5 B cells. The latter theory initially seemed particularly attractive in light of our previous studies showing greatly elevated constitutive and inducible IL-10 production by CD5 B cells compared to conventional B cells (8, 17). Recent studies have indicated, however, that CD5 B-cell depletion in anti-IL-10-treated mice is more likely to be another consequence of IFN~/elevation in these animals. When mice were treated from birth until 8 weeks of age with neutralizing anti-IFN~/antibodies in addition to neutralizing anti-IL-10 antibodies, they contained virtually normal numbers of peritoneal B cells (40), suggesting that neutralization of IFN~/prevented anti-IL- 10-induced depletion of CD5 B cells in these mice. Consistent with this notion, recent preliminary experiments have shown that IFN~/ suppresses in vitro proliferation of LPS-stimulated peritoneal cells but not LPS-stimulated spleen cells (A. O'Garra and M. Howard, unpublished observations).

In summary, the anti-IL-10-treated mouse con- firms several of the immunosuppressive in vitro properties of IL-10 described above (see Table I). Further work is required to evaluate whether the



immunostimulatory in vitro properties of IL-10 (see Table II) are also confirmed by the anti-IL-10- treated mouse; however, there is nothing yet to suggest that anti-IL-10 treatment decreases Ig secretion in vivo from the majority of conventional B cells, as would be anticipated from the B-cell stimulatory properties of IL-10. The phenotype of the anti-IL-10-treated mouse makes certain predictions for in vivo roles of IL-10. The elevation of monokines in these animals suggests that IL-10 may act as a potent antiinflammatory agent in vivo. Prelim- inary data indeed suggest that IL-10 is capable of directly protecting mice against death from endotoxin-induced shock in vivo (M. Howard and S. Menon, unpublished data). It is more difficult to speculate on IL-10 function in vivo based on the depletion of CD5 B cells in anti-IL-10-treated mice, since the contribution of this numerically small subset of B lymphocytes to the immune system is still unclear. The various models of CD5 B-cell function that have been advanced based on the specificities of antibodies they produce include roles in antibacterial immunity (57, 58), in clearance of host cellular debris such as senescent erythrocytes (59, 60), and in modulation of the antibody repertoire during development (61-64). Our recent finding that CD5 B cells are themselves potent producers of IL-10 (8, 17) indeed raises the possibility of a broader immunoregulatory role of CD5 B cells. Collectively, the observations that anti-IL-10- treated mice are deficient in IgM, IgA, specific antibacterial antibody responses, and a subpopulation of B cells believed to play a role in antibacterial immunity suggest that IL-10 may have the capacity to augment directly antibody-mediated antibacterial immunity. To this end, it will be interesting to evaluate the susceptibility of anti-IL-10-treated mice to a broad range of microorganisms.

P O T E N T I A L CLINICAL APPLICATIONS OF IL-10

In the absence of animal model experimentation or preclinical testing, it is difficult to predict accu- rately the clinical potential of IL-IO. Nevertheless, speculation may be justified on the basis of several of the striking biological properties described above. IL-IO is capable of suppressing IFN~/ and numerous monokines in a variety of in vitro assay systems. The elevation of these same cytokines in anti-IL-lO-treated mice strongly suggests that these immunosuppressive properties of IL-IO will occur in vivo. The suppression of monokine production

suggests that IL-10 may be a potent antiinflammatory reagent in a variety of clinical situations: bacterial sepsis, rheumatoid arthritis, psoriasis, etc. The ability of IL-10 to suppress a subset of T cell-derived cytokines (e.g., IFN-¢, IL-2, TNF[3) in addition to monokine suppression makes it an attractive candidate for prolonging allograft survival and in treating a variety of T cell-mediated autoimmune diseases, e.g., type I diabetes, multiple scle- rosis. Indeed, animal models already exist for some of these clinical conditions where suppression of IFN% IL-2, TNFa, or IL-6 using monoclonal antibodies causes a marked improvement in pathology. More information as to whether IL-10 also inhibits accessory and antigen presenting cell functions and proinflammatory cytokine production by other cells that have been implicated in inflammation, such as endothelial cells, epithelial cells, keratinocytes, Langerhans, and dendritic cells, together with additional information about its antiinflammatory effects and its ability to inhibit transplant rejection is required to determine whether IL-10 will indeed act as a bona fide antigen-nonspecific suppressor factor in vivo.

In other clinical situations, antagonism of IL-10 may be a more desirable choice. The expression of IL-10 activity (v-IL-10) by EBV presumably confers some survival advantage to the virus in terms of its ability to infect, replicate, and/or maintain itself within the host. The ability of (v) IL-10 to down- regulate IFN'y production by both T cells and NK cells, together with its B-ceU viability enhancing effects, suggests that (v) IL-10 can suppress antiviral immunity while at the same time enhancing the potential of EBV to transform human B cells. It is therefore feasible that IL-10 antagonists could effec- tively boost antiviral immunity in vivo to EBV and possibly other viruses. Similarly, IL-10 antagonists may also provide effective therapy in infectious disease situations where IL-10 has been shown to inhibit macrophage-mediated immunity against intra- and extracellular parasites. We therefore look for- ward with optimism to future experimentation where IL-10 is either administered directly or, alterna- tively, antagonized by neutralizing anti-IL-10 antibodies, in a variety of animal models of infectious disease, inflammation, autoimmunity, and transplan- tation. It is to be hoped that information obtained from these approaches will offer IL-10 numerous opportunities to diminish the clinical pathology as- sociated with these diverse disease situations.


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Biological properties of interleukin 10

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