Ia determinants on macrophages

Comp. Immun. Mierobiol. infect. Dis. Vol. 8, No. 2, pp. 89-97, 1985 Printed in Great Britain

Ia D E T E R M I N A N T S

0147-9571/85 $3.00+0.00 Pergamon Press Ltd

ON MACROPHAGES

PETER ERB Institute for Microbiology, University of Basel, Petersplatz 10, CH-4003 Basel, Switzerland Abstract--lt is well established that T cells cannot be activated by antigen alone but only if antigen is presented in context with I region associated (Ia) determinants. As a matter of fact, antigenpresenting cells or accessory ceils, which are obligatory for the induction of any type of immune response, all share the same major characteristic of la expression. Thus, there seems to be a direct correlation between accessory cell function and Ia expression. Originally, Ia determinants were only detected on a few cell types, B cells and macrophages being the first. However, during the course of time, more and more ceils were found to be la positive (Ia + ) and it is possible that most cells can express Ia, if appropriately induced. The regulation of la expression has been best studied in macrophages, where it has been found that positive induction elements include phagocytosis and gamma-interferon, while prostaglandin E and alpha-fetoprotein tend to down-regulate the expression of la. The regulation of la expression on accessory cells is thus an integrated part of immune regulation. It is highly likely, although not yet directly proven, that the Ia molecules are the products of the immune response (Ir) genes located within the major histocompatibility complex. They may even be the mediators of the Ir genes which determine whether an immune response can take place at all and/or the extent of the response. Recently, it has been shown that not all Ia + cells are able to activate every known T cell function. For example, for the activation of T helper cells which help B cells for antibody production only la + macrophages have so far been identified to be effective accessory cells. The activation of other T cell functions, e.g. lymphokine secretion or proliferation, is mediated by various types of other Ia + cells including macrophages. The biological significance of this restricted accessory cell function is not yet known, but it might be important in preventing immuno-pathological conditions, such as autoimmune reactions. Key words: Ia determinants, regulation of Ia expression, accessory cells, accessory cell heterogeneity, antigen-presentation, macrophages, T cell activation, T helper cells, T cell proliferation, T lymphokine production, T suppressor cells, T-B co-operation, Ir genes

DETERMINANTS Ia ET MACROPHAGES R~sum6---I1 est bien &abli que les cellules T n e peuvent pas ~tre activ6es par l'antig/~ne seul, mais le sont si l'antig6ne est present6 avec les d&erminants associ6s ~i la r~gion I (la). Les cellules pr6sentant l'antig+ne ou cellules accessoires, qui sont indispensables pour l'induction ou n'importe quel type de r6action immunitaire, partagent toutes la m~me caract6ristique majeure du d6terminant la. Ainsi, celle-ci semble &re un lien direct entre la fonction des cellules accessoires et rexpression de Ia. Initialement, les d&erminants Ia n'&aient d6tect+s que sur quelques types de cellules, les cellules B e t les macrophages ~tant les premi6res. Depuis, ont 6t6 trouv~es de plus en plus de cetlules Ia positives (Ia + ) et il est possible que la plupart des cellules puissent exprimer les d&erminants Ia si I'induction est appropri6e. La r~gulation de l'expression des antig6nes Ia a ~t6 le mieux 6tudi+e chez les macrophages, pour lesquels on a trouv~ que les 616ments inducteurs de Ia incluaient la phagocytose et l'interf~ron gamma tandis que les prostaglandines E et l'alpha-foetoprot+ine tendaient fi diminuer l'expression de ces d~terminants Ia. La r~gulation de l'expression de Ia sur les cellules accessoires fait donc partie int+grante de la r6gulation immunitaire. II est hautement probable, quoique pour l'instant non prouv+, que les molecules Ia sont les produits des g~nes de r~ponse immunitaire (Ir) situ~s darts le complexe majeur d'histocompatibilit& Ils pourraient mfime fitre les m+diateurs des g6nes Ir qui d6terminent si une r6ponse immune peut s'installer et/ou l'amplitude de cette r~ponse. R6cemment, on a montr6 que seules certaines cellules Ia + sont capables d'activer chacune des fonctions cellulaires T connues. Par exemple, pour l'activation des cellules T helpers qui coop~rent avec les cellules B pour la production d'anticorps, seuls les macrophages Ia + ont &~ identifi6s effectivement comme des cellules accessoires. L'activation des autres fonctions cellulaires T 89

90

PETER ERB (secr6tion de lymphokines ou prolif6ration) est m6di~e par diff6rentes autres cellules Ia + dont les macrophages. La signification biologique de cette fonction des cellules accessoires n'est pas encore eonnue, mais elle peut s'av6rer importante dans la prevention des pathologies immunitaires telles que les r6actions auto-immunes.

Mots-clefs: determinants Ia, r6gulation des d&erminants Ia, cellules accessoires, macrophages, pr+sentation de l'antig6ne, activation des cellules T, T helper, prolif6ration, lymphokine, T suppresseur, cooperation T-B, g6nes Ir

INTRODUCTION Since their discovery, macrophages were believed for a long time to only take part in the non-specific immunity, such as the defense against micro-organisms or the removal of foreign material, debris and dead cells by way of phagocytosis and intracellular degradation. However, in the late sixties it was recognized that macrophages were also involved in the development and expression of the specific immunity. Thus, Oppenheim et al. [1] reported in 1968 that the proliferation of human lymphocytes to specific stimuli was dependent on adherent cells, later identified to be monocytes, and Moshier [2] was the first to provide evidence for the requirement of macrophages for the development of antibody responses in vitro. Almost at the same time the picture of the dichotomy of the lymphocyte population into T and B cells emerged and it was soon clear that macrophages mainly affected T cell responses. However, macrophages are not the only cells that were found to function as accessory cells (AC) in immune responses. Other cells, which do not belong to the macrophage lineage, such as dendritic cells [3], P cells (a mast cell precursor) [4] and even B cells [5] were also identified as AC for the induction of at least some T cell functions. A list of cells which are reported to function as AC is given in Table 1. There is now general agreement that the induction of any immune response is strictly dependent on AC. The term "accessory cell" is therefore rather an undervaluation because these cells are equally as important as their partners, the lymphocytes.

Table 1. Types of accessory cells Accessory cell Peritoneal exudate cells (PEC) Splenic macrophages Alveolar macrophages Thymic macrophages Bone marrow derived Mph Langerhans cells Kupffer ceils Interdigitating ceils Follicular dendritic cells Astrocytes Endothelial cells Dendritic reticular cells P cells B cells Tumors L cells (Ia-gene transfected)

Type Mph.~ Mph Mph Mph Mph Mph-like Mph-like Mph-like Mph-like glial non-Mph non-Mph mast cell precur. lymphocyte B cell/Mph fibroblast

~ Ia pos.*

la regul.t

References

5-20 40-60 5-10 40-60 20-80 95 50-80 95 ? 0 95 95 95 95 95 0~

yes yes yes yes yes ?~ yes ? ? yes ? no no no yes/no ?

(~9 10 11, 12 13 14-16 17, 18 19, 20 21, 22 23, 24 25 26, 27 3 4 5 28-40 41

*~ Ia positive cells determined after isolation from tissue. tla expression dependent on appropriate stimulation. +*Mph: macrophages; ?: not known. §Negative before transfection with Ia genes, positive after transfection.

Ia determinants on macrophages

91

EXPRESSION AND FUNCTIONAL ROLE OF Ia DETERMINANTS ON MACROPHAGES AND OTHER AC In 1973, Rosenthal and Shevach [6] reported that in the guinea pig the activation of antigen-specific T cell proliferation required T cells and macrophages that were identical at the major histocompatibility complex (MHC). This important observation has been confirmed by us in the mouse using a different system, the induction of antigen-specific T helper cells (Thc) which help B cells for antibody responses [42]. Genetic mapping revealed that the interacting T cells and macrophages required identity at the I-A subregion of the H-2 complex [7]. Since then, the genetic restriction of T cell-macrophage interaction for the induction of a number of different T cell functions has been firmly established [43, 44]. The functions ascribed to macrophages for T cell activation were (1) taking up antigen, (2) processing it and (3) presenting antigen fragments to the appropriate T cells. However, the genetic restraints imposed on macrophage-T cell interactions indicated that T cells were not able to see the antigen fragments per se, but only in context with restriction elements. In 1975, we found that antigen-incubated macrophages secreted molecules which induced antigen-specific Thc in the absence of macrophages [42,45]. The immunochemical characterization of these molecules revealed that they consisted of I region associated products (Ia) non-covalently complexed with antigenic fragments [46]. This complex of Ia and antigenic fragments was called genetically related macrophage factor (abbreviated GRF) and it provided direct evidence that T cells respond to antigen only if presented in context with Ia. Since then, other groups found molecules with similar characteristics and functional properties [47, 48]. Nothing is known at present about the biological significance of G R F and its related mediators in vivo, but they will provide an excellent tool to better understand the mechanism of T cell activation. At the same time, when G R F was discovered, Unanue et al. [8] reported the expression of Ia products on a certain percentage of peritoneal exudate macrophages. Later on Ia was also detected on macrophages or macrophage-like cells located in other tissues such as the spleen, liver, lung, thymus, skin, etc. as well as on a number of cells not belonging to the macrophage-lineage [3, 4, 8-27] (Table 1). Moreover, in recent years a large number of Ia positive tumor cell lines of B cell or macrophage origin were found or made which expressed AC function like the macrophage [28-40]. Thus, AC function is not restricted to a single cell type, but is the property of many different cells. The prerequisite for being an AC, is the expression of Ia antigens. This well-known fact has been recently confirmed by elegant studies in which L-cell fibroblasts were transfected with genes coding for Ia antigens [41]. Non-transfected L-cells, which are Ia negative, do not function as AC for T cell activation. After transfection, the L-cells express Ia and activate T cells. However, as will be shown later, not every Ia positive cell of any type can activate all T cell functions. Therefore, heterogeneity exist not only in terms of the accessory cells available, but also in terms of accessory cell function. REGULATION OF Ia DETERMINANTS ON MACROPHAGES When Ia antigens were first detected on macrophages only a certain percentage of cells was made out to be Ia positive. The percentages reported ranged between 10 and more than 50% depending on the tissue location of the macrophages, with about 10% Ia +

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PETER ERB

macrophages in the peritoneal cavity and 50~ and more in the spleen or the thymus (Table 1). For a while it was thought that either two populations of macrophages exist, Ia positive and Ia negative, or that the expression of Ia is a differentiation-dependent process, with "young" macrophages being Ia positive, and "old" macrophages being Ia negative. However, studies performed by several groups clearly demonstrated that macrophages do not constitutively express Ia, but rather that Ia expression is subject to positive and negative regulation [10, 49-64]. Such regulatory principles were first defined in vitro. Macrophages taken out from the animal and cultured in vitro become Ia negative within 24-48h. However, the transition of I a + to I a - can be prevented if the macrophages are exposed to phagocytic stimuli such as bacteria, opsonized erythrocytes, antigen-antibody complexes or large soluble antigens like keyhole limpet hemocyanin (KLH) [49, 53]. More importantly, Ia negative macrophages become Ia + under the influence of certain T cell-derived lymphokines. For example, supernatants obtained from spleen cells incubated with ConA (ConA sup) or obtained from activated T helper cells induced the expression of Ia products on an Ia negative macrophage population [50-52, 54, 55, 57, 59]. One lymphokine, so far identified to induce Ia expression is gamma-interferon [54], but it is possible that other molecules expressing similar activities also exist. Negative regulation of Ia expression is mediated either by the removal of the enhancing stimuli, i.e. gamma-interferon, phagocytic stimuli, or by applying various inhibitory molecules, such as prostaglandins [60], corticosteroids [61], endotoxins [58], etc. A list of the positive and negative regulatory principles of Ia expression is given in Table 2. Regulation of Ia expression can not only be monitored in vitro but also in vivo [49, 53]. Thus, mice injected with certain bacteria or large multideterminant antigens showed a marked increase of Ia + macrophages in the peritoneal cavity. Another, very illustrative example of negative regulation in vivo has been demonstrated by Lu et al. [65-67] in Table 2. Regulation of Ia expression on macrophages Demonstration* References (A) Positive regulationt immunogenic stimuli (Listeria, C. parvum, latex, zymosan, opsonized red cells, antigen-antibody-complexes, KLH) gamma-interferon macrophage activating factor (MAF)$ S/N of ConA activated spleen cells$ lymphokines$ from T cells immune to Listeria, Mycobacteria, Trypanosoma, K L H (B) Negative regulationt removal of immunogenic stimuli prostaglandins class E alpha-fetoprotein corticosteroids endotoxins§ ultraviolet B light

yes

yes

49, 53

indirect no no yes

yes yes yes yes

54, 62 50, 54 50, 54, 59 50-52, 54, 55 59, 62, 63

yes indirect indirect yes no no

yes yes yes yes yes yes

49, 53, 63 60, 63 63 61 58 64

*Monitoring la on Mph either biochemically, serologically or functionally. tPositive regulation: maintainance of the number of la + Mph increase of the number of la + Mph conversion of Ia negative to la + Mph. Negative regulation: decrease of the number of Ia + Mph conversion of Ia + to la negative Mph. $Contains gamma-interferon; not clear if additional factors are also effective. §Acts by stimulation of macropbage prostaglandin E2 production.

Ia determinants on m a c r o p h a g e s

93

Table 3. W E H I - 3 tumor cells express AC function after preincubation with ConA sup for 3 days

T cell proliferation A C a d d e d to

primed BALB/c

ConA sup

T cells (3 x 105)

preincub.

~o Ia +

no K L H

K L H (40 # g / m l )

Stimuli.Index

-+(20°%)

10 0 90

477 1676 729 353

7783 3231 7972 725

16.3 1.9 10.9 --

BALB/c PEC WEHI-3 WEHI-3

104 5 × 103 5 × 103

125IUdR uptake

NIL

CPM

neonates, which have, compared to adults, a very low number of Ia + macrophages and a defect in antigen-presenting function, but in vitro, these macrophages can easily be induced to express Ia and AC function. The reason for the paucity of Ia + macrophages in vivo are increased levels of prostaglandins E and alpha-fetoprotein [63, 67]. Like normal macrophages, the expression of Ia antigen on a number of macrophagetumors is subject to regulation as well. These tumors represent an excellent example to demonstrate the correlation of Ia expression and AC function. For example, the monomyelocytic WEHI-3 tumor is Ia negative under in vitro culture conditions and does not induce antigen-specific T cell proliferation. However, preincubation of the WEHI-3 cells with Con A sup for 2-3 days results in Ia + cells which activate T cell proliferation in an antigen-specific manner (Table 3). la EXPRESSION AND FUNCTIONAL ACCESSORY CELL HETEROGENEITY There are a number of cells which in contrast to macrophages seem to be less prone to regulation of Ia expression. For example, dendritic cells [3, 68, 69], P cells [4], B cells [5, 70] as well as various B cell tumors and even some macrophage tumors [28-40] constitutively express Ia and are less subjected to positive or negative regulation. A comparison of the functional AC activity of several of these cells and non-transformed macrophages gave a surprising result [38-40, 71]. All cells tested activated antigen-specific T cell proliferation, induced T cells to secrete lymphokines or acted as AC in T-B co-operation cultures. The induction of T suppressor cells (Tsc) was variable, with some AC inducing strong

Table 4. Functional accessory cell heterogeneity Functions tested* Accessory cell Peritoneal exudate M p h Bone marrow derived M p h Dendritic cells P cells B cell tumors (eg. T A 3 , A202J) M p h t u m o r s (eg. W E H I - 3 , P 3 8 8 D I )

T prol

T lymph

T help

+ ÷ + + + +

+ + + + + +

+ + -

T suppr

+, +,

+ / + / ? -, + -, +

T - B co-op.

t t

nt

+

/ - ,+ / - ~

+ + + +

*T prol: antigen-specific T cell proliferation. T lymph: antigen-specific induction of lymphokine production by T cells. T help: antigen-specific activation of T helper cells. T suppr: specific and non-specific activation of T suppressor cells. T - B co-op: AC-activity in T-B co-operation. + : a c t i v a t i o n ; - : no activation; nt: not tested; ?: experiments are not yet completed.

The table does not reflect quantitative differences of activities of the AC tested. t B y increasing the number of M p h a d d e d to a constant number of T cells suppression can be induced. However, this does only partly reflect T cell-mediated suppression. +Suppression induced varies from strong ( + ), weak ( + / - ) to none ( - ) depending on the tumor cell line used.

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PETERER~

suppression and others inducing weak or no suppression at all. It is possible, but not yet demonstrated, that this variability of suppressor cell induction depends on the amount of Ia present on the AC. More importantly, only the non-transformed Ia + macrophages were able to activate antigen-specific T helper cells among the AC so far tested (Table 4). The reason for this differential AC activity is not clear and at present a matter of speculation. It does not seem to be dependent on whether the AC is transformed or not, as the non-transformed dendritic cells or P cells do also not activate Thc. Matis et al. [74] showed that the quantitative alteration of Ia on AC also lead to a quantitative alteration of the proliferative T cell responses. It is possible that quantitative variations of the Ia antigen on a per cell basis may even lead to the alteration of the functional activity of the AC. For example, AC expressing high amounts of Ia might induce tolerance or favour suppressor cell activation. Indeed, it has been recently demonstrated first, that, tolerance induction is MHC-restricted and, second, that tolerance induction of helper function at the level of a single clone approximately requires 100 times less antigen than necessary to tolerize proliferation ([72, 73], Feldmann and Lamb, personal communication). This can be explained by the requirement for an optimal concentration of Ia in relation to the antigen concentration for T helper cell activation. Cells such as dendritic cells or various tumor cells might express too much Ia and therefore be non-stimulatory for help but stimulatory for proliferation and other T cell functions, based on tolerance thresholds. This would also explain the observation that relatively undifferentiated (e.g. 3-7 days old) bone marrow derived macrophages (BMM0) which are between 20 and 40~ Ia positive, are excellent inducers of Thc, while differentiated (more than 30 days) BMM0, which.can be up to 80~ Ia positive, are virtually ineffective in inducing Thc [14, 59]. Based on this hypothesis it is evident that macrophages whose Ia expression can be regulated are optimally equipped for Thc induction. Clearly, other alternatives must also be considered. Thus, there might be differences at the T cell receptor level between Thc and other T cells, or macrophages and the other AC tested might differ in antigen handling and presentation. Ia EXPRESSION AND Ir GENES Immune responses to many, possibly all antigens are controlled by genes located v~ithin the I region of the H-2 complex of the mouse, respectively in the D / D R region of the HLA-complex in human. These genes which are responsible, whether an immune response to a given antigen takes place and/or the extent of the response are called immune response (Ir) genes. Ir gene control is intimately involved in the M H C regulation of cell interactions, such as T cell-macrophage interactions. Moreover, as has been already pointed out, T cells only recognize antigen in context of Ia products. The genes coding for the Ia glycoproteins are located in the very same region as the Ir genes. Thus, the conclusion is not far off that the Ia antigens are products of Ir genes and possibly the mediators of Ir gene function. There are various leads which support such a conclusion. First, as already discussed, AC function for T cell activation is intimately involved with the expression of Ia on the AC. Second, AC function can be blocked in vitro by adding appropriate anti-Ia antibodies into the cultures. Third, the response to bovine insulin is controlled by an I-A b encoded Ir gene [77]. It has now been demonstrated that the Ir gene control of the proliferative T cell response to bovine insulin is associated with the expression of Ia. W39 (a private specificity of I-A b) by the antigen-presenting macrophages [75, 76]. Thus, mice defective in the

Ia determinants on macrophages

95

expression of Ia.W39 on macrophages and B cells are non-responsive to bovine insulin. Fourth, the most stringent evidence for the close correlation of Ia and Ir gene products comes from studies of the macrophages from the B 6 . C - H - 2 bm~2 mouse, which bears a mutation in the beta-chain of the I-A b gene encoded molecule [78]. This mutation seems to consist of a very limited change in the beta-chain compared to the C57BL/6 mouse from which the mutant is derived [79, 80]. Despite these minor differences, macrophages obtained from this mutant lose the capacity to present bovine insulin to insulin-reactive T cells, while the ability to present another Ir gene-controlled antigen, (T, G ) - A - L , is fully retained [78]. Thus, a mutation slightly affecting the structure of the Ia molecule changes the restriction element in a way either that it cannot be seen any more by the T cells on the presenting AC or that it cannot appropriately interact with the antigen. The question whether Ia antigens are Ir gene products is not discussed primarily as one of academic interest but it bears significance to a number of pathological situations, such as autoimmunity, several HLA-linked diseases, inflammation processes and others. It is known, that under certain conditions Ia antigens are also expressed on epithelial, fibroblastic and other cells which are clearly not of the usual accessory cell lineage. The Ia expressions on these cells might even be subject to positive or negative regulation. The role of Ia on these cells is not clear, but it is likely that they are involved, like all other Ia + cells, in antigen presentation. This aberrant Ia expression might lead to the activation of autoreactive T cells with the consequences of inducing severe autoimmune diseases. For example, Bottazzo et al. [81] showed that the induction of Ia on thyroid epithelial cells may be an important step in the chain of events leading to autoimmune thyroid diseases. It is also conceivable that a continuous and enhanced Ia expression on macrophages in some tissue might lead to "hyper-activation" of T cells which in turn "hyper-stimulate" macrophages and other cells, an event leading to inflammation and tissue damage. Effects of this type have been shown by Schreiner et al. [82, 83] to take place in the kidney. CONCLUDING REMARKS It is now a well-established fact that the expression of Ia on macrophages is subject to positive or negative regulation by various means, while other, non-macrophage cell types, such as dendritic cells, B cells, express Ia constitutively. Moreover, it is likely that these latter cells express varying amounts of Ia from cell type to cell type. All this might be of considerable biological significance. The role of the AC expressing Ia constitutively might be to activate and expand certain types of antigen-reactive T cells, e.g. lymphokine producing T cells, and also to keep the immune system in an highly activated state. The lymphokines produced would induce macrophages to express the right amount of Ia in order to activate and expand T helper cells which stimulate B cells for antibody production or initiate the cytotoxic T cell pathway. As a feedback, the presence of high amounts of Ia in relation to the antigen (e.g. on dendritic cells) might lead to suppression or tolerance induction and interrupt the helper or cytotoxic pathway, thus preventing an overshooting response. It is at present open and a subject of further studies, whether the kind of T cell function activated depends on the type of the inducing AC, or whether its selection is determined by the amount of Ia expressed on the AC, or whether both mechanisms are operative. Acknowledgements--This work was in part supported by the Swiss National Science Foundation, grant no.

3.016.0.84.

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Ia determinants on macrophages 53. 54. 55. 56. 57. 58. 59. 60. 61. 62. 63. 64. 65. 66. 67. 68. 69. 70. 71. 72. 73. 74. 75. 76. 77. 78. 79. 80. 81. 82. 83.

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