Introduction
One of the amazing features of the corpus luteum (CL) is the rapidity with which a
very heterogeneous population of cells becomes organized into a functional unit. These
diverse cells then communicate both directly and through paracrine mediators to facilitate
the steroidogenic function and also the transient nature of the CL. Once the hormonal
regulators of luteal function and demise (for example, LH and prostaglandin F2α) had
been clearly delineated, considerable effort in the late 1970s and 1980s was spent
characterizing the morphological and functional characteristics of the large and small
steroidogenic cells. This was followed in the 1990s by increased interest in the roles
that nonsteroidogenic cells, including endothelial cells, fibroblasts, pericytes and
immune cells, might have in luteal function. It is now thought that the nonsteroidogenic
cells are very active participants in regulating the functional capacity and lifespan
of the CL. These cells communicate with the steroidogenic cells through the paracrine
signaling molecules that they produce, and also through direct cell contacts. One
form of direct cell-cell signaling that may serve to activate resident immune cells
is major histocompatibility complex (MHC) molecule-dependent interaction between luteal
cells with T lymphocytes. Expression of MHC molecules, and recognition of antigenic
peptides presented in the context of MHC molecules, serves as a means to regulate
the activation of T lymphocytes, thus controlling cytokine production and/or cytolysis.
Expression of MHC Molecules by Luteal Cells
Like the majority of other somatic cell types, luteal cells express class I MHC molecules.
What is surprising is that luteal cells of several species, including the cow, also
express class II MHC molecules [1-5]. However, the identity of cells expressing class
II MHC in the CL is somewhat in question. Class II MHC expression is typically limited
to cells of the immune system that are regarded as "professional" antigen presenting
cells, such as macrophages, dendritic cells, and to a lesser extent, B cells. Macrophages
present within the CL would therefore certainly account for a percentage of the class
II-positive cells. However, flow cytometric studies of dispersed bovine and ovine
luteal cells demonstrated the presence of class II MHC molecules on both large and
small cell populations [1,2], suggesting that, in addition to luteal macrophages (which
would be included in the small cell fraction), the steroidogenic luteal cells also
express class II MHC. Further, the bovine large luteal cell population was subdivided
into two groups, large dense cells and large less-dense cells [1]. Both populations
contain class II MHC-positive cells, but the large less-dense population contained
the highest percentage of class II MHC-positive cells. The identity of the cells comprising
these two populations is not known, but it is possible that one population represents
the large steroidogenic cells, and the other population is composed of aggregates
of luteal endothelial cells. A subpopulation of luteal endothelial cells expressing
class II MHC has recently been identified [6].
In the human, luteinization induces the expression of class II MHC molecules on granulosal
cells [7], and expression of both class I and class II MHC molecules increases on
granulosal cells in the late luteal phase [3]. In contrast, minimal class II MHC expression
is detected in developing bovine CL [1]. Class II MHC expression is elevated in the
bovine CL by midcycle, and in bovine and ovine CL, class II MHC expression is higher
near the time of luteal regression compared with midcycle [1,2,5]. In contrast to
the bovine and ovine CL, class II MHC expression in the equine CL is not elevated
until after the decline in circulating progesterone concentrations associated with
initiation of luteal regression [4]. Significantly, in each of these species, expression
of class II MHC is substantially less in CL from pregnant animals compared with non-pregnant
animals [1,2,4]. In the chicken, cells expressing class II MHC have been identified
in the theca layer of normally growing and pre-ovulatory follicles [8,9]. The percentage
of cells expressing class II MHC was greater in post-ovulatory follicles and atretic
follicles compared with pre-ovulatory follicles, and class II MHC-positive cells are
found in both the thecal and granulosal layers of post-ovulatory and atretic follicles
[9].
Given the information available describing the expression pattern of class II MHC
molecules in the CL, a role for class II MHC molecules in the process of luteal regression
has been proposed. The increase in expression of class II MHC molecules between early
and midcycle CL [1] coincides with the acquisition of luteolytic capacity. Expression
of class II MHC molecules on the surface of luteal cells increases near the time of
natural luteal regression and when luteal regression is induced by PGF2α [1,5] but
is lower in pregnant compared to non-pregnant animals [1,2,4]. From this observation
it can be inferred that class II MHC expression must be attenuated as part of the
mechanism that inhibits luteal regression during maternal recognition of pregnancy.
In addition, bovine luteal cells stimulate T lymphocyte proliferation in vitro, and
they are more potent stimulators of proliferation when derived from CL collected after
administration of PGF2α to the cow [10]. This indicates that there is a change in
the character of luteal cells that enhances their ability to stimulate the activation
of T lymphocytes.
A physiological role for MHC molecules in luteal function is supported by the observation
that both class I and class II molecules are expressed by luteal cells in vivo, and
the expression of class II molecules varies with functional state of the CL. These
observations have given rise to the hypothesis that the demise of the corpus luteum
may involve local autoimmune-response mechanisms facilitated by increased expression
of cell surface class II MHC at the time of luteal regression.
Major Histocompatibility Complex Molecules, Antigen Processing, and Autoimmunity
Stimulation of T lymphocyte activation is dependent on the specific interaction of
a T cell receptor for antigen (TCR) on the T cell with MHC molecules located on the
surface of the target cell [11]. The outcome of binding of the TCR to the MHC molecule
is determined by the peptides bound to the MHC molecules. In the context of T cell
responsiveness to a given cell or tissue, changes in the array of MHC-bound peptides
presented to T lymphocytes can determine whether T cells are activated by the MHC
molecules in a given tissue, or alternatively, whether T cells will remain in an unactivated
state, a condition known as immunological tolerance.
Antigenic peptides that are presented to T cells via MHC molecules are derived from
proteins that are proteolytically digested into short peptides prior to binding to
MHC molecules. Collectively, the proteolytic degradation of antigenic proteins and
the binding of the resulting short peptides to MHC molecules is called antigen processing.
Since antigen processing can determine the types of peptides bound to MHC molecules,
this process can impact whether cells within a tissue are able to activate T lymphocytes,
thereby stimulating an immune response.
Class I MHC
Processing of peptides for presentation in the context of class I MHC molecules is
carried out by the proteasome. The proteasome is a cytosolic protease complex composed
of multiple subunits that is responsible for the majority of intracellular protein
turnover, and also generates peptides that are presented to T cells via class I MHC
molecules (Figure 1; [12]). The catalytic core of the proteasome, referred to as the
20S proteasome, is composed of two heterologous families of subunits, termed α and
β subunits. The proteasome β subunits contain the active proteolytic sites [13]. The
composition of the 20S proteasome is balanced between expression of constitutive β
subunits under normal conditions, and expression of IFN-γ-inducible subunits during
inflammatory conditions. Interferon-γ induces the replacement of constitutively expressed
β-subunits with alternative β-subunits involved in antigen processing [14-18]. The
genes encoding two of these IFN-γ-induced subunits, LMP2 and LMP7, are located within
the class II MHC gene region [19-21] and these subunits have limited homology to the
constitutive subunits Y and X, respectively [14,22]. The third IFN-γ-inducible subunit,
originally termed MECL-1 and also referred to as LMP10 [17], is similarly homologous
to the constitutive β-subunit Z [23]. Thus, replacement of the constitutive β-subunits
X, Y and Z with the IFN-γ-inducible subunits (LMP7, 2 and 10) alters the peptide cleavage
patterns of the proteasome [24,25], resulting in alterations in the repertoire of
antigenic peptides presented by class I MHC molecules. Interestingly, the IFN-γ-inducible
subunits and their constitutive homologues seem to be reciprocally regulated. Tissues
expressing high levels of LMP2, LMP7, or LMP10 were observed to have low levels of
the constitutive homologous subunits [26]. Also, IFN-γ coordinately induces expression
of LMP2, LMP7 and LMP10 with concurrent reduction in expression of the constitutive
homologues [17].
Figure 1
Schematic representation depicting processing of antigens presented by class I MHC
molecules. 1) Intracellular proteins are proteolytically degraded within proteasomes.
2) Proteolytic degradation of intracellular proteins yields antigenic peptides of
nine to eleven amino acids. 3) Antigenic peptides generated by the proteasomes are
transported into the endoplasmic reticulum. 4) Newly synthesized class I MHC molecules
bind antigenic peptides within in the endoplasmic reticulum. 5) Class I MHC-antigenic
peptide complexes are exported through the Golgi and to the cell surface, for presentation
of antigenic peptide to CD8+ T cells.
The most notable instance in which IFN-γ-inducible proteasome subunits are suspected
to play a role in induction of an autoimmune response is autoimmune thyroiditis. The
thyrocytes themselves appear to act as antigen presenting cells during the progression
of the disease, stimulating the activation of T lymphocytes [27]. Studies of patients
with Grave's disease and Hashimoto's thyroiditis, both of which are autoimmune thyroid
disorders, found very high expression of LMP2 and LMP7 in the thyrocytes themselves
[28]. This implies that proteasomes in these cells generate peptides that, when bound
to class I MHC molecules, stimulate the activation of CD8+ cytotoxic T lymphocytes,
which infiltrate the thyroid tissue in massive numbers and exert cytotoxic effects
on the thyrocytes [29].
We have recently demonstrated that interferon-γ-inducible subunits of the proteasome
(mRNAs and protein) are expressed within the bovine corpus luteum [30]. Since bovine
luteal cells isolated from regressing CL more potently stimulate T lymphocyte proliferation
compared to luteal cells from midcycle CL [10], we hypothesized that IFN-γ-inducible
proteasome subunits would be upregulated in luteal tissue near the time of luteal
regression. Such a change in proteasome subunit expression could induce alterations
in the array of self-peptides presented to T cells in the context of class I MHC molecules
during luteal regression, which could explain the enhancement in the ability of luteal
cells to stimulate T cell activation following PGF2α administration. However, we discovered
that LMP7 and LMP10 were expressed in the CL at all stages of the estrous cycle, with
no upregulation following induction of luteal regression with PGF2α. No changes were
observed in LMP7 expression throughout the estrous cycle, but LMP10 expression was
less in early CL, and greater in midcycle and late CL [30].
Class II MHC
Class II MHC molecules, typically expressed on antigen presenting cells of the immune
system (ie. macrophages, dendritic cells and B lymphocytes), bind short peptides derived
from intracellularly processed proteins [31]. Class II MHC molecules are heterodimeric
proteins composed of non-covalently associated α and β chains. Assembly of the class
II MHC complex occurs within the endoplasmic reticulum (ER), and involves not only
α and β chains, but a third, non-polymorphic glycoprotein called the invariant chain
(Ii). As class II MHC molecules are synthesized, they immediately bind to Ii, a portion
of which occupies the peptide binding groove of the MHC molecule [32]. Invariant chain
serves a dual function, directing intracellular transport of class II MHC molecules
[33,34] and ensuring that binding of "inappropriate" peptides does not occur prematurely
[32]. The MHC class II-Ii complex is then transported into endosomes, in which proteolysis
by cathepsins and lysozomal proteases degrade the Ii into a peptide called class II-associated
invariant chain peptide (CLIP; [35,36]). Prior to binding of antigenic peptides by
class II MHC molecules, the CLIP fragment bound to the peptide binding groove must
be removed. A class II MHC-like heterodimeric protein generically referred to as DM
co-localizes with class II MHC molecules in endosomal compartments [37] and catalyzes
the removal of CLIP from the peptide binding groove of the class II MHC molecule [38,39].
Following CLIP removal, the class II MHC molecule is free to bind processed antigen,
before transport to the cell surface (Figure 2). The ability to process and present
antigen in a manner dependent on class II MHC can be conferred to non-antigen presenting
cells by transfection with a class II MHC molecule, Ii, and DM. These appear to be
minimal yet sufficient components required for reconstitution of the antigen processing
machinery [40].
Figure 2
Schematic representation depicting processing of antigens presented by class II MHC
molecules. 1) Extracellular and integral membrane proteins are internalized into endosomes
via endocytosis. 2) Lysozomes fuse with endosomes. 3) Proteolytic degradation of endocytosed
proteins occurs in the endolysozomal vesicle, resulting in the generation of antigenic
peptides. 4) A specialized subcellualr organelle containing the class II MHC molecules,
invariant chain, and DM fuses with the endolysozomal vesicle. This results in proteolytic
degradation of invariant chain to CLIP. DM then catalyzes removal of clip, and the
empty class II MHC molecules then bind antigenic peptides. 5) Class II MHC-antigenic
peptide complexes are then exported to the cell surface, for presentation of antigenic
peptide to CD4+ T cells.
Numerous autoimmune diseases are associated with aberrant expression of class II MHC
molecules, including insulin-dependent diabetes mellitus and autoimmune thyroiditis,
which are endocrine autoimmune disorders. In these diseases, the target cells express
Ii and DM molecules, indicating that they are acting as antigen presenting cells capable
of stimulating activation of CD4+ T cells. Both Ii and DM mRNAs are expressed within
bovine luteal tissue, with DMα and DMβ being elevated in midcycle compared to early
CL [41], and bovine luteal cells are potent stimulators of class II-MHC dependent
T cell proliferation [10]. Therefore, cells within the CL are able to process and
present antigens to T cells in the context of class II MHC, which may predispose luteal
cells to an autoimmune-type MHC-mediated response during luteal regression.
The local inflammation that occurs concurrently with autoimmune diseases is often
attributed to pro-inflammatory cytokines produced by Th1 cells. The mRNAs for several
pro-inflammatory T cell-derived cytokines, and in some cases the proteins themselves,
are present in the CL [5,42-46]. The presence of T cell-derived cytokines in luteal
tissue suggests that the T lymphocytes present within the tissue are activated. Activated
T lymphocytes produce IFN-γ and TNF-α, which increase expression of class I and class
II MHC molecules on luteal cells, as well as inhibit progesterone production [47,48].
Thus, a positive feedback loop of antigenic peptide presentation and T cell activation
could occur to facilitate the rapid demise of the tissue that occurs during luteolysis.
The Role of Costimulation In T Cell Activation
The presence of class I and class II MHC molecules on the surface of a cell allows
the cell to interact with CD8+ and CD4+ T lymphocytes, respectively. However, there
are two possible outcomes of MHC-mediated cellular interactions with T cells. In one
instance, binding of MHC molecules to the TCR can occur in the absence of accompanying
interactions between additional cell surface molecules. In this case an inactive state
known as anergy will be induced in the T cells [49,50]. Induction of anergy is one
means by which tolerance to antigens in peripheral tissues is induced, thus avoiding
an autoimmune response [51]. Anergic T cells are prevented from carrying out their
effector functions (cytokine secretion in the case of CD4+ T cells; cytotoxic activity
in the case of CD8+ cells).
Alternatively, MHC-TCR ligation can occur in conjunction with a second interaction
known as costimulation. Costimulatory signals are delivered to T cells by the cell
surface proteins B7-1 and/or B7-2, also known as CD80 and CD86, respectively [52-54].
These ligands, when present on the antigen presenting cell surface, bind to the CD28
cell surface molecule on T cells and provide the costimulatory signal that promotes
T cell survival [55] and induces T cell activation and clonal expansion [49,52,56],
allowing the T cell to carry out its effector functions. Therefore, while generation
of sets of self-derived peptides capable of binding to MHC molecules and stimulating
the activation of self-reactive T cells may predispose cells and tissues to an autoimmune
response, the absence of a costimulatory signal can result in induction of tolerance
to a tissue or cell type rather than activation of lymphocytes and induction of an
immune response [57].
We recently demonstrated the expression of CD80 and CD86 in bovine luteal tissue.
Similar to the pattern of DMα and DMβ expression, steady-state concentrations of CD80
and CD86 mRNA were greater in the bovine CL during midcycle as compared with early
CL [58]. In functional studies, antibodies against CD80 or CD86 inhibited luteal cell-stimulated
proliferation of T cells. These data indicate that bovine luteal cells express functional
costimulatory molecules, which enable them to provide the costimulatory signal necessary
for activation of T cells.
Conclusions
From the observation that expression of MHC molecules changes with functional state
of the CL, it may be inferred that these glycoproteins have a role in luteal physiology.
Since class II MHC molecules increase during luteolysis and are suppressed during
maternal recognition of pregnancy, it is likely that they are involved in luteal regression.
Presentation of peptides in the context of MHC molecules would result in activation
of T lymphocytes, with subsequent release of pro-inflammatory cytokines and cytolysis.
Luteal tissue contains the intracellular proteins that are necessary for processing
and presentation of antigenic peptides (LMP 7, LMP 10, Ii, DM), and the cell surface
molecules necessary for costimulation (CD80, CD86) are also expressed. Further, these
components are all apparently quite functional, since luteal cells are potent stimulators
of T cell proliferation. Collectively, these events closely resemble those that occur
in tissues undergoing autoimmune responses. Thus, we propose that the progression
of luteolysis involves a localized autoimmune response involving MHC-mediated antigen
presentation to T lymphocytes. The exact identity of the antigen-presenting cells
is yet to be determined. Finally, it must also be recognized that the role of MHC
molecules on luteal cells may be something other than to promote luteal regression.
An alternative hypothesis is that MHC molecules are present in the CL to promote tolerance,
particularly in the event of pregnancy. Clearly, the story of MHC molecules in the
CL is just beginning to unfold.