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684 Immunology—Autoimmunity

used to demonstrate the reformation of the nuclear lam- of the snRNP particles, fueling subsequent studies that
ina during telophase (Fig. 1a). Autoantibodies have also showed the snRNPs as components of the spliceosome
identified unexpected distributions of autoantigens, such complex that functions in pre-mRNA splicing.
as the distribution of the nucleolar protein fibrillarin to As the molecular and functional associations of au-
the outer surface of the chromosomes during cell division toantigens have become known, attempts to uncover the
(Fig. 1e, arrowheads). The localization of some autoanti- particular role of individual autoantigens have revealed
gens during the cell cycle has aided in their identification. that autoantibodies can directly inhibit the function of
Detection of proliferating cell nuclear antigen (PCNA) their cognate autoantigen. Although it remains to be de-
in S-phase cells (Fig. 1c) suggested its involvement in termined, it seems likely that such inhibition reflects the
DNA synthesis, while the distribution of speckles along involvement of conserved protein sequence or structure
the metaphase plate produced by other antibodies (Fig. 1f, in functional activity. An increasing number of autoanti-
arrowheads) was a significant contribution to their identi- bodies, many of unknown molecular specificity, recognize
fication as autoantibodies against the centromeric proteins their autoantigen only in a particular functional state or
A, B, and C. phase of the cell cycle. Of the several examples known, the
A feature of autoantibodies that underscores their best characterized is PCNA, which is the auxillary protein
uniqueness is their ability to recognize their target antigen of DNA polymerase delta and is recognized by autoanti-
not only from the host but also from a variety of species. bodies only during mitosis, even though PCNA is present
The extent of this species cross-reactivity is dependent throughout the cell cycle. When a population of cells at
on the evolutionary conservation of the autoantigen and different stages of the cell cycle is used in immunofluo-
is related to the conservation of protein sequence. One rescence anti-PCNA, autoantibodies produce varying de-
example is the snoRNP protein fibrillarin. Using autoanti- grees of fluorescence intensity, being negative for G 0 cells
bodies in a variety of techniques, this protein can be found and highly positive for S-phase cells (Fig. 1c). These
in species as diverse as humans and the unicellular yeast intriguing features of some autoantibodies have added
Saccharomycescerevisiae.cDNAcloningoffibrillarinhas new dimensions to their biological usefulness and have
confirmed the expected high degree of conservation of the suggested that functionally active macromolecular com-
protein sequence. plexes may play a role in the elucidation of autoantibody
Autoantibodies react with the conserved sequence and responses.
conformational elements of their cognate antigens; these
features have made them useful regents in the cloning of
cDNAs of expressed proteins from cDNA libraries from II. DETECTION OF AUTOANTIBODIES
a variety of species. However, because of their reactivity AND AUTOANTIGENS
with the human protein, they have been used primarily
to clone the cDNAs and characterize the primary struc- The most commonly used methods for the detection and
tures of numerous human cellular proteins. The diversity characterization of autoantibodies, whether in clinical
of the targets that have been exploited by this approach is medicine or molecular/cellular biology, fall under sev-
illustrated in Tables I and II. eral broad biophysical areas and include fluorescent, en-
Elucidation of the structure of the autoantigens that are zymatic, and radiographic techniques. As described below
the targets of autoantibodies from systemic autoimmune some of these techniques have been specifically developed
diseases has revealed that many are functional macro- for antibody detection, while others have been borrowed
molecular complexes involved in nucleic acid or protein and/or adapted from the biological/biophysical sciences.
synthesis. A distinguishing feature of many of these com- The methodology employed in these techniques has been
plexes of nucleic acid and/or protein is that autoantibodies described in limited detail to afford the reader the op-
do not recognize all the components of the complex. An portunity of understanding how the technique is put into
extreme, but useful, example is the ribosome, which in eu- practice at the laboratory bench, particularly in the study
karyotes may contain more than 70 proteins. However few of autoimmunity.
of these proteins are recognized by autoantibodies, the ma-
jor targets being the sequence-related P proteins (P 0 ,P 1 , A. Immunofluorescence
P 2 ). Nonetheless, the use of autoantibodies that identify
1. History
specific components of such complexes has aided in iden-
tifying other subunits of these complexes, with profound Characterization of the antigenic specificity of serum an-
consequences. Thus the initial identification of anti-Sm tibody by immunofluorescent methods dates to the early
and anti-nRNP autoantibodies in SLE led to the observa- 1950s. In what was the forerunner of current technology,
tion that they recognize some of the protein components Weller and Coons, in 1954, used human serum to identify