Shan-Ren Zhou

The effects of citrullination or variable amino-terminus acylation on the encephalitogenicity of human myelin basic protein in the PL/J mouse

The post-translational modifications of myelin basic protein (MBP) in the form of citrullination and varying length of amino-terminus acylation may modify the biological functions and immunological features of MBP. Both modifications influence the reaction of antibodies and specific T cells recognizing MBP. The present study was undertaken to compare the encephalitogenicity of the citrullinated isomer of MBP (MBP-C8) with the unmodified isomer of MBP (MBP-C1) and to determine if the length of amino-terminal acylation of MBP peptide 1-21 altered an encephalitogenic epitope. MBP-C8, whether from patients with or without multiple sclerosis (MS), and MBP-C1 could induce active experimental allergic encephalomyelitis (EAE) in PL/J mice. A trend of reduced severity of EAE was observed in MBP-C8-injected animals. An increase in the length of amino-terminus fatty acid decreased the encephalitogenicity of MBP peptide 1-21 for both active and adoptive EAE in PL/J mice. Only lymph node cells sensitive to MBP peptide acetyl 1-21 and butyl 1-21 could transfer clinical EAE. In adoptive EAE, MBP peptides hexyl and octyl 1-21 induced moderate histopathological but no clinical change, whereas MBP peptide decyl 1-21 caused neither. A broadening in the antibody response could be detected in the sera of mice with active EAE induced by MBP-acylated peptides 1-21. Our findings demonstrate that encephalitogenicity is retained in the presence of citrullination but that the length of amino-terminus acylation diminishes the encephalitogenicity of MBP in the PL/J mouse.(ABSTRACT TRUNCATED AT 250 WORDS)

An immunochemical comparison of human myelin basic protein and its modified, citrullinated form, C8

An immunochemical analysis was conducted to compare the C1 isomer of human myelin basic protein (MBP) with the newly described and less cationic, citrullinated isomer of MBP referred to as C8. Ten polyclonal antisera directed at multiple epitopes or restricted regions of MBP were used in radioimmunoassays to examine MBP-C1 and MBP-C8. Antisera reactive with MBP peptide 1-14 clearly distinguished MBP-C1 from MBP-C8. Antisera to human MBP peptides 10-19 and 90-170, but not to MBP peptide 69-89, showed modest differences between MBP-C1 and MBP-C8. The MBP-C8s from multiple sclerosis (MS) and non-MS brain reacted essentially the same. With murine monoclonal antibodies and enzyme-linked immunosorbent assay (ELISA), differences between MBP-C8 and other isomers were shown for anti-MBP 10-19 but not for anti-MBP 1-9 or anti-MBP 80-89. These findings imply differences in sequence or conformation in the structure of MBP-C7 compared to MBP-C1, most notably near the amino terminus.

Immunological analysis of the amino terminal and the C8 isomer of human myelin basic protein

The citrullination and N-terminus acylation of myelin basic protein (MBP) increases the heterogeneity among the MBP isoforms. The present study was undertaken to further characterize the immune response to the citrullinated form (C8) of MBP as well as to the variably acylated N-terminus of MBP. Six well-characterized murine monoclonal antibodies (mAbs) to human MBP-C8 or MBP peptides (four mAbs to MBP acetyl 1-9, one mAb to MBP 10-19 and one mAb to MBP 80-89), one murine T cell line (PL11) to human MBP peptide acetyl 1-9 and one Lewis rat T cell line (RT-1) to guinea pig (GP) MBP peptide 68-88 were used to assess reactivity with MBP-C1, MBP-C8, and MBP peptides including a series of MBP peptide 1-21 containing 0, 2, 4, 6, 8 or 10 carbon fatty acids. Enzyme-linked immunosorbent assay (ELISA) results revealed that all of the mAbs reacted with human MBP-C1 and MBP-C8 except anti-MBP 10-19 and anti-MBP-C8. The former reacted only with MBP-C1 and the latter only with MBP-C8. The presence and length of acylation of MBP peptide 1-21 modified reactivity. Three mAbs to MBP acetyl 1-9 reacted only with acetyl 1-21, and one mAb anti-MBP actyl 1-9 reacted with all of MBP 1-21 preparations whether acylated or not. mAb anti-MBP-C8 generally reacted better with acylated MBP 1-21 having longer fatty acids. The PL11 T cell line strongly proliferated to human MBP-C1, MBP-C8 and MBP acetyl 1-9, responded, but less well, to MBP 1-21 with longer fatty acids and failed to respond to nonacylated MBP peptide 1-21. The RT-1 cell line responded strongly to GP MBP peptide 68-88, marginally to MBP-C8 and failed to respond to MBP-C1 or any of the other MBP peptides. Specific immune responses to different MBP charge isomers and different N-terminal acylating groups of MBP may play a role in immune-mediated demyelination.

Interstrain cross-reactive idiotypes on monoclonal antibodies to an encephalitogenic myelin basic protein peptide.

In order to assess the role of idiotype (Id) and the anti-Id network in murine experimental autoimmune encephalomyelitis (EAE), Id-bearing monoclonal antibodies (mAb) to human myelin basic protein (MBP) peptide acetyl 1-9, as well as mAb anti-Id, were developed in EAE-susceptible PL/J mice (H-2u). These mice recognize MBP residues acetyl 1-9 as an encephalitogenic determinant. Reactivities of PL/J Id-bearing mAbs to MBP and to MBP peptides were identical to those of mAbs generated against the same MBP peptide in EAE-resistant BALB/c mice (H-2d), even though isotypes of the mAbs differed. By using an inhibitory ELISA and immunoblotting, it was demonstrated that one PL/J mAb anti-Id recognized a public or framework Id, whereas another PL/J mAb-anti Id was directed to a private Id more restricted to the paratopic site. Two Id-bearing PL/J mAbs shared a cross-reactive Id (IdX) on the light chain, and an interstrain IdX was present on both the heavy and light chains of mAbs raised in PL/J and BALB/c mice to the same MBP peptide. The PL/J mAb anti-Id was capable of cross-regulating the production of Id-bearing mAbs by hybridomas across murine strains. These findings suggest that a restrictive family of germ-line genes encode for these Id-bearing antibodies to MBP peptide, irrespective of whether the MBP peptide is encephalitogenic in the murine strain immunized. Manipulation of the Id network may provide a means for modifying autoimmune demyelinating diseases of the central nervous system.

Changes Induced in Astrocyte Cathepsin D by Cytokines and Leupeptin

Cathepsin D is widely, but unevenly, distributed among cells and is capable of degrading a number of neural peptides and proteins. The present study was undertaken to examine the level of cathepsin D in astrocytes that might be relevant to its induction in inflammatory demyelination. Primary astrocytes were cultured from neonatal rat cerebrums according to the method of McCarthy and de Vellis. Based on staining for cell markers, cultures were >95% astrocytes and <3% microglia. Under serum‐free conditions, leupeptin induced a 1.4‐ to 2.0‐fold increase, maximal by 48 hours, in cathepsin D protein quantified by a radioimmunoassay. Cathepsin D enzymatic activity, inhibitable by pepstatin, also increased. Northern blot analysis demonstrated that leupeptin also increased cathepsin D mRNA expression. Kinetic analysis indicated that maximal cathepsin D mRNA levels are detected 24 h after stimulation with leupeptin. Exposure of astrocytes under the same conditions to rat recombinant interferon‐γ, human recombinant tumor necrosis factor‐α, human recombinant interleukin‐1β, lipopolysaccharide, calcium ionophore, or a combination of these reagents did not increase the level of cathepsin D above controls. These results indicate that astrocytic cathepsin D mRNA and protein can be induced by selected materials. Furthermore, the effects attributed to leupeptin as a proteinase inhibitor may be modified by its ability to increase cathepsin D activity.

The structure of a myelin basic protein-associated idiotope.

A cross-reactive idiotope (CRI) has been previously described on monoclonal antibodies (mAbs) specific for encephalitogenic peptides from myelin basic protein (MBP). The anti-CRI mAb, F25F7, binds an idiotope (Id) localized to the light chains of an anti-MBP peptide 1-9 mAb, denoted F23C6, and an anti-MBP peptide 80-89 mAb, denoted 845D3. It is the purpose of this study to further delineate the CRI being recognized by F25F7. To this end, we have found a structural correlation between the CRI and the antigen, a small synthetic peptide, denoted PBM 9-1, used to elicit the anti-Id mAb. Sequence comparison between the light chain of F23C6 and PBM 9-1 reveals a region of homology in CDR 2/FWK 3. The configuration of this site in the VL, as determined by comparison with a mAb, HyHEL-10, whose structure has been determined and is 97% homologous to the light chain of F23C6, conforms to the rules used to define antigenic determinants or Ids. A synthetic peptide having the F23C6 VL CDR 2/FWK 3 sequence inhibited the binding of F25F7 to F23C6 and 845D3. Taken together, these data suggest the Id recognized by F25F7 is defined, in part, by the PBM 9-1-like sequence of F23C6.

The modulatory effect of anti-idotypic antibody on hybridoma cells secreting antibody to human myelin basic protein peptide 80–89

The idiotype (ID) of an antibody is postulated to be involved in immunoregulation by means of the immune network. This hypothesis was examined by studying the effect of a monoclonal antibody (MAb) anti-ID on hybridoma cells secreting an ID-bearing MAb, 845D3, to human myelin basic protein (MBP) peptide 80-89. The monoclonal anti-ID (IgM/kappa), reacted with heavy and light chains of 845D3, an IgG1/kappa MAb, but not with a control MAb of the same isotype specific for an MBP peptide differing by one amino acid residue. Fluorescence-activated cell sorting revealed IgG heavy and kappa light chains on the surface and in the cytoplasm of 90% or more of the 845D3 cells. The anti-ID did not react with the surface of the 845D3-secreting cells, and reacted with the cytoplasm of 14-18% of these cells. In spite of the absence of cell surface ID, the anti-ID significantly decreased antibody production by the ID-secreting cells. There was no effect of the anti-ID on control hybridoma cells secreting MAb to another MBP cells. There was no effect of the anti-ID on control hybridoma cells secreting MAb to another MBP peptide. Anti-ID exerted no cytotoxic effect on ID-bearing hybridoma cells and in fact caused a marginal increase in their proliferation compared to control MAb. These results indicate that an anti-ID may alter the antibody production of ID-secreting cells in a specific manner and without cytotoxicity. This may be one of the means for controlling an immune response against MBP generated by the cells of the immune system or in situ within the nervous system. The cellular mechanism(s) for this effect remains to be defined.