Soluble adhesion molecules in juvenile rheumatoid arthritis.

OBJECTIVE: To determine serum levels of soluble (s) adhesion molecules in patients with juvenile rheumatoid arthritis (JRA), and to determine whether differences exist in these levels among the 3 subtypes of JRA, and whether levels of these molecules correlate with other measures of disease activity. METHODS: Serum levels of soluble forms of intercellular adhesion molecule-1 (ICAM-1), ICAM-3, vascular (V) CAM-1, L-selectin, and E-selectin were determined by sandwich ELISA in 16 patients with JRA (6 systemic, 6 polyarticular, 4 pauciarticular). Differences in levels among JRA subtypes were determined by ANOVA, and correlations between levels and the following clinical variables were assessed by linear regression analysis: erythrocyte sedimentation rate (ESR), total white blood cell count (WBC), hematocrit (HCT), platelet count (PLT), and total swollen joint count (JC). RESULTS: sE-selectin levels were significantly higher in patients with systemic disease compared to other subtypes (p<0.04). Furthermore, there was a trend toward higher levels of sICAM-1 in systemic disease, which did not reach statistical significance. Significant correlations were found between sE-selectin and ESR (r = 0.68, p<0.006), WBC (r = 0.70, p<0.003), and PLT (r = 0.54, p<0.05) and between sL-selectin and WBC (r = 0.55, p<0.03). CONCLUSION: Because of the small number of patients studied, and the lack of age matched control data, our results must be interpreted with caution. Nonetheless, levels of sE-selectin, and possibly ICAM-1 appear to be relatively elevated in systemic JRA, and may indicate cytokine induction and endothelial cell activation in that subtype. Several molecules, especially sE-selectin, correlate with hematologic variables in JRA. These results suggest that serum levels of these molecules may provide a useful additional marker for disease activity in certain patients.

Isoaspartyl post-translational modification triggers autoimmune responses to self-proteins.

The normal functioning immune system is programmed to attack foreign pathogens and other foreign proteins while maintaining tolerance to self-proteins. The mechanisms by which tolerance is broken in the initiation of autoimmunity are not completely understood. In the present study, mice immunized with the murine cytochrome c peptide 90-104 showed no response by the B or T cell compartments. However, immunization with the isoaspartyl form of this peptide, where the linkage of Asp(93) to Leu(94) occurs through the beta-carboxyl group, resulted in strong B and T cell autoimmune responses. Antibodies elicited by immunization with the isoaspartyl form of self-peptide were cross-reactive in binding to both isoforms of cytochrome c peptide and to native cytochrome c self-protein. In a similar manner, immunization of mice with the isoaspartyl form of a peptide autoantigen of human systemic lupus erythematosus (SLE) resulted in strong B and T cell responses while mice maintained tolerance to the normal aspartyl form of self-antigen. Isoaspartyl linkages within proteins are enhanced in aging and stressed cells and arise under physiological conditions. These post-translationally modified peptides may serve as an early immunologic stimulus in autoimmune disease.

Binding of Ku protein to DNA. Measurement of affinity for ends and demonstration of binding to nicks.

Ku, also known as nuclear Factor IV, is an abundant nuclear DNA-binding protein which requires free DNA ends for the initial interaction with double-stranded DNA (dsDNA) and can bind at multiple sites along dsDNA in an energy-independent manner. Its function in vivo is unknown, but it has been implicated in both DNA replication and repair and in transcriptional control. We have used an electrophoretic mobility shift assay to further define the DNA binding properties of the Ku protein. Titration of Ku to a fixed amount of any of several target linear dsDNA fragments produced ladders of shifted bands proportional to the length of DNA, confirming the multiple binding activity of Ku and demonstrating its sequence-independent nature. Using a short DNA fragment with one Ku binding site, the binding constant of Ku for dsDNA ends was calculated to be 2.4 x 10(9) M-1. Competitive inhibition experiments confirmed the requirement of a free DNA end for binding by Ku and demonstrated that Ku binds isolated nicks in dsDNA. Nick binding was also observed directly using radiolabeled singly nicked circular DNA. The relative affinities of Ku for specific nick sites and free DNA ends were approximately equal, and nick binding was sequence-independent. Finally, in a study of a possible role for Ku in protecting or repairing damaged DNA, Ku was shown to inhibit the ability of T4 DNA ligase to circularize linear dsDNA molecules, demonstrating that some Ku molecules remain at the DNA terminus rather than translocate. A similar inhibition was not observed at nicks. These experiments document a new DNA binding specificity for Ku and further suggest that the high affinity end and nick binding activity is biologically relevant to its functions in vivo.

Ku polypeptides synthesized in vitro assemble into complexes which recognize ends of double-stranded DNA.

The Ku protein is composed of two polypeptide subunits, p70 and p80, and binds DNA ends in vitro. Previous studies suggested that p70 and p80 are physically associated in vivo, although such an association may have been mediated by DNA. We have now utilized full-length Ku polypeptides synthesized in vitro to examine the association of p70, p80, and linear DNA to form a complex. In gel filtration chromatography, p70 migrates as a 70-kDa structure, whereas p80 migrates at 150 kDa. Co-translation of the two cDNAs yields complexes which migrate at 300 kDa and contain equimolar quantities of the p70 and p80 polypeptides, providing direct evidence that p70 and p80 assemble into a complex in the absence of DNA. To demonstrate that this recombinant protein complex binds DNA, we developed a radiolabeled protein electrophoretic mobility shift assay. When radiolabeled proteins synthesized in vitro were incubated with linear DNA and fractionated in a nonreducing, nondenaturing gel, a band representing a complex of p70, p80, and the DNA was seen. Formation of this Ku-DNA complex required free DNA ends, and binding to DNA ends was not observed with individual p70 or p80 subunits. DNA binding was not reconstituted by mixing the individual subunits together. These studies thus demonstrate that it is the complex of p70 and p80, not individual p70 or p80, which possesses the DNA binding properties previously described for native Ku protein. These results provide new information about the assembly, structure, and DNA binding properties of the Ku protein.

An autoantibody to single-stranded DNA: comparison of the three-dimensional structures of the unliganded Fab and a deoxynucleotide-Fab complex.

Crystal structures of the Fabs from an autoantibody (BV04-01) with specificity for single-stranded DNA have been determined in the presence and absence of a trinucleotide of deoxythymidylic acid, d(pT)3. Formation of the ligand-protein complex was accompanied by small adjustments in the orientations of the variable (VL and VH) domains. In addition, there were local conformational changes in the first hypervariable loop of the light chain and the third hypervariable loop of the heavy chain, which together with the domain shifts led to an improvement in the complementarity of nucleotide and Fab. The sugar-phosphate chain adopted an extended and "open" conformation, with the base, sugar, and phosphate components available for interactions with the protein. Nucleotide 1 (5'-end) was associated exclusively with the heavy chain, nucleotide 2 was shared by both heavy and light chains, and nucleotide 3 was bound by the light chain. The orientation of phosphate 1 was stabilized by hydrogen bonds with serine H52a and asparagine H53. Phosphate 2 formed an ion pair with arginine H52, but no other charge-charge interactions were observed. Insertion of the side chain of histidine L27d between nucleotides 2 and 3 resulted in a bend in the sugar-phosphate chain. The most dominant contacts with the protein involved the central thymine base, which was immobilized by cooperative stacking and hydrogen bonding interactions. This base was intercalated between a tryptophan ring (no. H100a) from the heavy chain and a tyrosine ring (no. L32) from the light chain. The resulting orientation of thymine was favorable for the simultaneous formation of two hydrogen bonds with the backbone carbonyl oxygen and the side chain hydroxyl group of serine L91 (the thymine atoms were the hydrogen on nitrogen 3 and keto oxygen 4).

The T-cell independent antigen, NP-ficoll, primes for a high affinity IgM anti-NP response.

In a number of different strains of inbred mice, immunization with a hapten coupled to a protein carrier results in production of homogeneous serum antibodies. At the genetic level this corresponds to the use of a very limited set of variable region genes in the actively secreting B-cells. In contrast, immunization with the same hapten coupled to a T-cell independent (TI) carrier produces a heterogeneous antibody response. Here we show that successive immunizations of C57BL/6 mice, first with the hapten NP coupled to ficoll, a TI carrier, and then one month later with a subliminal dose of the same hapten coupled to a protein carrier, generate a novel set of hybridomas. These hybridomas produce antibodies which are of the IgM isotope and which lack somatic mutation. Some of these antibodies have a much higher affinity for NP than do antibodies which use the prototypical gene combination (VH186.2-lamda 1) of the strain specific response in C57BL/6 mice.

A limited number of B cell lineages generates the heterogeneity of a secondary immune response.

We have studied the cellular basis for heterogeneity in the secondary immune response by creating a large set of B cell hybridomas from a single C57BL/6 mouse immunized with the hapten (4-hydroxy-3-nitrophenyl)acetyl coupled to chicken gamma-globulin, and searching among the cells for all possible sets of clonally related lines. Among 28 independent cell lines from a single animal we find that 21 of them fall into seven small families of from two to five members; only seven lines have no matches among the set. A statistical analysis determines that the number of genetically distinct B cell precursors whose progeny were isolated during this secondary response is 18, with 95% confidence limits of 14 and 26. The distribution of family sizes implies that each activated clone has proliferated to approximately the same extent. Complete sequencing of the variable region of immunoglobulin heavy chain mRNA from 19 of the cell lines reveals many shared somatic mutations among related lines, implying a pronounced founder effect has occurred to eliminate most of the progeny of each precursor B cell. This may be the result either of a high frequency of debilitating somatic mutation or from selection against cells still expressing the idiotype of primary anti-(4-hydroxy-3-nitrophenyl)acetyl antibodies.