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We have investigated whether sequence 67 to 74 shared by beta chains of rheumatoid arthritis (RA)-associated HLA-DR molecules imparts a specific pattern of peptide binding. The peptide binding specificity of the RA-associated molecules, DRB1*0401, DRB1*0404, and the closely related, RA nonassociated DRB1*0402 was, therefore, determined using designer peptide libraries. The effect of single key residues was tested with site-directed mutants of DRB1*0401. The results have demonstrated striking differences between RA-linked and unlinked DR allotypes in selecting the portion of peptides that interacts with the 67-74 area. Most differences were associated with a single amino acid exchange at position 71 of the DR beta chain, and affected the charge of residues potentially contacting position 71. The observed binding patterns permitted an accurate prediction of natural protein derived peptide sequences that bind selectively to RA-associated DR molecules. Thus, the 67-74 region, in particular position 71, induces changes of binding specificity that correlate with the genetic linkage of RA susceptibility. These findings should facilitate the identification of autoantigenic peptides involved in the pathogenesis of RA.

The major histocompatibility complex (MHC) class II molecules are highly polymorphic membrane glycoproteins that bind peptide fragments of proteins and display them for recognition by CD4+ T cells. To understand the effect of human MHC class II polymorphism on peptide-MHC interaction, we have isolated M13 phage from a large M13 peptide display library by selection with DRB1*0401 and DRB1*1101 molecules, as recently described for DRB1*0101. Sequence analysis of the peptide-encoding region of DR-bound phage led to the identification of position-specific anchor residues, defining motifs for peptide binding to DR molecules. The three DR motifs share two anchor residues at relative positions 1 and 4, while allele-specific anchor residues have been identified at position 6. These results provide a biophysical basis for both the promiscuity and the specificity of peptide recognition by DR molecules.

Murine macrophage clones were generated from thymus, spleen, brain, and bone marrow by in vitro immortalization with recombinant retroviruses carrying an avian v-myc oncogene. The cloned cell lines express F4/80 molecules, exert phagocytosis, have nonspecific esterase activity, and express class II molecules after interferon gamma activation. The macrophage clones are diploid and their karyotypes have remained stable for greater than 3 years in culture. After the macrophage clones were activated, their pattern of cytokine production was investigated. Functional heterogeneity in cytokine transcription was demonstrated: one of six liposaccharide-activated macrophages was unable to transcribe interleukin 1 alpha, whereas all of the liposaccharide-activated clones were able to transcribe tumor necrosis factor alpha. Interleukin 6 production was detected in three of six clones. The production of nitrite and tumor necrosis factor alpha as effector molecules of cytotoxicity was detected in all clones, thus showing that a single macrophage can exert more than one cytotoxic mechanism. The results indicate that immortalized and cloned macrophages have a differentially regulated expression of cytokine genes, adding further evidence for the existence of functional heterogeneity among cloned macrophages. This heterogeneity seems to derive from differentiation-related mechanisms rather than from external constraints.

We have recently immortalized murine brain macrophages (microglial cells) with a complex of retroviruses (3RV) transducing separately the myc and mil oncogenes. Surprisingly, the immortalized cells harboured an exogenous v-myc oncogene, but no v-mil sequences. The transformed macrophage cell lines grew in vitro without the addition of exogenous growth factors and were also able to grow in vivo in nude mice. In addition, they released oncogenic retroviruses able to immortalize mouse macrophages from primary splenic or thymic cultures. Molecular cloning of the provirus (VN-11) harboured in a microglial clone demonstrated that no cell-derived sequences apart from an avian v-myc gene were transduced by the recombinant retrovirus. When cells were tested for production of myeloid growth factors, they were found to transcribe and synthesize the Macrophage-Colony Stimulating Factor (M-CSF). The correlation between viral infection and activation of the M-CSF gene was tested using a M-CSF dependent cell line from which growth factor independent clones could be readily obtained after infection. The synthesis of M-CSF was detected only in cells expressing the avian v-myc protein. These data support the hypothesis that, in our conditions, macrophages can be immortalized by the expression of v-myc and the concomitant establishment of an autocrine loop triggered by viral infection.

Cytokine-mediated communication between the immune system and the nervous system has been shown in the past few years. The precise cellular sources of these molecules in the brain is still a controversial issue. We have thus immortalized primary cell cultures from mouse embryonic brains to analyze cloned cells involved in cytokine production. The cell clones obtained were identified as microglial cells and shown to produce several monokines. Among these, TNF alpha was detected by molecular analysis and cytotoxicity assays and shown to be expressed by microglial cells, after activation with LPS. Surprisingly, the TNF alpha-mediated cytotoxic activity, which was neutralized by specific antisera, was not detected in the cell supernatants but was mediated through cell-to-cell contact. Using antibodies to TNF alpha in FACS analysis, specific cell membrane staining on live microglial cells was shown. The results suggest that in the brain the form of TNF alpha detectable by standard procedures is the cell bound form and not the most common form, secreted TNF alpha. In addition, the effects of recombinant TNF alpha in vitro and in vivo were evaluated. In vitro, rTNF alpha stimulated beta-endorphin, GH, and PRL release from cultured cells prepared from rat anterior pituitary glands. In vivo, the administration of rTNF alpha to rats was able to modify analgesic responses. The concomitant administration of naloxone, an opiate receptor antagonist, or monoclonal anti-IL-1 antibody decreased the analgesic effects induced by rTNF alpha. This indicates that the analgesic effect might not be mediated directly by rTNF alpha but by other mediators, whose action is under the control of TNF alpha.

Yeast DNA primase and DNA polymerase I can be purified by immunoaffinity chromatography as a multipeptide complex which can then be resolved into its functional components and further reassembled in vitro. Isolated DNA primase synthesizes oligonucleotides of a preferred length of 9-10 nucleotides and multiples thereof on a poly(dT) template. In vitro reconstitution of the DNA primase:DNA polymerase complex allows the synthesis of long DNA chains covalently linked to RNA initiators shorter than those synthesized by DNA primase alone. The SS (single-stranded) circular DNA of phage M13mp9 can also be replicated by the DNA primase:DNA polymerase complex. Priming by DNA primase occurs at multiple sites and the initiators are utilized by the DNA polymerase moiety of the complex, so that almost all the SS template is converted into duplex form. The rate of DNA synthesis catalyzed by isolated yeast DNA polymerase I on the M13mp9 template is not constant and is characterized by distinct pausing sites, which partly correlate with secondary structures on the template DNA. Thus, replication of M13mp9 SS DNA with the native primase:polymerase complex gives rise to a series of DNA chains with significantly uniform termini specified by the primase start sites and the polymerase stop sites.

A 5600-base pair segment spanning the coding region of the Saccharomyces cerevisiae DNA polymerase I gene was sequenced and found to contain an open reading frame of 1468 codons, corresponding to a polypeptide of Mr 166,794. A pol1 temperature-sensitive mutation, encoding a DNA-polymerase-primase complex with altered stability, has a single base-pair substitution that changes the glycine at position 493 to a positively charged arginine. Protein sequence comparison with other prokaryotic and eukaryotic DNA polymerases reveals three major regions of homology. This observation suggests that certain DNA polymerases might require the conservation of critical amino acid residues for activity.

The immunopurified yeast DNA-polymerase-I--DNA-primase complex synthesizes oligo(rA) and oligo(rG) molecules that are used as primer for replication of poly(dT) and poly(dC). Neither initiation nor DNA synthesis is observed with poly(dA) and poly(dI). Nitrocellulose-filter binding shows that the enzyme complex binds to deoxypyrimidine polymers, but not to deoxypurine polymers. Although the yeast complex initiates DNA synthesis on deoxypyrimidine homopolymers, it prefers to elongate pre-existing primer molecules rather than to initiate de novo DNA replication. The size of the oligo(rA) and oligo(rG) primer molecules has been determined by urea/polyacrylamide gel electrophoresis: longer oligoribonucleotides are synthesized when their utilization is prevented by omitting dNTP. An oligodeoxythymidylate template with a chain length as short as five residues can support oligo(rA) synthesis catalyzed by the yeast DNA-polymerase--DNA-primase complex and the size of the oligoribonucleotide products synthesized with oligodeoxythymidylate of differing chain length has also been determined. The mechanistic properties of the DNA-polymerase--DNA-primase complexes, purified from different eukaryotic organisms, appear to be very similar. The possible biological implication of the studies on the mechanism and specificity of initiation of DNA synthesis in a well-defined model template system has been discussed.