A. Arthur Gottlieb

Macrophage regulation of DNA synthesis in lymphoid cells: Effects of a soluble factor from macrophages

Abstract Addition of rat peritoneal macrophages to nonadherent rat spleen cells in culture results in enhancement or suppression of DNA synthesis depending on the ratio of macrophages to lymphocytes. At high ratios of macrophages to lymphocytes (1:5), suppression can be observed as early as four hours. Macrophages suppress incorporation of thymidine (TdR) by nonadherent spleen, thymus and bone marrow cells, in most instances, to less than 5% of that observed in culture to which macrophages were not added. In the presence of macrophages, incorporation of [3H]uridine and [14C] amino acids by spleen cells was also moderately suppressed. Based on 51Chromium release and dye exclusion assays, it appears that suppression is not due to cytotoxicity. Furthermore, suppression of [3H]TdR incorporation by nonadherent spleen cells is reversible, in the presence of an antigenic stimulus, following removal of the macrophages from the cultures. The suppressive effects are not elicited by extracts of macrophages, freeze-thawed or heated macrophages, but appear to be due to a low molecular weight, heat stable factor released into the macrophage culture fluid.

Two ribonuclease H activities from the murine myeloma, MOPC-21

Abstract Two distinct enzymatic activities capable of degrading the RNA strand of an RNA · DNA hybrid have been isolated from the murine myeloma, MOPC-21. These activities can be distinguished by their respective chromatographic and sedimentation properties as well as pH optima and divalent cation requirements.

Lymphoid cell RNA's and immunity.

Publisher Summary This chapter discusses how the biochemists are puzzled by the apparently crude nature of the systems with which the immunologists work, the failure to define precisely the components and products of the system, and the semi-quantitative nature of many immunologic assays. The interactions of “RNA” with the “lymphoid system” lacks form. The chapter focuses on certain phenomena that justifiably stimulate further investigation. The chapter describes that many of these phenomena should be defined, and the reasons for conflicting results from one laboratory to another require clarification. The chapter showsconcern over the mode of action of the various RNA species, whose effects are not easily explained in terms of conventional functions of RNA. This indicates that it may have underestimated the mechanisms by which RNA may serve in the differentiation of antibody-forming cells, and suggests that RNAs may operate by as yet undetermined mechanisms in such systems, in addition to serving conventional roles as tRNAs, mRNAs or as components of the ribosome. It is likely that the future will yield a much clearer picture of many of the phenomena described herein.

Binding of synthetic copolymers to the macrophage ribonucleoprotein.

Abstract The binding of the optical enantiomorphs, poly(Glu 60 Ala 30 Tyr 10 ) ( l -GAT) and poly( d Glu 60 d Ala 30 d Tyr 10 ) ( d -GAT), to rat peritoneal exudate ribonucleoprotein (RNP) was investigated. the l -GAT copolymer exhibited an average of twenty times the binding of the d -GAT copolymer. This difference was paralleled by a difference in the degree of conversion of the two enantiomorphs to a trichloroacetic acid (TCA)-soluble form. On the other hand, no discrimination in binding was observed when radiation-degraded fragments of both copolymers were used. These results support the conclusion that peritoneal exudate cells must carry out a sterospecific degradation step before antigen can bind to the RNP molecule. Because the quantitative difference inbinding of the two optical enantiomorphs seemed to correlate with their difference in immunogenicity, further experiments were performed to determine if such a correlation were a general rule. Conversion of the d -GAT copolymer to a more immunogenic form by complexing it with methylated bovine serum albumin (MBSA) did not increase the amount of binding to the RNP molecule. In addition, the binding of an immunogenic, neutral analogue of the l -GAT copolymer, poly[N 5 -(3-hydroxypropyl)Gln 60 Ala 30 Tyr 10 ], was less than the binding of the weakly immunogenic d -GAT copolymer. Although attachment in all four cases was correlated with the amount of TCA-soluble fragments, no definite quantitative correlation between immunogenicity and binding to the macrophage RNP could be established. However, these results do not preclude a role for the RNP molecule in the induction of an immune response as factors other than the absolute quantitative amount of binding might be important.

Evidence for double stranded RNA region in macrophage antigen-binding RNP

Abstract Peritoneal macrophages contain a ribonucleoprotein (RNP) which is unique to this cell type. Upon exposre of macrophages to antigens, fragments of the antigens are found to be associated with the RNP molecule. In this report, we provide evidence that the RNP molecule contains a double stranded RNA component. The RNP moiety can be degraded by treatment with a combination of ribonucleases A, T 1 and III followed by heating. It is suggested that both the polypeptide component and the double stranded RNA component of the complex contribute to the resistance of the complex to ribonucleases.

THE NATURE OF ANTIGEN-RIBONUCLEOPROTEIN COMPLEXES

President Conant of Harvard once said, “Behold the turtle-he makes progress only when he sticks his neck out.” It is our purpose to do just that in regard to the serious question raised by many immunologists about the role of antigen-RNA complexes in immunity. First we must determine whether such complexes really do exist in nature, or whether they are, as Dr. Goodman would have us believe, only laboratory artifacts. The history of the development of these studies is well known. The contributions of Garvey and Campbell * and of Fishman and Adler regarding macrophage RNA, and the subsequent work of Friedman and colleagues as well as that of Askonas and Rhodes on the presence of antigen in such RNA extracts have been adequately reviewed in the literature. The important issue in this discussion is the relationship of antigen(s) to RNA species in the macrophage. Are there some guidelines for this interaction indicating that genuine, and probably important, associations do occur in vivo: and, how can we distinguish such interactions from random associations of antigen with RNA, either through Mg++ ion chelation of negative charges on the antigen with phosphate groups on the RNA or through some other mechanism? The approach that we have adopted to answer these questions is well known, but let us review the salient features. RNA extracted from adherent peritoneal cells, when banded in cesium sulfate, displays a minor light density ( p = 1.58 g/cc) species we have referred to as macrophage ribonucleoprotein (RNP).s This species has an estimated molecular weight of 12,000, contains 28% protein, has considerable double-stranded structure in the RNA component, and, curiously, when antigens are presented to peritoneal exudate (p.e.) cells in vitro, fragments of antigen are “captured” into this complex. We can readily calculate that the fragment of antigen in this complex cannot exceed 3,600 daltons.“. This immediately raises the question of whether such a fragment of antigen can be “relevant” to the immune response, since it is clear that such a fragment cannot preserve many of the gross features of the tertiary configuration of native antigen. In this regard, we note that this minor light density band or RNP species carried all of the immunological activity of the Fishman system. It is important, before proceeding further, to settle the issue of whether RNP per se is a laboratory artifact. This question can easily be resolved by reference to a study of macrophage ribosomes by Bishop and Gottlieb.R In that work it was demonstrated that RNP was distributed in a 2: 1 ratio between cellular supernatant and ribosomes. The fraction on the ribosomes was released under conditions of high ionic strength. When macrophage ribosomes were subjected to density gradient ultracentrifugation, the RNP species could be seen to band at an appropriate density ( p = 1.54 g/cc) relative to the ribosome. In this instance, no phenol or other reagent was introduced to extract RNA.

Antigen Capture by a Unique Ribonucleoprotein of Macrophage Cells

The association of antigens with polynucleotides has been a subject of intense interest in the past years. In particular, several reports have appeared which suggest that the association of antigen(s) with ribonucleic acids in vitro leads to an augmentation of the immunogenic properties of the substances so complexed (Fishman et al. 1961, 1963; Askonas and Rhodes, 1965). Polynucleotides have also been implicated as nonspecific stimulators of the immune response (Braun and Nakano, 1967).

DISCUSSION PAPER: FUNCTIONS OF RNA IN IMMUNITY

This meeting has been an interesting blend of biochemistry, immunology, and medicine. The subject of “RNAs and immunity” seems to please neither the immunologists nor the biochemists. The former find it uncomfortable to work with molecules whose functions are not easily understood in relatively simple terms. RNAs are an oppressive burden and, from this point of view, are most conveniently dealt with as artifacts. The biochemist, on the other hand, finds this area lacking for other reasons: The complexity and heterogeneity of the cell populations involved, the failure in many cases to precisely define and identify the components and products of a reaction mixture, the lack of a more rigorous application of stoichiometric principles, and the semiquantitative nature of many of the assays make the biochemist uncomfortable. These comments would suggest that we abandon this area as having little potential. Nevertheless, the practical applications of these phenomena, as in the presentations this afternoon, and the imaginative application by Dr. Lawrence of “transfer factor” to the solution of intractable clinical problems suggest that we should pursue these studies; indeed, we would be negligent if we did not do so. Moreover, there are certain observations evolving that suggest that something important is now occurring. I shall mention only five, but there are others: