The Journal of Experimental Medicine

Thompson, R. A.; Lachmann, P. J.

doi: 10.1084/jem.131.4.629pmid: 4193934

This paper describes the characteristics of the indicator factor (I) which takes part in reactive hemolysis and its identification as the seventh component of complement. I was shown to be a beta globulin with a sediment coefficient of 5.7S and a molecular weight of about 140,000. Experiments on the depletion of I activity with anti-I antiserum or with activated R euglobulin showed that I was a late acting complement component necessary for the lysis of cells after the EAC142 stage. Complement component analysis of purified I fractions excluded all known components except C7. The physicochemical characteristics of I are compatible with published data on C7. The method of quantitation described represents a convenient method of testing for C7. Footnotes Submitted: 25 August 1969

Lachmann, P. J.; Thompson, R. A.

doi: 10.1084/jem.131.4.643pmid: 4193935

It has been shown that the "activated reactor" that is produced in certain human sera by complement activation is a stable complex of the fifth and sixth component of complement (C56). On interaction with C7, the indicator factor, a complex C567 is formed which for a short time (half-life less than 1 min) has an activated binding site and can attach itself to normal red cell membranes, conferring on them the hemolytic properties of the "heat stable" complement intermediate EC 1 ∼ 7, the capacity to be lysed by C8 and C9. These cells have neither antibody nor the complement components up to C3 bound on them. The binding site—activated C567c—can similarly bind to other hydrophobic surfaces, including agarose gel where it forms a "stainable line". If the complex is not bound to a surface, the binding site decays and the resulting complex will no longer give rise to lysis. However it will still inactivate C8 and C9 in solution. The sera that can generate activated reactor apparently do so because they have an excess of C5 and C6, compared to their content of C7. The phenomenon of reactive lysis thus represents complement-mediated lysis of unsensitized cells initiated at the C5 stage by a stable complex (C56) which was generated by complement activation at a distance. The immunochemistry of the phenomenon is described and some of its implications discussed. Footnotes Submitted: 25 August 1969

Bowman, Catharine G.; Bonventre, Peter F.

doi: 10.1084/jem.131.4.659pmid: 5430783

The effect of diphtheria toxin on subcellular components of protein synthesis was determined. Polyribosomes prepared from intoxicated guinea pigs functioned normally in an in vitro assay system, while the activity of soluble enzymes (transferases) from toxin-treated animals was significantly reduced. At high toxin dosages, this reduction was widespread, but when levels of toxin comparable to those which might be generated in a natural infection were given, inhibition of soluble enzyme activity was found only in extracts from heart and skeletal muscle. Possible nonspecific inhibition in the assay system due to interference by free toxin or by a serum component was eliminated. Since it was possible to demonstrate reactivation of soluble enzyme activity with nicotinamide and toxin, it was suggested that diphtheria toxin acts in the intact sensitive animal in a manner analogous to its action in tissue culture or in cell-free systems. It was hypothesized that the lethal biochemical lesion of the toxin in sensitive animals was the inactivation of transferase enzymes, principally in the heart. It was also suggested that the lethal lesion induced in diphtheria-sensitive and resistant species may not be identical. Footnotes Submitted: 26 October 1969

Miller, J. F. A. P.; Mitchell, G. F.

doi: 10.1084/jem.131.4.675pmid: 5464380

Collaboration between thymus-derived lymphocytes, and nonthymus-derived antibody-forming cell precursors occurs during the immune response of mice to sheep erythrocytes (SRBC). The aim of the experiments reported here was to attempt to induce tolerance in each of the two cell populations to determine which cell type dictates the specificity of the response. Adult mice were rendered specifically tolerant to SRBC by treatment with one large dose of SRBC followed by cyclophosphamide. Attempts to restore to normal their anti-SRBC response by injecting lymphoid cells from various sources were unsuccessful. A slight increase in the response was, however, obtained in recipients of thymus or thoracic duct lymphocytes and a more substantial increase in recipients of spleen cells or of a mixture of thymus or thoracic duct cells and normal marrow or spleen cells from thymectomized donors. Thymus cells from tolerant mice were as effective as thymus cells from normal or cyclophosphamide-treated controls in enabling neonatally thymectomized recipients to respond to SRBC and in collaborating with normal marrow cells to allow a response to SRBC in irradiated mice. Tolerance was thus not achieved at the level of thelymphocyte population within the thymus, perhaps because of insufficient penetration of the thymus by the antigens concerned. By contrast, thoracic duct lymphocytes from tolerant mice failed to restore to normal the response of neonatally thymectomized recipients to SRBC. Tolerance is thus a property that can be linked specifically to thymus-derived cells as they exist in the mobile pool of recirculating lymphocytes outside the thymus. Thymus-derived cells are thus considered capable of recognizing and specifically reacting with antigenic determinants. Marrow cells from tolerant mice were as effective as marrow cells from cyclophosphamide-treated or normal controls in collaborating with normal thymus cells to allow a response to SRBC in irradiated recipients. When a mixture of thymus or thoracic duct cells and lymph node cells was given to irradiated mice, the response to SRBC was essentially the same whether the lymph node cells were derived from tolerant donors or from thymectomized irradiated, marrow-protected donors. Attempts to induce tolerance to SRBC in adult thymectomized, irradiated mice 3–4 wk after marrow protection, by treatment with SRBC and cyclophosphamide, were unsuccessful: after injection of thoracic duct cells, a vigorous response to SRBC occurred. The magnitude of the response was the same whether or not thymus cells had been given prior to the tolerization regime. The various experimental designs have thus failed to demonstrate specific tolerance in the nonthymus-derived lymphocyte population. Several alternative possibilities were discussed. Perhaps such a population does not contain cells capable of dictating the specificity of the response. This was considered unlikely. Alternatively, tolerance may have been achieved but soon masked by a rapid, thymus-independent, differentiation of marrow-derived lymphoid stem cells. On the other hand, tolerance may not have occurred simply because the induction of tolerance, like the induction of antibody formation, requires the collaboration of thymus-derived cells. Finally, tolerance in the nonthymus-derived cell population may never be achieved because the SRBC-cyclophosphamide regime specifically eliminates thymus-derived cells leaving the antibody-forming cell precursors intact but unable to react with antigen as there are no thymus-derived cells with which to interact. Footnotes Submitted: 28 October 1969

Hahn, Helmut H.; Cheuk, S. Fai; Elfenbein, C. Dianne S.; Wood, W. Barry

doi: 10.1084/jem.131.4.701pmid: 5430784

Only intact exudate granulocytes from rabbits generated large amounts of endogenous pyrogen when incubated in 0.15 M NaCl. No matter how whole-cell lysates or combinations of subcellular fractions were incubated, their yields of pyrogen never approached those of whole cells; at most, only minimal amounts of pyrogen were formed, once the integrity of the cells had been destroyed. Some pyrogen could be extracted from disrupted cells, but never more than a fraction (<25%) of that released from incubated whole cells. The yield could be slightly improved by lowering the pH (to 3.5) and by increasing the volume of extraction fluid. Virtually all of the preformed pyrogen that could be extracted from sucroselysed cells was found in their cytoplasmic fraction. Contrary to the results of Herion et al. (3), none could be detected in the granular (or lysosomal) fraction. Likewise, all efforts to recover pyrogen from the membrane-nuclear fraction were unsuccessful. In keeping with the finding that preformed pyrogen is contained in the cytoplasmic fraction were the observations that practically all of the aldolase, a cytoplasmic enzyme, and very little of the acid phosphatase, a granular enzyme, were lost from the cells during the release of pyrogen. Lysozyme, an enzyme stored in both the granules and the cytoplasm, was partially released from the cells under the same circumstances. Neither the release of pyrogen nor its slight intracellular buildup that precedes release (4) were affected by concentrations of puromycin that block protein synthesis in the cells and prevent their activation. Hence, it is concluded that the release process, which also involves the formation of active pyrogen (4), does not require protein synthesis, whereas activation of the cells, which may involve the synthesis of an inactive precursor (2), does. Footnotes Submitted: 9 November 1969

Unanue, Emil R.; Cerottini, Jean-Charles

doi: 10.1084/jem.131.4.711pmid: 5430785

Macrophages were cultured for several hours after a brief exposure to radio-iodinated keyhole limpet hemocyanin. Most of the hemocyanin taken up by the macrophages was rapidly catabolized and eliminated from the cell. A few molecules were retained on the plasma membrane of the cells for prolonged periods and were not subject to endocytosis and catabolism. These few molecules of hemocyanin bound to the plasma membrane were identified by observing the fixation of antibody fragments to macrophages at low temperature. The membrane-bound antigen, which could be removed by trypsin or EDTA, was of large molecular size, though heterogeneous. A great part of the immune responses of mice to hemocyanin bound to live macrophages could be abrogated by treatment of the macrophages in vitro with antibody or trypsin. Hence, most of the immunogenicity of hemocyanin bound to macrophages was attributed to the few molecules of antigen bound to the plasma membrane. Footnotes Submitted: 23 November 1969

Nordlund, James J.; Root, Richard K.; Wolff, Sheldon M.

doi: 10.1084/jem.131.4.727pmid: 5430786

Release of the protein molecule, leukocytic pyrogen, is one of the many reactions exhibited by leukocytes after phagocytosis. After the ingestion of heat-killed S. albus , a 3–4 hr latent period exists, during which human peripheral leukocytes release no pyrogen, yet cellular metabolism is altered in such a way that pyrogen output may subsequently occur in the absence of further phagocytosis. Transcription of messenger RNA and translation of new protein are initial events in the. activation process, since addition of the inhibitors, actinomycin D, and cycloheximide or puromycin, during this period markedly depressed or abolished subsequent pyrogen release. These effects were noted to be dependent upon the time of addition of the inhibitors. None of the inhibitor drugs interfered with cell viability as measured by phagocytosis and hexose monophosphate shunt activity, nor did they alter the pyrogenicity of preformed leukocytic pyrogen. Vincristine did not inhibit pyrogen formation, consistent with its reported failure to alter RNA synthesis in mature human granulocytes. The glycolytic inhibitor, sodium fluoride, blocked pyrogen release both when added prior to particle ingestion or 1 hr after the initiation of phagocytosis. Whereas inhibition of phagocytosis would explain the sodium fluoride effect prior to 1 hr, this was not observed in leukocyte preparations incubated for 1 hr with S. albus before adding sodium fluoride. When sodium fluoride was added to preparations 2 hr after the start of incubation, the LP production was unimpaired. Potassium cyanide had no effect on cell activation or pyrogen release. These findings suggest that the primary energy supply for the activation process is derived from high energy phosphate bonds provided by anaerobic glycolysis. Since the major amount of cell activation appears to occur in the 1st hr after phagocytosis, this energy might be involved in the induction of a genome leading to the transcription of m-RNA and its translation into new protein or is required for polysome integrity during protein synthesis. It is suggested that this new protein may be leukocytic pyrogen itself, or an enzyme responsible for cleaving it from an inactive precursor. Footnotes Submitted: 9 October 1969

Thompson, A.; Halbert, S. P.; Smith, U.

doi: 10.1084/jem.131.4.745pmid: 5430787

Pulsating mammalian myocardial cells were found to be highly susceptible in tissue culture to rapid destruction by streptolysin O. Cessation of beating occurred almost immediately, followed within minutes by multiple cell membrane bleb formation. Parallel with these changes, the cytoplasm became intensely granular and the nuclear membrane apparently thickened when viewed by phase microscopy. At the ultrastructural level, the cell membrane blebs were found to contain relatively small numbers of granular fragments. The endoplasmic reticulum of damaged heart cells was quite swollen, and its contents were considerably condensed. The myofibers were not strikingly altered, but cytoplasmic and mitochondria vacuoles were rather abundant. Cardiac endothelial, kidney epithelial, and fibroblast cells were also susceptible to lysis by this toxin, but the reactions occurred more slowly or bleb formation was less evident. An antiserotonin drug known to be protective against streptolysin-O in vivo (UML-491), did not protect against killing of cardiac cells at the tissue culture level. Serotonin could not be detected in the culture fluid after lysis of cardiac cells by streptolysin O. Footnotes Submitted: 14 October 1969

Rossi, Giovanni B.; Cudkowicz, Gustavo; Friend, Charlotte

doi: 10.1084/jem.131.4.765pmid: 4914375

Proliferation and erythroid differentiation of transplanted DBA/2 marrow cells and Friend virus-induced leukemic cells were assessed in syngeneic, allogeneic ( H-2 compatible), and (BALB/c x DBA/2)F 1 hybrid mice (CDF 1 ). Measurements were made 5 days after transplantation of donor cells into nonirradiated or X-irradiated mice by the spleen colony or the 125 IUdR- 59 Fe uptake methods. Growth of DBA/2J (Jackson subline) marrow grafts was poor in irradiated CDF 1 J hybrids as compared with growth in syngeneic and allogeneic hosts. The DBA/2J transplants proliferated, however, without impairment in irradiated CDF 1 hybrids which were the progeny of DBA/2 male parents of other sublines, e.g. DBA/2Ha, DBA/2Cr, and DBA/2Cum. In contrast, tissue-cultured Friend leukemic cells of DBA/2J origin grew deficiently in all CDF 1 hybrids tested, regardless of irradiation and of the DBA/2 parent's subline. The growth pattern of transplanted DBA/2J cells was a manifestation of hybrid resistance. The results with DBA/2J and other DBA/2 subline grafts suggested that hybrid histocompatibility alleles were expressed to a greater extent in leukemic than in normal marrow cells, for the former were consistently recognized as "nonself" by CDF 1 mice, but not the latter cells. The property of deficient growth in irradiated CDF 1 Ha hybrids was acquired by DBA/2J hemopoietic cells within 6 hr from infection in vivo with Friend leukemia virus, and persisted during the following 8 days. It was ascribed to enhanced expression of hybrid histocompatibility gene(s) ( Hh ) induced by the virus. Autonomous growth potential of hemopoietic cells, manifested by proliferation in nonirradiated recipients, was first detected 24 hr from infection, and likewise persisted at the later intervals. At the same time, the infected cells grew deficiently also in nonirradiated CDF 1 Ha mice. The two irreversible cellular changes were regarded as the earliest signals of virus-induced transformation. Footnotes Submitted: 24 October 1969

Hyslop, N. E.; Dourmashkin, R. R.; Green, N. M.; Porter, R. R.

doi: 10.1084/jem.131.4.783pmid: 5430788

Hapten-antibody complexes prepared at equivalence with the bivalent hapten bis -DNP-octamethylene-diamine and purified rabbit anti-DNP antibody were fractionated by Sepharose gel-filtration and the fractions examined by electron microscopy. Individual fractions were tested for whole-complement fixation and C1 fixation. Dimer forms did not show this type of biological activity, while fractions containing tetramers and larger polymers exhibited both C and C1 fixation, which could be inhibited by prior exposure of the complexes to the univalent hapten epsilon-DNP-caproic acid. The dose-response result indicated that the C-fixation observed was not due to interpolymeric cooperative effects. It was concluded that in the generation of biological activity by soluble antigen-antibody complexes made with complement-fixing antibody, quaternary structural changes following specific combination with antigen may be as important as any tertiary structural alterations that occur in the individual immunoglobulin molecule. Footnotes Submitted: 13 November 1969