The Journal of Experimental Medicine

Theofilopoulos, Argyrios N.; Dixon, Frank J.; Bokisch, Viktor A.

doi: 10.1084/jem.140.4.877pmid: 4139225

In the present work we studied the expression of membrane-bound Ig (MBIg) as well as receptors for IgG Fc and complement on nine human lymphoblastoid cell lines. When MBIg and receptors for IgG Fc were compared, four categories of cell lines could be distinguished: ( a ) cell lines having both MBIg and receptors for IgG Fc, ( b ) cell lines having MBIg but lacking receptors for IgG Fc, ( c ) cell lines lacking MBIg but having receptors for IgG Fc, and ( d ) cell lines lacking both MBIg and receptors for IgG Fc. Two types of receptors for complement could be detected on the cell lines studied, one for C3-C3b and one for C3d. When sensitized red cells carrying C3b or C3d were used for rosette tests, three categories of cell lines could be distinguished: ( a ) cell lines having receptors for C3b and C3d, ( b ) cell lines having receptors only for C3d and ( c ) cell lines lacking both receptors. However, when a more sensitive immunofluorescent method was used instead of the rosette technique, it was found that cell lines unable to form rosettes with EAC1423b hu were able to bind soluble C3 or C3b which indicated the presence of these receptors on the cell surface. Inhibition experiments showed that receptors for C3-C3b and receptors for C3d are distinct and that receptors for C3-C3b and C3d are different from receptors for IgG Fc. A cell line (Raji) without MBIg but with receptors for IgG Fc, C3-C3b, and C3d was selected for use in studying the binding mechanism of soluble immune complexes to cell surface membrane. Aggregated human gamma globulin was used in place of immune complexes. Immune complexes containing complement bind to Raji cells only via receptors for complement, namely receptors for C3-C3b and C3d. Binding of immune complexes containing complement to cells is much greater than that of complexes without complement. Immune complexes bound to cells via receptors for complement can be partially released from the cell surface by addition of normal human serum as well as isolated human C3 or C3b. We postulate that such release is due to competition of immune complex bound C3b and free C3 or C3b for the receptors on Raji cells. Footnotes Submitted: 13 June 1974

Fu, S. M.; Kunkel, H. G.

doi: 10.1084/jem.140.4.895pmid: 4139226

Hemagglutination and fluorescent antibody studies have provided strong evidence for the unavailability or absence of specific antigenic sites on membrane-bound IgM which are present in serum and intracellular IgM. Antisera specific for different parts of the molecule indicated that a portion but not all of the Fc was involved. Absorption experiments with normal and leukemic viable B lymphocytes failed to remove a population of Fc antibodies found in IgM-specific antisera. Similar findings were made for IgD, the other major membrane immunoglobulin of human peripheral blood B cells. Various interpretations of these observations are discussed. The most likely possibility appears that the C-terminal portion of the heavy chains of the immunoglobulin molecule is buried in the membrane. Footnotes Submitted: 27 June 1974

Julius, Michael H.; Herzenberg, Leonard A.

doi: 10.1084/jem.140.4.904pmid: 4139227

Cells binding DNP groups conjugated to fluoresceinated mouse gamma globulin ( F DNP-MGG) were isolated from spleens of unprimed mice using a fluorescence-activated cell sorter (FACS). The isolated cells were specifically enriched at least 100-fold for anti-DNP precursor activity in an adoptive transfer assay as compared to unfractionated spleen. The fraction depleted of binding cells, although depleted of anti-DNP precursor activity, responded as well as unfractionated spleen when assayed for anticarrier (keyhole limpet hemocyanin KLH) precursor activity. High avidity binding cells were stained using low concentrations of F DNP-MGG. Medium and low avidity binding cells were stained using high concentrations of F DNP-MGG in the presence of free hapten which selectively blocked staining of the high avidity binding cells. Cells were supplemented with an excess of carrier-primed (KLH), nylon-purified splenic T cells and transferred to irradiated recipients. DNP-KLH was given at transfer and 5 days later. The anti-DNP plaque-forming cell (DNP-PFC) response and the avidities of the DNP-PFC in the irradiated recipients were measured by hapten inhibition of direct PFC plaque formation 12 days after transfer. At this time, very few indirect PFC were found. There was a positive correlation between the avidity of the DNP-binding cells and the avidity of the anti-DNP antibody secreted by their progeny. High avidity DNP-binding cells gave rise to predominantly high avidity anti-DNP-PFC. Medium and low avidity binding cells gave rise to medium and low avidity DNP-PFC. Footnotes Submitted: 7 May 1974

Pierce, Carl W.; Kapp, Judith A.; Solliday, Susan M.; Dorf, Martin E.; Benacerraf, Baruj

doi: 10.1084/jem.140.4.921pmid: 4610076

The effects of alloantisera against leukocyte alloantigens on plaque-forming cell (PFC) responses to sheep erythrocytes and the terpolymer of L -glutamic acid 60 - L-alanine 30 - L -tyrosine 10 (GAT) by mouse spleen cells in vitro have been investigated. Polyspecific antibodies against both H-2 and non- H-2 alloantigens on responding spleen cells suppressed both IgM and IgG PFC responses; antisera against alloantigens coded for by the K and I regions, but not the D region, of the H-2 complex also effectively suppressed PFC responses. The suppression was not due to cytotoxicity to the spleen cells or anti-immunoglobulin activity in the sera and was directly related to the amount of antiserum added to the cultures. The suppression was specific for spleen cells against which the alloantiserum was directed. The alloantisera suppressed responses most effectively when present during the first 24 h of incubation, and although not rendering lymphoid cells incapable of developing PFC responses after removal of noncell-bound antibody, did act by interfering with successful initiation of the PFC response. The alloantisera suppressed both IgM and IgG PFC responses when directed against alloantigens only on macrophages, but selectively suppressed IgG responses when directed against alloantigens only on lymphoid cells. The alloantisera did not interfere with the ability of macrophages to bind GAT or to support the viability of the lymphoid cells, but did interfere with the ability of macrophage-associated antigen to effectively stimulate antibody responses by the lymphoid cells. Possible mechanisms for the effects of alloantisera on macrophages and the selective suppression of IgG responses when the antisera are directed against alloantigens on lymphoid cells are discussed with reference to our current understanding of genetic restrictions governing cell interactions in the development of antibody responses in mice. Footnotes Submitted: 11 June 1974

Stackpole, Christopher W.; Jacobson, Janet B.; Lardis, Michael P.

doi: 10.1084/jem.140.4.939pmid: 4610077

The modulation or loss of thymus-leukemia (TL) antigenicity from the surfaces of mouse RADA1 leukemia cells and normal thymocytes during incubation with TL antibody in vitro at 37°C was investigated by cytotoxicity, immunofluorescence, and immunoelectron microscopy. The fate of bivalent and monovalent antibody during modulation was visualized by fluorescence microscopy. Considerable antibody remained bound to the cell surface after modulation, bivalent antibody being displaced topographically into "patches" and "caps" while monovalent antibody was only slightly aggregated on the cell surface. Some antibody was internalized, presumably by pinocytosis, and was sequestered into the Golgi region of the cell. Capping usually occurred over the pole of the cell opposite from the Golgi region, which may explain the lack of extensive pinocytosis of modulating bivalent antibody. Since modulation with monovalent antibody occurs without patch or cap formation, gross topographical redistribution of TL antigen-antibody complexes is not required for modulation, although more subtle displacement of these complexes may be involved. Modulation was demonstrable by cytotoxicity with guinea pig C' but not with absorbed rabbit C', indicating that modulated TL antigens remain bound to the cell surface. A heat-labile factor in TL antiserum and in mouse serum in general is responsible for "blocking" the cytolytic interaction of guinea pig C' with modulated TL antigen-antibody complexes. Footnotes Submitted: 10 June 1974

Bodel, Phyllis

doi: 10.1084/jem.140.4.954pmid: 4427091

The characteristics of pyrogen production and release by human blood monocytes were investigated. A dose-response assay of monocyte pyrogen in rabbits indicated a linear relationship of temperature elevation to dose of pyrogen at lower doses. Monocytes did not contain pyrogen when first obtained, nor did they release it spontaneously even after 5 days of incubation in vitro. Pyrogen production was apparent 4 h after stimulation by endotoxin or phagocytosis, and continued for 24 h or more. Puromycin, an inhibitor of protein synthesis, prevented both initiation and continuation of pyrogen production and release. Pyrogen-containing supernates retained most pyrogenic activity during overnight incubation even in the presence of activated cells. Lymphocytes appeared to play no role in either initiation or continuation of pyrogen production in these studies. Footnotes Submitted: 10 June 1974

Jones, Patricia P.; Cebra, John J.

doi: 10.1084/jem.140.4.966pmid: 4139228

Fluorescent antibody staining with antibodies to the f and g locus allotype markers present on rabbit α-chains revealed that the α-chain is the heavy chain on the Peyer's patch lymphocytes which previously had been shown to be the precursors of IgA-producing plasma cells. In addition, lymphocytes which had been stripped of membrane Ig with pronase and then cultured overnight to allow the sole expression of endogenous membrane Ig were found to have either the µ-chain or the α-chain on their membranes, but not both. These results suggest that most lymphocytes are restricted to the synthesis of one class of heavy chains at a time and that the commitment to synthesizing that particular heavy chain is maintained during the differentiation of lymphocytes into plasma cells. The proportion of lymphocytes with membrane α-chains is higher in the Peyer's patch and appendix, two gut-associated lymphoid tissues (GALT), than in other lymphoid tissues. Since the GALT are enriched sources of precursors for IgA-producing plasma cells compared to nongut-associated tissues, the presence of cells bearing membrane α-chains correlates well with the relative abilities of these tissues to generate IgA plasma cells. Footnotes Submitted: 23 May 1974

Lonai, Peter; McDevitt, Hugh O.

doi: 10.1084/jem.140.4.977pmid: 4547782

In vitro antigen-induced tritiated thymidine uptake has been used to study the response of sensitized lymphocytes to (T,G)-A--L, (H,G)-A--L, and (Phe,G)-A--L in responder and nonresponder strains of mice. The reaction is T-cell and macrophage dependent. Highly purified T cells (91% Thy 1.2 positive) are also responsive, suggesting that this in vitro lymphocyte transformation system is not B-cell dependent. Lymphocytes from high and low responder mice stimulated in vitro react as responders and nonresponders in a pattern identical to that seen with in vivo immunization. Stimulation occurs only if soluble antigen is added at physiological temperatures; antigen exposure at 4°C followed by washing and incubation at 37°C fails to induce lymphocyte transformation. Stimulation is specific for the immunizing antigen and does not exhibit the serologic cross-reactivity which is characteristic of these three antigens and their respective antisera. The reaction can be inhibited by anti- H-2 sera but not by anti-immunoglobulin sera. The anti-immunoglobulin sera did, however, inhibit lipopolysaccharide or pokeweed mitogen stimulation. These results suggest that the Ir-1A gene(s) are expressed in T cells, and that there are fundamental physiologic differences between T- and B-cell antigen recognition. Footnotes Submitted: 25 June 1974

Gordon, Saimon; Unkeless, Ay C.; Cohn, Z. A.

doi: 10.1084/jem.140.4.995pmid: 4427092

The injection of thioglycollate medium into the peritoneal cavity of the mouse induces high levels of macrophage fibrinolytic activity due to the production and secretion of a plasminogen activator, a trypsinlike serine protease, which is absent in unstimulated macrophages. Intraperitoneal injection of endotoxin or mineral oil can stimulate only a fraction (<10%) of the fibrinolytic activity of thioglycollate cells, similar to the partial stimulation (<10%) seen 1–2 days after phagocytosis of latex or SRBC by unstimulated macrophages. The endotoxin-stimulated macrophages contain and release relatively low levels of plasminogen activator, but these primed cells can be triggered to produce and secrete high levels of enzyme, by phagocytosis of latex. Under conditions where the plasminogen activator is induced and secreted, there are no effects on the production and/or release of lysozyme or intracellular acid hydrolases, Discovery of a two-stage procedure for inducing macrophage plasminogen activator made it possible to study the role of cell priming and phagocytosis separately. Endotoxin was a more effective priming agent, weight for weight, than lipid A:BSA complex. Secretion of the plasminogen activator was induced only by thioglycollate, or endotoxin and latex. In situ fibrinolysis was induced by these agents and mineral oil, BCG, and fetal calf serum, in decreasing order of effectiveness. Phagocytosis of latex in all cases except thioglycollate stimulation, increased fibrinolytic activity from three- to sixfold. Latex and a variety of other particles such as M. lysodeikticus , aggregated γ-globulin and immune complexes showed dose-dependent stimulation of fibrinolysis by endotoxin-primed macrophages. Although the initial phagocytic trigger was not specific for the substance employed, the ability to induce a sustained response depended on the persistence of the phagocytized particle within the cell. Fibrinolysis and secretion of plasminogen activator continued at high levels for at least 9 days after uptake of latex, a nondigestible particle, whereas plasminogen activator was secreted only transiently after ingestion of rapidly digested M. lysodeikticus . The induction of plasminogen activator secretion provides a mechanism by which the activated macrophage can exert a selective effect on its extracellular environment. Footnotes Submitted: 24 June 1974

Yoshiki, Takashi; Mellors, Robert C.; Strand, Mette; August, J. T.

doi: 10.1084/jem.140.4.1011pmid: 4279268

The use of monospecific antisera for the analysis by radioimmunoassay and immunofluorescence study of two major viral proteins, gp69/71 and p30 of murine leukemia virus, that could be of significance in the pathogenesis of immune complex glomerulonephritis of mice, particularly NZB and B/WF 1 hybrid mice, yielded the following conclusions. A remarkably high concentration of viral envelope glycoprotein, gp69/71, was detected in the spleen and serum of New Zealand mice (NZB, NZW, B/WF 1 , and W/BF 1 ); the concentration in the spleen was 10-fold greater than that found in AKR mice and 30-fold greater than that present in C57BL/6 mice. The gp69/71 was deposited along with bound immunoglobulins, apparently as an immune complex, in the diseased kidneys of mice, and the glomerular site and extent of deposition of gp69/71 was related to the severity of the glomerulonephritis. This study suggests that the pathogenesis of immune complex glomerulonephritis (and vasculitis) in mice is related to the expression of this specific viral envelope glycoprotein and to the host immune response to this protein. Footnotes Submitted: 9 May 1974