Renal homotransplantation in rats. I. Allogeneic recipients.

Within 3-6 hr after the reestablishment of the circulation, a characteristic pathology developed in renal homotransplants. Blood monocytes and lymphocytes adhered to large thin-walled vessels of the septa carrying interlobular arteries, traversed their walls, and aggregated in the connective tissue spaces around them. Within 3 days, the number and size of the extravascular cells markedly increased, filling the septa completely and spreading from them centrifugally to occupy the intertubular spaces throughout the cortex. The composition of these aggregates at first was a mixture of lymphocytes and monocytes, and later consisted of large blast cells, macrophages, a few plasma cells, and polymorphonuclear leukocytes. Mitotic activity was seen 2 days after surgery among the large blast cells and increased to a maximal level a day later. Coevally with these changes, the thin-walled septal vessels, intertubular veins and capillaries, and finally, arteries and arterioles, in that order, were damaged. Focal injury of tubules was slight 24 hr after homografting; widespread cortical necrosis had developed 5-7 days later. At no time up to 7 days were concentrations of immunoglobulins detected by fluorescence microscopy in the transplanted kidneys. The morphologic manifestations and temporal sequences of renal homograft destruction suggested that several mechanisms acted synergistically to eliminate the transplant. The initial injury appeared to be the result of an interaction between host lymphoid cells and target endothelium, a phenomenon akin to allogeneic inhibition; followed by spreading ischemia; additional contact injury to tubules; and nonspecific inflammation associated with necrobiotic tissue.

Cytology of immunologic memory. A morphologic study of lymphoid cells during the anamnestic response.

Pairs of rats were immunized with keyhole limpet hemocyanin (KLH) and simultaneously labeled with thymidine-methyl-(3)H or 5-iodo-2'-deoxyuridine-(125)I. From 10-50 days later, their lymphoid organs were examined 3 days after anamnestic stimulation with KLH or after primary injection of BGG. Light and electron microscopic study of the labeled cells revealed that immunologic memory resided in the mature resting monoribosomal lymphocyte which, upon stimulation, transformed to an immature polyribosomal lymphocyte and mitotically active blast cell. These latter elements differentiated into plasma cells directly or after mitosis.

Density of surface immunoglobulin and capping on rat B lymphocytes. I. Changes with aging.

The rate of capping and shedding of cross-linked surface immunoglobulins (SIg) was slower in old Lewis rats (greater than 24 mo) than in young Lewis rats (3-4 mo). Analysis of spleen cell populations with the fluorescence-activated cell sorter indicated that with aging there was a loss of cells with a high density of SIg. Cells with the highest density of SIg capped and shed cross-linked SIg faster than cells with a low density of SIg. The alteration in density of SIg may account for the difference in capping kinetics. Colchicine treatment increased the rate of capping of lymphocytes from young animals, but had no effect on the capping kinetics of lymphocytes from old animals.

Cell-mediated cytotoxicity during rejection and enhancement of allogeneic skin grafts in rats.

Cell-mediated cytotoxicity (CMC) in spleens and lymph nodes of allografted rats was determined by release of (51)Cr from labeled target cells incubated with aggressor lymphoid cells. CMC was first detected in grafted adult rats on day 5, peaked on days 7 and 8, and declined rapidly to background levels by days 9 to 11. In allografted neonates and in cyclophosphamide-treated or neonatally thymectomized adults CMC was a fraction of that observed in normal adult rats. Enhancing antibodies deferred in vivo peak activity of CMC in allografted neonates for 3-4 days, and blocked in vitro the action of aggressor lymphocytes by binding to target cells. Enhancing antibodies had no effect on the cytotoxicity of aggressor cells, but horse antibodies to rat thoracic duct cells inhibited in vitro CMC of aggressor cells.

Immunologic competence of thoracic duct cells. I. Delayed hypersensitivity.

Delayed hypersensitivity was produced in donor Lewis rats by sensitization with soluble protein antigens emulsified in complete Freund's adjuvant. Cells of their thoracic duct lymph were collected for varying periods of time and transferred intravenously to isogenic Lewis recipients. With this model the following conclusions were reached: 1. Delayed hypersensitivity was transferred by thoracic duct cells. 2. The longer the drainage of the thoracic duct, the fewer cells were needed to achieve a successful transfer. With continuing drainage the proportion of small lymphocytes decreased and large cells increased. There was, therefore, a better correlation between successful transfer of delayed hypersensitivity and the number of large cells transfused than between positive skin reactions and transfer of small lymphocytes. 3. Prolonged fistula of the thoracic duct did not diminish the skin reaction of sensitized donors to specific antigen. 4. Delayed hypersensitivity was elicited in recipients 3 wk after transfer of sensitized cells. There was evidence that delayed hypersensitivity was enhanced in recipients, possibly because of prior skin testing. 5. Total body X-irradiation abolished the lesions of passively transferred delayed hypersensitivity. Recovery of positive skin tests was observed 19 to 20 days later. 6. The lesions of delayed hypersensitivity were probably mediated by cells. There was no evidence that a circulating high affinity antibody played a role in this type of immunologic reaction.

Heterogeneity of antibody-forming cells. An electron microscopic analysis.

Cell suspensions from draining lymph nodes of immune and nonimmune rats were reacted in vitro with (125)I-labeled antigens. In light microscopic radioautographs of smears, 17% of the immunized cells were tagged by specific antigen; 2.0% of control cells were positive. In electron microscopic radioautographs, 90% of the labeled elements from immune donors were lymphocytes, blast and plasma cells; 10% were monocytes-macrophages or other elements, including naked nuclei. 15% of the labeled cells from control materials were lymphocytes and plasma cells, while 85% were monocytes-macrophages and naked nuclei. Within cell suspensions derived from immunized animals there were almost twice as many lymphocytes marked by isotope as plasma cells, and the lymphocytes ranged in morphology from mature monoribosomal elements to immature polyribosomal cells. Antibody-forming cells fixed labeled antigen at their surfaces. The monocyte-macrophage class was distinguished by a high mean grain count and by distribution of grains within cytoplasmic vacuoles and lysosomes.

The role of thymus and bone marrow cells in delayed hypersensitivity.

Adoptive transfer experiments were performed to define the immunological role of thymus and bone marrow cells in the induction of delayed hypersensitivity (DH). The results indicated the following, (a) Bone marrow from immune donors contained cells capable of being stimulated by antigen to initiate the expression of DH. (b) Bone marrow from nonimmune or tolerant donors contained cells that were needed to complete the expression of DH after the infusion of immune lymph node cells. (c) Normal bone marrow and thymus cells cooperated in the irradiated recipient to induce the most vigorous skin reactions to specific antigen; these reactions were seen only when the recipients were stimulated by antigen. Either cell type alone was ineffective. (d) In the presence of tolerant bone marrow cells, thymus cells from immune donors gave a more vigorous response than did thymus cells from normal or tolerant donors. (e) There was suggestive evidence that thymus cells were the source of trigger elements that initiated DH. (f) Antigen in the irradiated recipient was necessary to induce DH after infusion of bone marrow cells alone, or bone marrow and thymus cells together.

IgE-mediated chemotaxis of rat basophilic leukemia cells towards specific antigen.

We evaluated chemotactic properties of four sublines of rat basophilic leukemia cells using blindwell Boyden chamber assays. After sensitization with a mouse monoclonal IgE directed against dinitrophenyl (DNP), cells from sublines 2H3-C and 926a underwent chemotaxis toward DNP-bovine serum albumin (BSA) and sublines RBL-1 and 4A did not. Chemotactic responses required specific IgE and were determined by the IgE antigen specificity used for sensitization. The threshold for chemotaxis was on the order of 10(-10) M DNP-BSA. Release of incorporated [3H]-serotonin did not always parallel chemotactic responses, which suggests that chemotaxis and secretion may be two unlinked processes that occur during basophil activation. Our results predict a possible in vivo mechanism whereby specific chemotactic responses of basophils and other FcR epsilon-bearing cells are mediated via specific IgE bound to membrane FcR epsilon.

Elimination of syngeneic sarcomas in rats by a subset of T lymphocytes.

Established subcutaneous Moloney sarcomas (MST-1) of large size and long duration were eliminated from syngeneic rats by intravenous infusion of varying numbers of specific syngeneic effector T lymphocytes. Spleen cells from BN rats in which tumor had regressed were cultured in an in vitro mixed lymphocyte tumor cell culture (MLTC) to augment cytotoxicity of effector cells. In the MLTC a T cell subset was expanded in response to MST-1 antigens and transformed into blast elements. With these changes, there was an increase in the W3/25 antigen on the T cell surface, a decrease of W3/13 antigen, and an increase in the number of T cells with Ia antigens. The subset associated with elimination of established tumors was a blast T cell W3/25+, W3/13+, as detected by monoclonal antibodies to rat T antigens. The W3/25+ subset was poorly cytotoxic in vitro for MST-1 and apparently functioned in vivo as an amplifier or helper cell in the tumor-bearing host. The W3/25- population was a melange of cells that included (W3/13+, W3/25-) T cells, null cells, Ig+ cells, and macrophages, and was associated with enhancement of tumor in vivo, suggesting the presence of suppressor cells.

The production of vesicular stomatitis virus by antigen- or mitogen-stimulated lymphocytes and continuous lymphoblastoid lines.

A variety of lymphoid cell populations were examined in terms of their ability to replicate vesicular stomatitis virus (VSV), a lytic, RNA-containing virus maturing at the cell surface. The number of cells capable of producing VSV was estimated in terms of infectious centers by the virus plaque assay (VPA), and morphologically by electron microscopy (EM). The lymphoid cells examined in this study included: (a) lymph node cells from delayed hypersensitive guinea pigs stimulated by specific antigen, (b) mouse spleen cells activated by selective bone marrow-derived (B) cell and thymus derived (T) cell mitogens, and (c) cells of human and murine continuous lymphoblastoid or lymphoma lines. In unstimulated cultures of guinea pig lymph node cells there is a background of approximately 1 in 1,000 cells which produces VSV; in purified protein derivative (PPD)-stimulated cultures the number of cells producing virus was 1.6% in the VPA and 1.9% by EM. These cells were large lymphocytes with some morphological features of transformed lymphocytes but were not typical blast cells. A few macrophages were associated with virus in both stimulated and control cultures. These observations indicate that (a) cells responsive to antigens, as detected by a marker virus, were lymphocytes; (b) cells other than lymphocytes (macrophages) were capable of replicating VSV even without antigenic stimulation; and (c) the correlation of results obtained by VPA and morphologic examination was usually quite good. Of the total number of mouse spleen cells stimulated with concanavalin (Con A), a T cell mitogen, 4.5 (EM)-5.7% (VPA) were associated with VSV. These were characteristic transformed lymphocytes, similar to phytohemagglutinin (PHA)-stimulated human lymphocytes. In contrast Escherichia coli lipopolysaccharide (LPS)-treated mouse spleen cultures contained lower numbers of virus plaque-forming cells. The majority of such cells associated with virus displayed extensive rough endoplasmic reticulum. Two cultured murine lymphomas containing lymphocytes with the theta surface marker (L5178Y and EL-4) showed a 15-100-fold higher incidence of virus-producing cells than leukemias (L1210 and C57Bl/6) which did not carry this marker. Similarly, the L2C guinea pig leukemia, a known B cell leukemia, yielded a low percent of virus plaque-forming cells (<2%). However, MOPC-104, a plasma cell tumor presumed to be of B cell origin, was found to be an efficient virus producer. There was a wide variation in the efficiency of VSV replication among human lymphoblastoid lines. One line, Wil-2, produced 80% infectious centers after 24 h of exposure to VSV, and all cells were associated with virus at the EM level. The relationship between the virus-producing cells and different lymphocyte subpopulations as well as the efficiency of the two assays for studying virus-producing lymphocytes is discussed.

Thymus-dependent lymphocytes in tissue sections of rejecting rat renal allografts.

Lewis kidneys were grafted into BN recipients and examined at daily intervals up to 6 days after grafting with immunofluorescent reagents. A horse antiserum specific for T lymphocytes revealed an increasing number of T lymphocytes in the cellular infiltrates of rejecting allografts. These were detectable 1 day after grafting, reached a maximum 3 days later, and were relatively diminished at 6 days. In control isografts and nonimmunological inflammations of kidney, a small number of dispersed T lymphocytes was seen. A rabbit antirat thymocyte antiserum, given to allografted BN rats, prolonged survival of the grafts and decreased the cellular infiltrate and the number of T lymphocytes in the infiltrates. We conclude that in graft rejection there is a flow of T lymphocytes into areas of tissue damage and these T lymphocytes are immunologically reactive to graft antigens.

Experimental allergic glomerulonephritis induced in the rabbit with homologous renal antigens.

Rabbits immunized to several homologous renal antigens developed a variety of autoantikidney antibodies. Some of these antibodies reacted with the host's glomeruli and appeared to cause glomerulonephritis. Passive transfer of sera from some of these nephritic rabbits into normal, unilaterally nephrectomized rabbits led to the induction of nephritis. The production of autoantibody to glomeruli was transitory in most instances in spite of continued immunization. In some rabbits immunized to whole kidney homogenate, extracts or sediment, antibodies were found fixed to renal tubular basement membranes where an ultrastructural lesion was demonstrated. Rabbits also produced antikidney antibody apparently to tubular cytoplasmic components which did not fix to kidney in vivo and were of no pathogenetic significance. Immunization with autologous renal basement membranes induced a small autoantibody response in half the rabbits. This response was not associated with detectable renal injury.

Studies on the mechanism of fever accompanying delayed hypersensitivity. The role of the sensitized lymphocyte.

Experiments have been carried out to investigate the possible role of the sensitized lymphocyte in mediating the fevers of delayed hypersensitivity. Rabbits were made delayed hypersensitive to one of several heterologous proteins (bovine gamma globulin, bovine serum albumin, or human serum albumin) by footpad injection of antigen or antigen conjugated with dinitrophenol and incorporated in complete Freund's adjuvant. At intervals after sensitization, various tissues were removed, and single cell suspensions were incubated overnight with either carrier protein or conjugate in vitro. Release of an endogenous pyrogen (EP) was assayed by intravenous injection of the supernatant fluid into unsensitized rabbits. Of the tissues tested only those containing both lymphocytes and pyrogen-producing cells, blood, spleen, and draining lymph nodes, released detectable amounts of EP when incubated with antigen in vitro. Incubation of normal blood cells with specifically sensitized lymphocytes and antigen also resulted in significant release of pyrogen. Similarly, blood leukocytes released EP in vitro after mixture with supernates derived from incubation of sensitized lymphocytes and antigen. Cells and supernatant fluids from draining lymph nodes were usually effective in activating normal blood leukocytes earlier after sensitization than were those from mesenteric lymph nodes, suggesting that such cells, or antigen, had migrated from the original site of sensitization. The activator was soluble, nonpyrogenic in the dosages tested, and required incubation of viable cells with specific antigen for its production. These properties suggest that it may belong to the class of "lymphokines," biologically active agents released from lymphocytes that have been activated by immunologic or certain nonimmunologic stimuli.

Production of ultrastructural membrane lesions by the fifth component of complement.

A direct quantitative relationship has been demonstrated between the number of cell bound C4,2 complexes or C5 molecules and the number of ultrastructural lesions visualized on the cell membrane subsequent to immune hemolysis. When bound C4,2 complexes exceeded bound C5 molecules, the number of ultrastructural lesions seen corresponded to the number of C5 molecules. However, in the reverse situation, with bound C5 molecules in excess of bound C4,2 complexes, the latter determined the number of lesions. During the complement-reaction sequence, the lesions first became visible in the nonlytic intermediate complex EAC1,4,2,3,5 and their number was unaffected when lysis was induced by C6-C9. Since the lesions were also demonstrable on the intermediate complex EC5,6,7, it is concluded that the protein C5 is responsible for their production. Once formed, the physical presence of the C5 molecule is no longer required for the manifestation of the lesions as indicated by persistence of lesions after removal of C5 protein by trypsin. The C5-dependent ultra-structural phenomenon has therefore been interpreted to represent a true structural change of the membrane which, however, is not accompanied by a permeability defect.

Production of macrophage migration inhibition factor by continuous cell lines.

Macrophage migration inhibitory factor (MIF) was found in media of human and mouse lymphocyte and fibroblast cell lines that were continuously growing. Its release was dependent on activation of the cells to enter the mitotic cycle, particularly on cells in S phase. The greatest quantity of MIF was detected in supernatants of lymphocytes collected during S phase after the cells were synchronized in G(1) and in supernatants of growing fibroblasts. When the latter were contact inhibited little or no MIF was found in media. MIF was also released into media of cells proliferating in homologous serum in the absence of fetal calf serum and into media lacking any protein. The MIF produced by lymphocyte lines eluted from Sephadex G-100 in the same fashion as MIF produced by the interaction of sensitized guinea pig cells and antigen. The results indicated that MIF is not a specific mediator of delayed hypersensitivity and cellular immunity and that MIF released by sensitized lymphocytes incubated with antigen merely reflects that fraction of cells activated by antigen to enter the mitotic cycle.

Mobility of surface proteins on normal rat macrophages and on a "macrophagelike" rat tumor.

Peritoneal macrophages endocytosed their histocompatibility antigens (RT1), Fc receptors (FcR), and concanavalin A (Con A) receptors after cross-linking by ligands, but did not cap these membrane proteins. The 323N cell, a "macrophage like" tumor cell, under identical conditions capped its surface proteins. Experiments measuring fluorescence recovery after photobleaching showed that the mobile fraction of RT1 was significantly greater in 323N cells than in normal peritoneal macrophages. Presumably, the membrane proteins of 323N are not as tethered to the cytoskeleton, or, if so, are in a nexus that is not the same as that which occurs between membrane proteins of normal macrophages and the cytoskeleton. The mobility of RT1 on normal lymphocytes was also different from that of macrophages. These observations suggest that the movement of membrane molecules is determined by cell type and is regulated by the cytoskeleton which varies in structure and function from cell type to cell type.

Autoradiographic plaques for the detection of antibody formation to soluble proteins by single cells.

Single cells that synthesize antibody to soluble proteins can be detected and counted by an autoradiographic procedure. The method is based on the specific binding of I(131)-labeled antigen to the antidogy which has been produced by and has diffused around cells in an agarose medium and which has been precipitated by an antibody to gamma globulin. The antigen-binding zones appear as dark spots on high-speed x-ray film exposed to the dried agarose preparations.

Alloantigen expression of a rat Moloney sarcoma.

Cells of a brown Norway (BN) rat Moloney sarcoma (MST) failed to express certain BN alloantigen specificities and bound only about 30-50% the amount of labeled alloantibody bound by normal BN spleen cells. MST cells lacked antigen specificities shared by BN and WF rats, but expressed some of the antigen shared by BN and AUG rats. Further loss of alloantigens that occurred with prolonged in vitro culture was associated with reduced virulence of MST cells for syngeneic hosts and with increased expression of tumor-associated antigens. The LEW rats, which are resistant to MST cells, might have rejected the tumor on the basis of factors other than Ag-B antigens.

Genetic control of immune responses to Moloney leukemia virus in rats.

When BN and LEW rats were immunized with untreated or with inactivated Moloney murine leukemia virus (M-MuLV), BN rats produced high antibody responses to the p15, p30, and gp70 antigens of the virus, whereas LEW rats were low responders to these antigens. BN rats also exhibited a high response and LEW rats a low response when the two strains were immunized with purified p30. Studies of (LEW X BN)F1 and backcross rats suggested that factors associated with AgB exerted major influences on responses to antigens of M-MuLV and that other factors were also important. When other rat strains representing 5 AgB alleles were tested, some were high and some were low responders to M-MuLV, and responses to p15, p30, and gp70 were not always parallel. Since M-MuLV replication was greater in cells of BN rats than in cells of LEW rats, replication of M-MuLV may have influenced the levels of responses to some viral antigens. Control of virus replication appeared to be due to cell mechanisms rather than to the environment of the host.

Distribution of viral glycoprotein gp 69/71 on cell surfaces of producer and nonproducer cells.

The distribution of the viral glycoprotein, gp 69/71, was studied on the cell surfaces of virus-producing and nonproducing cells. gp 69/71 was distributed evenly on cell membranes and on viral membranes of cells producing murine leukemia virus. gp 69/71 was distributed uniformly on cell membranes of thymocytes from NZB and strain 129 mice but was not detected on cell membranes of BALB/c thymocytes. Viral particles were associated with NZB and BALB/c thymocytes but were not in or on 129 thymocytes. We conclude that the presence of gp 69/71 on thymocyte membranes is not related to virus production. In the strains showing gp 69/71 on their thymocyte surfaces (NZB and 129), a large percentage of the thymocytes are positively labeled, all more or less equally. Immunochemical analysis of cell surface proteins labeled with 125I corroborated the ultrastructural observations. Thus, gp 69/71 can be expressed on the surface of thymocytes independently of virus particle production.