In vitro inhibition of lymphoproliferative responses to tumor associated antigens and of lymphoma cell proliferation by rat splenic macrophages.

Spleens from W/Fu rats bearing a syngeneic progressively growing (C58NT)D tumor contain cells which can inhibit lymphoproliferative responses in a mixed lymphocyte-tumor interaction designed to demonstrate suppressor activity. Spleens from rats having rejected (C58NT)D tumors also contained suppressor cells but to a lesser degree. The growth inhibition assay, which measures inhibition of proliferation of tumor cells, was evaluated as a simple assay system to screen for suppressor cell activity. The effector cells in both assays had the same characteristics, indicating a predominant role of macrophages. Normal rat spleens were found to contain growth inhibition activity which led to the demonstration of suppressor cell activity in spleens of normal animals. Removal of suppressor cells from the spleens of immunne rats results in consistently higher lymphoproliferative responses to tumor associated antigens on the tumor cells.     

Inhibition of the mixed lymphocyte culture by peritoneal exudate cells.

Inhibition of mixed lymphocyte cultures (MLC) by macrophages and by supernatants of short term cultured macrophages was assessed by incorporation of ³H-thymidine (TdRH3) and also by blast cell counts and by determination of cellmediated lympholysis. Peritoneal exudate cells (PEC) induced by thioglycollate, at concentrations >10%, inhibited all three parameters of MLC. Lower concentrations of PEC, and supernatants from cultured PEC, inhibited TdRH3 incorporation, but had no significant effect on blast cell counts or on generation of cytotoxic effector cells. Inhibition by the supernatants could be reversed by dialysis or by use of low specific activity TdRH3. These data indicate that macrophages can inhibit proliferative responses in MLC, but that this must be carefully distinguished from selective inhibition of TdRH3 incorporation.     

Inhibition of in vitro lymphoproliferative responses by in vivo passaged rat 13762 mammary adenocarcinoma cells. I. Characteristics of inhibition and evidence for an infectious agent.

Small numbers of X-irradiated 13762 cells added as third-party cells to mitogen response assays or mixed lymphocyte cultures caused a significant reduction in viability of the cocultivated lymphocytes, and completely inhibited the expected lymphoproliferative responses. Results showed that the factor(s) responsible for the inhibitory effect was preserved after ultrasonic disruption of the tumor cells, could be sedimented by ultracentrifugation, and was sensitive to treatment with ultraviolet light. Further, cytopathic effects could be serially propagated using cell-free supernatants obtained from sonicated 13762 tumor cells. The results suggest that the 13762 adenocarcinoma line, as carried in vivo in this laboratory, harbors an infectious particle which can affect the proliferative responses of lymphocytes in vitro.     

The influence upon mitogenic and cellular immunologic reactive systems in vitro by poly(I:C) and BCG murine interferons induced in vivo.

We have previously reported that lymphocytes from W/Fu rats immunized with syngeneic (C58NT)D tumor cells were cytotoxic against these cells in a 4-hr ⁵¹Cr release assay. We have investigated the feasibility of cryopreserving lymphocytes and target cells and have selected freezing conditions which provide good yields of viable cells and functional activity. Lymphocytes from different animals had a recovery of 60–80% viability which resulted in a corresponding 55–75% recovery of cytotoxic activity. Repeated testing of lymphocyte cytotoxicity from a pool of frozen spleen cells against either fresh or frozen (C58NT)D cells gave reproducible cytotoxicity. In addition, recovery of high levels of lymphocyte function was also demonstrated when cryopreserved cells were employed in long-term cytotoxic assays, i.e., ³H-proline and ¹²⁵IUdR release assays, in the lymphoproliferative response to mitogens (PHA and Con A)³ or tumor cells (MLTI) as measured by ³H-thymidine incorporation, and in the in vitro generation of secondary cytotoxicity.By employing these cryoprotective techniques it is possible to have: 1) a population of lymphoid cells with known functional activity and 2) a pool of target cells with known susceptibility to lysis and antigenic content. Furthermore, the use of frozen cells as internal standards in each test also permits the analysis of assay variation as well as the study of variation in various cell types.     

Author index for volume 31

Kilham rat virus (KRV) was isolated from the lymphocyte cytopathic 13762 summary adenocarcinoma tumor line described in part I of this report, as well as three other in vivo passaged rat tumors maintained in the same animal room. It could not, however, be isolated from the noncytopathic (CS8NT)D tissue culture line. Tests done with CsCl₂-purified KRV preparations showed that the virus could replicate in rat lymphocytes and could profoundly depress lymphocyte viability and lymphoproliferative responses. Heterologous anti-KRV antiserum could reverse the inhibitory effects of the purified virus preparation and the inhibitory effects of ultrasonically disrupted KRV-infected tumor cells, but could only partially reverse the inhibitory properties of X-irradiated whole 13762 tumor cells. The results suggest that KRV could account for some, if not all, of the inhibitory properties of the 13762 tumor line.     

Studies of the T-lymphocytes resident in the spleens of thymectomized, irradiated, bone marrow-reconstituted (TXB) mice.

The T-lymphocytes resident in the spleens of thymectomized, lethally irradiated mice that had been reconstituted with syngeneic bone marrow (TXB) were characterized. Both recently reconstituted N-TXB, (approximately 3 weeks after bone marrow injection) and aged (>6 months after reconstitution) A-TXB animals were studied. The T-lymphocytes from spleens of recently reconstituted N-TXB donors did not respond to PHA but did react significantly to Concanavalin A (Con A). The lack of PHA sensitivity was not due to dilution of reactive cells by other cell types. Removal of adherent cells, likewise, did not restore N-TXB spleen cell PHA responsiveness. N-TXB splenic T-cells were cortisone resistant. N-TXB spleen cells by themselves did not cause a graft vs host response. However, N-TXB spleen cells amplified the graft vs host response of normal lymph node cells but not N-TXB lymph node cells. Addition of cyclic GMP enhanced [³H]thymidine uptake of N-TXB spleen cells caused by Con A. N-TXB spleen cells were exclusively spleen seeking. The Con A reactive cell within N-TXB spleens was demonstrated to be of donor origin. Fetal liver as well as syngeneic bone marrow contained cells capable of reconstituting the Con A response. Spleen cells from aged. (>6 months) A-TXB were found to be PHA sensitive. Competitive inhibition assays measuring θ expression in A-TXB spleen cells indicate a significant increase in the θ positive lymphocyte population occurred with time. The data indicate that considerable reconstitution of θ positive cells had occurred in A-TXB donors. The results also suggest that the T-lymphocyte population of the TXB spleen may be a unique subpopulation of T-lymphocytes that resides exclusively in spleen and bone marrow.     

Cell-mediated immunity in experimental filariasis: lymphocyte reactivity to filarial stage-specific antigens and to B- and T-cell mitogens during acute and chronic infection.

To study immunological responses in chronic filarial infections, a model utilizing inbred Lewis rats infected with Brugia pahangi was developed. Microfilaria were found in the bloodstream of over 90% of the rats by 16 weeks of infection. Using in vitro lymphocyte blastogenesis, cell-mediated immune responses of blood, splenic, and mesenteric node lymphocytes were followed during 1.5 years of infection. Lymphocyte responses to antigen prepared from infective stage filarial larvae were detectable in the early weeks of infection, whereas responses to microfilarial antigen only developed late as microfilaremia waned. Lymphocyte responses to antigen from adult filaria vacillated during the infection. With the mitogens, phytohemagglutinin, pokeweed mitogen, and bacterial lipopolysaccharide, periods of B and T-cell hyporesponsiveness were demonstrable. Between 16 and 36 weeks of infection node lymphocytes from many rats were unresponsive to all mitogens and antigens. The model of B. pahangi in inbred rats offers advantages for immunological studies of filarial infections.     

Ability of dialysates containing transfer factor to induce lymphocyte activating factor by human mononuclear cells.

Dialysates of human leukocyte lysates containing transfer factor (TF_d) stimulated human mononuclear cells (MNL) to produce lymphocyte activating factor (LAF). Both unfractionated and adherent MNL cultures were stimulated by TF_d to produce a factor which was mitogenic for murine thymocytes and had the biochemical characteristics of LAF as determined by Bio-Gel P-100, DEAE cellulose, and hydroxylapatite chromatography. Fractionation of TF_d on Sephadex G-25 showed that the specific transfer factor activity of converting in vivo skin tests was present in the major uv-absorbing peak, whereas the substance(s) that induced LAF activity was present in a number of the other fractions. Therefore, the capacity of TF_d to induce monocytes to produce LAF is not a measure of classical transfer factor activity. However, this effect of TF_d may instead participate in the nonspecific immunoenhancing effects of TF_d.     

Immunological reactivity of the lung: VII. Effect of corticosteroids and cyclophosphamide on the Fc receptor function of alveolar macrophages

The effects of various in vitro and in vivo regimens of either corticosteroid or cyclophosphamide administration on guinea pig alveolar macrophages were studied. Corticosteroid- and cyclophosphamide-induced immunosuppression was assessed by the effect of drug administration on the capacity of alveolar macrophages to attach to and/or ingest antibody-coated sheep red blood cells (SRBC). In vitro hydrocortisone (up to 20 μg/ml) had no effect on either the binding or ingestion of antibody-coated SRBC. Two separate regimens of in vivo corticosteroids were given: a single dose of iv hydrocortisone (100 mg/kg), which is a short-acting soluble preparation, and sc doses of cortisone acetate (100 mg/kg for 7 days), which is a depot preparation resulting in sustained levels of plasma cortisol of the magnitude of that found for a brief period of time following iv injection of hydrocortisone. Both regimens resulted in similar degrees of peripheral blood lymphocytopenia and monocytopenia 4 and 24 hr, respectively, following injection. The regimen of hydrocortisone has previously been reported to have no effect on alveolar macrophage cytotoxic effector function in antibody-dependent cellular cytotoxicity (ADCC), whereas the cortisone acetate regimen markedly suppressed ADCC. In the present study, hydrocortisone had no effect on either the binding or ingestion of antibody-coated SRBC by alveolar macrophages. In contrast, cortisone acetate caused a marked decrease in both the binding and ingestion of antibody-coated SRBC. This suppressive effect was maximal at suboptimal concentrations of antibody on the SRBC and could be overcome by increasing the concentrations of anti-SRBC antibody. Alveolar macrophages from animals treated with daily cyclophosphamide (a regimen which suppresses ADCC) were capable of binding and ingesting antibody-coated SRBC normally. Thus, prolonged exposure to corticosteroids in vivo causes an alteration in membrane Fc receptor function of alveolar macrophages, which can explain this impaired ability to kill target cells. Since cyclophosphamide therapy did not interfere with the binding and ingestion of antibody-coated target cells, it is concluded that the impairment in killing of target cells by alveolar macrophages is not directly related to an alteration of Fc receptor function but to a defect in the actual killing process.     

Induction of anti-hapten antibody response by hapten-isologous carrier conjugate. II. Specificity of hapten-reactive helper T cells.

Anti-hapten antibody production was elicited by the immunization of hapten-isologous carrier conjugate (PAB-MGG) in mice. Spleen and lymph node cells taken from these primed mice could demonstrate their helper activity for anti-DNP antibody production when transferred intravenously into 600R X-irradiated recipient mice along with DNP-primed B cells and the double hapten conjugated carrier, DNP-MGG-PAB. Isologous carrier (MGG)-primed cells could not demonstrate this helper activity. Accordingly, helper cells reactive for a haptenic group are considered to develop by the immunization of hapten-isologous carrier conjugate. Hapten-reactive helper activity was also induced by the immunization of other hapten-isologous carrier conjugates, e.g., MAB-MGG, PABS-MGG or PAB-MSA. These hapten-reactive helper cells were T lymphocytes, as the helper activity of PAB-MGG-primed cells was completely abolished by in vivo ATS-treatment. Helper activity of PAB-MGG-primed cells for DNP-primed B cells was also demonstrated through the double hapten conjugated heterologous carrier DNP-HGG-PAB to be the same as with DNP-MGG-PAB, but weakly through DNP-KLH-PAB. As HGG but not KLH resembles MGG in composition, almost all hapten-reactive helper T cells can be considered to recognize not only haptenic groups but also physicochemical properties of the hapten-conjugated carrier site. However, these helper T cells could discriminate structural differences among related haptenic groups, because PAB-MGG-primed cells clearly responded to DNP-MGG-PAB to demonstrate their helper activity for DNP-primed B cells, but responded only weakly to DNP-MGG-PABS or DNP-MGG-MAB. When the specificity restrictions of T and B cells to the same haptenic group were compared by responsiveness measured after the antigenic stimulation (B cell function by anti-hapten antibody production and T cell function by helper activity), differences were noted, as PAB-MGG-primed T cells could respond not only to DNP-MGG-PAB but also fairly well to DNP-MGG-MAB to demonstrate their helper activity, but PAB-MGG-primed B cells responded to only PAB-MGG. Thus, hapten specificity appears to be much more restricted for B cells than T cells. The difference of this responsivity between B cells and helper T cells was thought to derive from the specificity difference of B cell and helper T cell receptors rather than from any sensitivity differences of the experimental procedure. The differences in the specificity restrictions of receptors of B and helper T cells were discussed in the light of hapten-specificity.     

Production of macrophage migration inhibitory factor and lymphotoxin by leukocytes from normal and Wiskott-Aldrich syndrome patients

The production of macrophage migration inhibitory factor (MIF) and lymphotoxin (LT) by cultured leukocytes from patients with Wiskott-Aldrich syndrome (WAS) and normal controls was studied. The presence of these lymphokines in leukocyte culture supernatants usually correlated directly with the dose of stimulant used. Doses of nonspecific mitogens and specific antigens, which produced maximal in vitro lymphocyte transformation, stimulated maximal production of these mediators. When the incorporation of tritiated thymidine by stimulated leukocyte cultures from patients with Wiskott-Aldrich syndrome (WAS) was deficient, they usually produced less MIF and lymphotoxin than normal. However, when their in vitro lymphoproliferative responses were normal, the lymphotoxin activity in supernatants of WAS leukocyte cultures was normal.     

Cerebrospinal fluid flow and iodide131 transport in the spinal subarachnoid space

Free Na¹³¹ I and ¹³¹I-Albumin were injected in the cisterna magna of rhesus monkeys. The dynamics of descent into the spinal subarachnoid space and transport out of the cerebrospinal fluid were determined by gamma scintigraphy. ¹³¹I-Albumin moved slowly caudally, reaching the sacral CSF in three hours. Free Na¹³¹I was rapidly absorbed locally and did not descend. When its transport out of cerebrospinal fluid was inhibited by the addition of unlabeled isotonic Na I, ¹³¹I descended slowly at a rate parallel to that of tagged albumin. Injection of Na¹³¹I in hypertonic solutions caused immediate descent. Two minute periods of tumbling activity caused rapid movement of Na¹³¹I and ¹³¹I-Albumin into the lumbar spinal fluid. Na¹³¹I dynamics may serve as a model for other molecules actively transported out of cerebrospinal fluid, such as 5-hydroxy-indoleacetic acid; descent into caudal spinal fluid may depend on the degree of efflux from cerebrospinal fluid and on the animal's activity.     

Species differences in hepatic glutathione depletion, covalent binding and hepatic necrosis after acetaminophen

Acetaminophen, a widely prescribed analgesic that causes fulminant hepatic necrosis in overdosed humans, produced varying degrees of hepatotoxixity in mice, rats, hamsters, guinea pigs and rabbits. The severity of hepatic injury paralleled the rate of activation of acetaminophen by hepatic microsomal enzymes to a potent arylating agent. The severity of hepatic damage in various species also correlated directly with the rate of hepatic glutathione depletion after acetaminophen. These findings support the hypothesis that the electrophilic arylating agent formed from acetaminophen $\text{in}$$\text{vibo}$ is preferentially detoxified by conjugation with glutathione and that arylation of hepatic macromolecules occurs only when glutathione availability is exceeded. Since N-hydroxylation of another N-acetylarylamine (2-acetylaminofluorene) occurs to a much greater extent in the species that are susceptible to acetaminophen-induced hepatic necrosis, the data also are consistent with the hypothesis that the toxic metabolite of acetaminophen results from N-hydroxylation.     

Suppression of lymphokine production: I. Macrophage-mediated inhibition of MIF production

Macrophages have been found to suppress the in vitro production by stimulated T lymphocytes of a lymphokine, migration inhibitory factor. When macrophages isolated from primary MSV-induced tumors were added to antigen-stimulated MSV-immune spleen cells, a complete suppression of MIF production was observed. This suppression was nonspecific, since MIF production by antigen-stimulated alloimmune spleen cells and by PHA-stimulated normal spleen cells was also inhibited. Suppressor macrophages could also be induced by inoculation with Corynebacterium parvum, whereas light mineral oil-induced peritoneal macrophages had no detectable effect on MIF production. The failure to detect MIF in the supernatants of stimulated cultures containing activated macrophages appeared to be due to inhibition of lymphokine production rather than to absorption or inactivation of MIF or to interference with the assay for detection of MIF. Macrophages were able to suppress MIF production only when added during the first 4–5 hr of culture and they had no effect when added later. These data show that activated macrophages can nonspecifically suppress lymphokine production and that this appears to be due to inhibition of an early step in lymphocyte stimulation.     

Mechanisms involved in the expression of Jones-Mote hypersensitivity: II. Lymph node morphology and in vitro correlates

Lymph node morphology, in vitro lymphocyte transformation, and inhibition of macrophage migration were studied at varying intervals after sensitization for Jones-Mote hypersensitivity (JMH) with ovalbumin (OA) in Freund's incomplete adjuvant (FIA). The effect of cyclophosphamide (CY, 300 mg/kg), given 3 days before sensitization with OA in FIA, was also studied in an attempt to clarify further its role in increasing the intensity of skin reactions and its effect on the passive transfer of skin reactivity described in the preceding paper. There were increased numbers of large pyroninophilic cells in paracortical areas of draining lymph nodes and increased in vitro DNA synthesis, by lymph node cells, in animals treated with CY 3 days before sensitization with OA in FIA. There was no inhibition of macrophage migration of PEC from animals sensitized with OA in FIA, whether or not these guinea pigs had been treated with CY before sensitization.     

Activation of the alternative complement pathway by lymphoblastoid cell lines derived from patients with Burkitt's lymphoma and infectious mononucleosis.

Cultured human lymphoblastoid cell lines derived from patients with Burkitt's lymphoma or infectious mononucleosis were shown to activate the alternative pathway of complement fixation. This reaction does not require any conventional antibody directed against the cells. Although the reaction showed an absolute dependence on the presence of factor B it was relatively independent of the presence of factor D or of properdin. To this extent activation of the alternative pathway by lymphoblastoid cells resembles that produced by “C3-nephritic factor.” Rat and mouse complement were activated in a manner similar to human complement, but guinea pig complement was inactive. Chicken complement, unlike any of the mammalian complements tested, was able to bring about lysis of the lymphoblastoid cell lines by the alternative pathway.     

Activity and distribution of lysosomal enzymes during collagenous matrix-induced cartilage,bone, and bone marrow development

The appearance of the lysosomal enzymes acid phosphatase, arylsulfatase, and β-glucuronidase was studied during endochondral bone and bone marrow formation induced by implantation of demineralized bone matrix. The activities of acid phosphatase and β-glucuronidase gradually increased from the stage of mesenchymal cell proliferation on Day 3 onward to reach a peak on Day 13, during maximal bone remodeling. However, arysulfatase activity exhibited a sharp increase on Day 9, associated with the onset of cartilage hypertrophy and chondrolysis. The peak of arylsulfatase activity was also attained on Day 13. The activities of all three enzymes declined on Day 15 but acid phosphatase again exhibited an increase during hematopoietic bone marrow differentiation on Days 19–21. Histochemical and ultrastructural studies revealed intense lysosomal enzyme activity in macrophage-like cells on Day 7 and thereafter. During chondrolysis and bone remodeling, these cells were present in a perivascular location. Osteoclasts also exhibited strong reactivity for the lysosomal enzymes. Due to its characteristic temporal appearance during development of endochondral bone, arysulfatase may be used as a marker enzyme for chondrolysis and bone resorption.     

The distribution of cryoprotective agents into lipid interfaces

Capillary rise measurements indicate that the cryoprotective agents dimethyl sulfoxido, glycerol, sucrose, hydroxy ethyl starch, polyvinyl pyrrolidone, and dextran are surface active, suggesting the possibility that they distribute into the lipids of the cell membrane. However, Langmuir trough experiments and surface pressure-temperature measurements using aliphatic alcohols and cephalin show that these more efficient surfactants exclude the glycerol and DMSO from the interface. We conclude that DMSO and glycerol must exert their influence on the aqueous side of the monolayer. In contrast, the data for PVP indicate that it enters the lipid monolayer, but can easily be excluded by surface pressure.     

The stimulation of collagen secretion by ascorbate as a result of increased proline hydroxylation in chick embryo fibroblasts.

Collagen secretion by chick embryo fibroblasts was measured by incorporating [¹⁴C]proline into proteins and then analyzing the amount of collagen in the cell and medium separately by using purified bacterial collagenase. In order to produce varying levels of hydroxylation, cells were incubated with varying concentrations of ascorbate or with varying concentrations of α,α′-dipyridyl in the presence of saturating ascorbate. Ascorbate stimulated both the hydroxylation of proline in collagen and the secretion of collagen; the concentration of ascorbate required for half-maximal stimulation of both proesses was approximately 4.5 × 10^?7, m. Since the cells could concentrate ascorbate 10-fold, this K_M for proline hydroxylation is 100-fold lower than values reported for purified prolyl hydroxylase (Abbot, M. T., and Udenfriend, S. (1974) in Molecular Mechanisms of Oxygen Activation (Hayaishi, O., ed.), p. 173, Academic Press New York; Kivirikko K. I., et al. (1968) Biochim. Biophys. Acta, 151, 558–567). Conversely, α,ga′-dipyridyl inhibited both proline hydroxylation and collagen secretion; half-maximal inhibition of both processes was observed at 7 × 10^?5, m. The results of the two types of experiments show that the secretion of collagen becomes directly proportional to proline hydroxylation when approximately 30% of the proline residues in collagen have been hydroxylated compared to maximal hydroxylation of 50%. Since the stability of triple-helical collagen at 37 °C has been shown to be dependent on the hydroxyproline content of the molecule (Rosenbloom, J., et al. (1973) Arch. Biochem. Biophys., 158, 478–484), we suggest that the observed proportionality between secretion and hydroxylation is a reflection of the increased amount of stable triple helical collagen at 37 °C. When the cells were incubated with a concentration of ascorbate that was saturating for secretion and hydroxylation, there was no significant activation of prolyl hydroxylase as measured in a cell-free extract. These experiments suggest that ascorbate effects collagen secretion by acting at the site of proline hydroxylation but not by increasing the activity of prolyl hydroxylase.     

The effects of corticosteroids on immunoregulation in sarcoidosis.

The effects of in vivo hydrocortisone administration on the kinetics and functional capabilities of cells involved in the immune response in sarcoidosis were examined. Untreated sarcoidosis patients have a decrease in the absolute numbers of circulating T lymphocytes (P < 0.05). However, with regard to the proportions of T lymphocyte subpopulations, there is an increase in the relative proportions of IgG Fc receptor positive T cells (T_G) (P < 0.01), which have suppressor capabilities in certain in vitro systems of mitogen-induced antibody production, and a relative decrease in IgM Fc receptor positive T lymphocytes (T^M) which have helper effects in this system (P < 0.05). Additionally, sarcoidosis patients have circulating “suppressor” monocytes capable of suppressing anti-sheep red blood cell (SRBC) plaque-forming cell (PFC) responses by pokeweed mitogen (PWM)-stimulated lymphocytes. The in vitro removal of this cell abrogated this depressed response (P < 0.01). Intravenous administration of hydrocortisone produced a transient absolute T lymphocytopenia (P < 0.01) accompanied by a relative increase in T^G cells (P < 0.01) and a relative decrease in T_M cells (P < 0.02). Four hours after hydrocortisone therapy, at the point of maximal hydrocortisone-induced monocytopenia (P < 0.01), the suppressed ability of sarcoidosis lymphocytes to synthesize and secrete in vitro anti-SRBC antibody after polyclonal activation was corrected (P < 0.01), and PFC responses comparable to those seen in untreated normal subjects were obtained. These studies demonstrate that corticosteroid administration has profound effects on certain in vitro demonstrable immunoregulatory abnormalities in sarcoidosis.