Nonadherent Spleen Cells Research Articles

Effect of lipopolysaccharide on the stimulation of macrophage Fc-Dependent phagocytosis by splenic B lymphocytes

Macrophage phagocytic activity is regulated by a variety of products derived from activated lymphocytes. It has been reported that nonactivated splenic B and T lymphocytes enhance macrophage glucose metabolism. In addition, the enhancement of macrophage glucose metabolism was further increased by direct effects of bacterial lipopolysaccharide (LPS) on B, but not T, lymphocytes. In the present study, the effect of purified murine splenic B and T lymphocytes on Fc-dependent phagocytosis by thioglycollate-elicited peritoneal macrophages in the presence or absence of LPS has been investigated. Fc-dependent phagocytosis was assayed by measuring the ingestion of 51Cr-labeled, IgG-tagged sheep erythrocytes. After 3 or 4 days in culture, nonadherent spleen cells (NASC) and B and T lymphocytes from C3H/HeN (LPS-responder) mice produced 92 ± 27%, 83 ± 13%, and 147 ± 33% increases in C3H/HeJ (LPS-hyporesponder) macrophage phagocytic activity, respectively. A similar effect was observed in Balb/c mice. Cell-free supernatant from NASC and B lymphocytes precultured for 2 or 4 days produced a 74 ± 20% and 157 ± 42% increase in phagocytosis respectively. At concentrations which have been previously shown to markedly enhance the ability of splenic B lymphocytes to stimulate macrophage glucose metabolism, Escherichia coli K235 LPS (10 μg/ml) did not alter the stimulatory effects of any of the splenic lymphocyte populations on macrophage Fcdependent phagocytosis. These data suggest that B lymphocytes produce a soluble factor(s) which stimulates macrophage phagocytosis. In addition, LPS has different effects on the regulation of macrophage phagocytic activity and metabolism by B lymphocytes.

B cell growth factor activity of immunoaffinity-purified and recombinant human interleukin 2.

We investigated the effect of recombinant and affinity-purified human interleukin 2 (IL2) on human B cell proliferation. Five X 10(4) nonadherent spleen cells that had been depleted twice of T cells were activated by 3-day culture with formaldehyde-killed Staphylococcus aureus Cowan strain I (SAC) prior to addition of tested growth factors. Cultures were harvested 72 h later. It was found that both IL2 preparations led to optimal cell proliferation compared with a control supernatant obtained by 36 h phytohemagglutinin stimulation of spleen mononuclear cells. Moreover, the effect of such spleen supernatant on B cell proliferation correlated with the IL2 activity since its B cell growth factor activity (BCGF) was not greater than that of purified IL2 and no residual BCGF activity could be detected after absorption of all IL2 activity by the IL2-dependent cytotoxic T lymphocyte line cells. T cells, enumerated as E-rosetting cells as well as T3+ cells, represented 0.2 to 2% of the cells recovered at termination of the cultures (day 6) and there were less than 1% E-rosetting cells in freshly purified or SAC-activated (day 3) B cell populations. Therefore, we conclude that IL2 is a growth factor not only for activated T cells but also for activated human B cells.

Effect of N-acetyl-muramyl-L-alanyl-D-isoglutamine on interferon production in mice by Newcastle disease virus.

The activity (carbon clearance) of the reticuloendothelial system (RES) of mice inoculated intraperitoneally with N-acetyl-muramyl-L-alanyl-D-isoglutamine (muramyl dipeptide, MDP) was greatly stimulated 1 day, but not 7 days after MDP treatment. No enhancement of resistance to ectromelia virus infection and influenza virus infection in mice treated with MDP was observed. In mice splenectomized 1 week after MDP pretreatment, normal levels of circulating interferon were produced in response to Newcastle disease virus (NDV), whereas in the mice treated with MDP after splenectomy, circulating interferon levels were reduced to the same level as produced in the MDP-untreated and splenectomized mice. Interferon production in response to NDV was augmented in non-adherent peritoneal and spleen cell cultures derived from MDP-pretreated mice, whereas it was reduced in peritoneal and splenic macrophage cultures. These results suggest that the non-adherent spleen cells activated with MDP were disseminated from the spleen to other organs, that the lack of enhancement of interferon production in mice pretreated with MDP might be due to reduced interferon production in macrophages, and that the activation of the RES of the whole body by MDP did not correlate with the enhancement of interferon production in spleen cells or with the reduction of interferon production in macrophages.

Induction of suppressor T cells in culture — I. Cell-cell interactions

The present study was designed to examine the cellular requirements for the generation of the suppressor T cells induced in the presence of fetal calf serum in culture. When C57B1/6 mouse spleen cells were cultured for 4–5 days, these precultured cells were shown in mixing experiments to suppress the generation of cytotoxic effector cells (CTL) against allogeneic P815 cells or the generation of anti-SRBC humoral response by freshly explanted C57B1/6 spleen cells. Spleen cells cultured in the presence of silica (0.5 mg) for 4 days, did not develop suppressor activity. However, when silica was added 3 days after the start of the suppressor generation culture, the development of suppressor cells was only slightly affected, although the phagocytic activity of these spleen cells was still totally abolished. When plastic or G-10 Sephadex column nonadherent spleen cells were cultured alone for 4–5 days, these cells did not suppress the generation of CTL or anti-SRBC humoral response. When the nonadherent spleen cells were cultured with plastic adherent spleen cells, however, suppressor cells developed and the suppressor activity of these cells was dependent on the number of adherent spleen cells co-cultured with the non-adherent spleen cells. This activity of the adherent spleen cells was insensitive to treatment with anti-Thy 1.2 serum plus complement and to X-irradiation. Furthermore, adherent PEC could not substitute for adherent spleen cells, indicating a possible tissue specificity for the macrophages in the adherent cell fraction which can function in supporting and/or accelerating the differentiation of “immature” suppressor T cells. Finally, culture-induced suppressor T cells were sensitive to X-irradiation and their activity was refractory to IL2 (TCGF), whereas the activity of alloantigen-induced suppressor cells was sensitive to IL2.

Separation and isolation of lymphocyte subpopulations of different affinity for the antigen

In order to separate, isolate, and determine the number and distribution of the subpopulations of lymphocytes of diverse affinities that are present in an immune response toward a single hapten, antitrinitrophenyl (TNP) lymphocytes from immunized animals were purified by cell chromatography. Non-adherent spleen cells were passed through a column consisting of TNP-substituted polyacrylamide beads. The retained cells were eluted by applying a linear concentration gradient of TNP-lysine. Elution profiles having a limited number of peaks were obtained in all cases. The avidity of the cells in each fraction was measured by inhibition of formation of immune rosettes by free hapten. Results showed that each peak was located along the gradient according to its affinity since there was a direct correlation between the affinity and the concentration of hapten needed for the elution. The cells in each peak appeared to belong to a homogeneous subpopulation as shown by the slope of the curves obtained in the determination of avidity, suggesting that each peak corresponded to one expanded clone.

Manganese chloride enhances murine cell-mediated cytotoxicity: effects on natural killer cells.

Natural killer (NK) cell activity of mice given a single injection of manganese chloride (MnCl2) was significantly enhanced as measured in a 4-hr in vitro 51Cr release assay. Enhanced activity persisted for several days after injection. This cytotoxic activity was associated with nonadherent spleen cells and was completely eliminated by injecting MnCl2-treated mice with anti-asialo GM1 serum. Manganese-enhanced natural cytotoxicity was observed in several mouse strains with differing NK cell reactivity (CBA/J, C57BL/6, A/J, C3H/HeJ, and C57BL/6 beige mice) and with several tumor target cells with differing sensitivity to NK cytolysis (YAC-1, RBL-5, EL-4, and P815). The growth of B16-F10 melanoma lung tumors was inhibited in mice injected with MnCl2 one day before tumor challenge. Manganese chloride enhancement of NK cell activity appeared to be mediated by interferon (IFN). Low levels of IFN were detected in the serum of mice as early as 4 hr after MnCl2 injection. Rabbit anti-mouse IFN alpha, beta but not anti-mouse IFN gamma completely eliminated the MnCl2-enhanced NK cell activity in the spleens of mice. The observed enhancement of NK cell activity by MnCl2 is similar to that reported for more complex molecules that act by inducing IFN production.

Immunoreactive beta-endorphin in a subpopulation of mouse spleen macrophages.

Using radioimmunoassay and immunofluorescence with antibodies to beta-endorphin (beta EP) and ACTH, we have shown that a subpopulation of mouse spleen cells, expressing Mac-1, a marker of macrophage differentiation, contains immunoreactive (ir)-beta EP, ir-ACTH, and smaller amounts of presumptive higher molecular weight forms of both. Neither nonadherent spleen cells, nor adherent or nonadherent cells from peripheral blood, contained detectable levels of these peptides. These findings suggest that beta EP and ACTH may be synthesized in a subpopulation of spleen macrophages, and are consistent with the possibility that these or related peptides may modulate lymphocyte function in the specific microenvironment of the spleen.

The identification and characterization of antimacrophage serum (AMS) and its application to murine fibrous histiocytoma.

The preparation of antimacrophage serum (AMS) and its application to murine fibrous histiocytoma are reported. AMS, prepared by inoculating peritoneal macrophages from mice into rabbits, was purified by repeated absorption with kidney homogenate, red blood cells, thymus cells, non-adherent spleen cells and finally, L-cells. The molecular weight was determined as 170,000 daltons using SDS-polyacrylamide gel electrophoresis. AMS diluted to 1:32 reacted with 61.4% of peritoneal macrophages, but not red blood cells, lymphocytes, granulocytes and fibroblasts, as judged by membrane immunofluorescence. 57.1% of monoclonal B-10 macrophages and 75.6% of 28-12 macrophages transformed with SV40 reacted with AMS. B-10 and 28-12 cell tumors simulating human malignant fibrous histiocytoma gave a distinctive diffuse pattern of immunofluorescence with AMS which affected both round- and spindle-shaped cells, although positive fluorescence of spindle-shaped cells was somewhat weaker than that of round-shaped cells. However, tumor tissues from a murine fibrosarcoma showed no reaction with AMS. Based on these findings, AMS may be useful in determining the histogenesis of some soft tissue tumors of doubtful origin.

Surface properties of lymphocyte subpopulations in autoimmune NZB/NZW F 1 hybrid mice: Alterations correlated with the immunodeficiency of aging

Partitioning in a two-polymer aqueous phase system was used to probe the surface properties of lymphoid cell subpopulations in aged male NZB/NZW F 1 hybrid (B/W) mice, an important model of autoimmunity, immunodeficiency, and lymphoid malignancy. Spleen cells were fractionated by countercurrent distribution (CCD, a multiple-step extraction procedure) in a charged dextran-polyethylene glycol system. CCD of spleen cells from young, clinically normal male B/W mice yielded several broad distribution patterns which frequently had two or more peaks. Analysis of differentiation antigens and functional properties of cells from different parts of the distribution revealed a subfractionation of the three major lymphocyte subpopulations. B lymphocytes had a low partition coefficient ( K); T cells had an intermediate K and null cells had the highest K. To examine the partitioning behavior of T lymphocytes, spleen cells which were nonadherent to nylon wool columns were subjected to CCD. Nonadherent cells from young B/W mice consistently gave a single peak with high K. Aged mice (18 months) usually had nonadherent cells with a predominantly low K. In some experiments a systematic increase in the number of these cells could be demonstrated with increasing mouse age. An analysis of the adherence and partitioning behavior of lymphocyte subpopulations revealed no change in the adherence properties or proportions of B lymphocytes in aged mice. The large proportion of cells having a low partition coefficient in the nonadherent spleen cell population of old mice appears to be due to an increase in the number of null cells and in a decrease in the K of some T lymphocytes.

Increase of MLC sensitivity by elimination of a non-adherent responder cell subpopulation with anti-macrophage serum

A specific rabbit anti-rat macrophage antiserum (SAM) was prepared with a cytotoxic reactivity pattern complementary to that of a specific anti-lymphocyte serum. This was used to characterise adherent and non-adherent spleen cell subpopulations in mixed lymphocyte cultures. Adherent SAM + cells reacted as accessory cells whereas non-adherent SAM + cells were suppressors. Selective elimination thus achieved resulted in a highly significant increase of MLC reactivity in certain strain combinations and in conversion from non-reactivity to reactivity in others.

Suppression of lymphocyte proliferative responses: demonstration of two stages occurring in the in vitro generation of suppressor macrophages.

The suppressor macrophages generated by in vitro culture of spleen cells are shown to be derived from nonadherent splenic precursors. The induction of suppressor macrophage generation requires the presence of both plastic-adherent and plastic-nonadherent spleen cells during the first 24 to 48 hr of culture. After this induction period, the primed nonadherent cells can continue to generate suppressor macrophages through several serial transfers. The primed nonadherent spleen cell population is predominantly comprised of cFcR+ cells (40 to 50%) and Thy-1+ cells (40%), many of which appear to be undergoing blastogenesis. The generation of suppressor macrophages appears to proceed in two stages. The first stage is radiosensitive and has a minimum duration of 1 to 2 days. Although this stage initially coincides with the inductive period, it is also required for the continuous propagation of the suppressor macrophages upon serial transfer of the nonadherent spleen cells. The second stage is radioresistant and has a maximum duration of 2 to 3 days. The in vitro events thus bear similarities to events occurring during in vivo differentiation and activation of effector macrophages. It is suggested that the in vitro generation of suppressor macrophages may be one component of syngeneic mixed lymphocyte responses when spleen cells are used as the responding population, and that the suppressors represent part of a regulatory system controlling lymphocyte and macrophage differentiation and activation.

Correlation between in vivo and in vitro studies of modulation of resistance to experimental Candida albicans infection by cyclophosphamide in mice.

Mice receiving a single injection of cyclophosphamide (150 mg/kg) 1 to 6 days before inoculation with viable Candida albicans showed an increased susceptibility to the challenge accompanied by a reduction in peripheral blood polymorphonuclear leukocytes and lymphocytes as well as in spleen cellularity. Several immunological in vitro functions also appeared to be dramatically depressed. Most of these hematological and functional parameters returned to control values by day 9 after cyclophosphamide administration, at a time when resistance to C. albicans infection appeared to be unchanged. However, when exposure to cyclophosphamide occurred 12 to 21 days before inoculation with the live yeast, enhanced resistance was observed with the majority of the animals surviving challenge. To gain some insight into the mechanisms underlying this late increase in resistance to C. albicans infection after cyclophosphamide administration, we analyzed a series of immunological functions, including the in vitro candidacidal activity of polymorphonuclear neutrophils and plastic-adherent and nonadherent spleen cells as well as the activity of natural killer cells and alloreactive T lymphocytes. The results show that a numerical rebound of blood polymorphonuclear neutrophils and the appearance of a highly candidacidal cell population in the spleen may be among the factors underlying the late increase in resistance to C. albicans after administration of cyclophosphamide.

Cellular immunosenescence in F344 rats: Decline in responsiveness to phytohemagglutini involves changes in both T cells and macrophages

Spleen cells from F344 male rats showed decreased DNA synthetic responsiveness ([ 3H]thymidine incorporation) with age to the T cell mitogen phytohemagglutinin (PHA). Decreased proliferative responses were associated with decreased production of interleukin-2 (IL-2) and could be partially restored by providing exogenous IL-2. Responses of spleen cells from aged rats could also be enhanced by removal of Sephadex G-10 adherent cells. Furthermore, co-culture of adherent cell-containing spleen cells from aged rats with nonadherent spleen cells from young rats resulted in suppression of responses. Purified peritoneal exudate macrophages (PEM) from aged rats showed potent regulatory effects of PHA responses of young and old nonadherent spleen cells resulting in suppression above 2.5% macrophage/nonadherent spleen cell ratios. PEM from young rats enhanced the response of young T cells but failed to affect the response of aged T cells. T cell proliferation in the presence of prostaglandin (PGE 1) showed age-dependent differences in regulation such that 10 −6 to 10 −7 M young responses were enhanced and old responses were suppressed. These results suggest that decreased responsiveness of T cells to PHA with age is a complex phenomenon involving changes in both production of regulatory mediators by T cells (IL-2) and macrophages (PGE) as well as changes in T cell responsiveness to these signals.

The immunological response of Wistar rats to the intracranially implanted C-6 glioma cell line.

The immune response of Wistar rats to the intracranial inoculum of 1 X 10(5) C-6 glioma cells was evaluated. The growth of these cells disrupted the blood-brain barrier by day 9. The rats with the implanted tumor cells died between three to four weeks following injection. No significant cell-mediated cytotoxicity against 51Cr labelled C-6 cells was seen in the short term (4 hours) cytotoxicity assay with spleen cells obtained from glioma-bearing rats at any stage of tumor growth. In the long term (18 hours) cytotoxicity assay, significant activity was detected using whole, adherent and nonadherent spleen cells from glioma-bearing rats at every assessment point during the growth of the tumor, but this cytotoxicity was also seen in normal rat splenocytes. The lack of cell mediated cytotoxicity above normal values was not due to a generalized immunosuppression since splenocytes from glioma bearers were found to have responses to Con A comparable to normal controls. However, normal or glioma-bearer splenocytes showed augmented cytotoxicity in the presence of serum obtained from rats bearing a glioma tumor starting by day 13 of tumor growth and rising in cytotoxic activity until death. This activity was not seen with normal serum. The glioma-bearer serum, though not cytotoxic to the C-6 cells alone, became cytotoxic with the addition of rabbit complement. These data indicate that the growth of intracranially implanted glioma cells in rats elicits primarily a humoral cytotoxic immunity without a significant cell-mediated immunity. This humoral immunity develops after the breakdown of the blood-brain barrier.

Helper function of rat spleen cells reactive with guinea pig serum.

Treatment of rat spleen cells with normal guinea pig serum (GPS) has been shown to produce a significant loss of responsiveness to stimulation with either Con A or PHA. This phenomenon was seen even when the spleen cells were triggered with mitogens up to 96 hr after treatment with GPS, suggesting that GPS had produced a long-lasting alteration in some cells or removed a cell subpopulation. Glass-wool non-adherent spleen cells, known to produce greater responses that unfractionated spleen cells, also had their responses to Con A and PHA reduced by GPS treatment though the response was still greater than that of untreated, unfractionated cells, suggesting that the actual responder cells had been spared by GPS. Suppressor cells did not appear to be the target of GPS because such an effect would have resulted in increased responsiveness and the opposite result was obtained. That a helper cell was affected by GPS was suggested by the following observations: a) the virtually unresponsive GPS-treated spleen cells produced greater than normal responses after removal of glass-wool-adherent suppressor cells; b) the response of glass-wool-nonadherent spleen cells was significantly decreased after GPS treatment; and c) mixtures containing equal numbers of glass-wool-nonadherent and GPS-treated spleen cells also showed reduced responsiveness after GPS treatment.

In vitro release of lymphotoxin by spleen cells from C3H/HEJ and C57BL/6 mice infected with Trypanosoma cruzi.

Abstract. Lectin (PHA-P) activated nonadherent spleen cells from uninfected inbred strains of mice known to exhibit high parasitemias when infected with Trypanosoma cruzi (A/J and C3H/HeJ), released in vitro significantly less lymphotoxin (LT) than did a mouse strain (C57BL/6) known to exhibit low parasitemia when infected with T. cruzi. The capacity of mice to release LT in vitro changed upon infection with T. cruzi. Cells from infected C57BL/6 mice released LT levels well above that from uninfected C57BL/6 animals within 4 days after initial infection, and their capacity to release LT remained high even after the parasites were not detectable in the blood. Cells from infected C3H/HeJ mice were not able to respond as rapidly as those from C57BL/6 animals; however, they were able to release LT at the same levels as the infected C57BL/6 by day 18. The parasitemia induced by Trypanosoma cruzi in C3H/HeJ and C57BL/6 mice was compared with the in vitro ability of their spleen cells to spontaneously release LT. Spontaneous release of LT was significantly higher by spleen cells from infected C57BL/6 mice than by cells from infected C3H/HeJ mice. The kinetics of natural LT release differed from that of mitogen-(PHA-P)-stimulated LT release; LT activity peaked at 3–6 hours of incubation in the former and then declined whereas LT activity in the latter reached a plateau after 3 hours of incubation and did not decline for at least 18 hours in the presence of the inducer. Supernatants from infected C57BL/6 splenocytes, when concentrated 5× by ultrafiltration, significantly inhibited both bloodstream trypomastigote motility in vitro and their infectivity (by 98%) for WI38 human embryonic lung cell cultures. Similar preparations from C3H/HeJ splenocytes only slightly inhibited bloodstream trypomastigote motility and delayed, but did not prevent, infection of WI38 cells. No agglutination of immobilized parasites was noted. Splenocyte supernatants did not affect culture epimastigote motility or growth. This is the first report of direct action by lymphokine-containing supernatants against T. cruzi bloodstream trypomastigotes.

Extrarenal Erythropoietin Production by Macrophages

Murine bone marrow and adherence-separated spleen cells cultured on hydrophobic, gas-permeable Teflon foils (petriperm dishes) can be shown to synthesize and secrete erythropoietin (Epo) and colony-stimulating activity (CSA) simultaneously into the surrounding medium. The Epo activity in the supernatants of primary cultures as measured by the fetal liver erythroid colony-forming technique, from adherent and nonadherent spleen cells, increases over the first 7 days in culture, followed by a plateau until 14 days. Use of the macrophage-specific cytotoxic agent, crystalline silica, as a tool to release residual Epo contained in these cells produces a similar time-Epo activity curve to that found in the primary supernatants. This, together with functional and morphological examination of the cells, indicates that macrophages are responsible for this activity. The total Epo activity released from adherent and nonadherent spleen cells at plateau levels was estimated to be 25 mU/ml culture/day. Weekly subcultivation of bone marrow and adherence-separated spleen cells initiated from primary cultures demonstrated a massive increase in both Epo activity and CSA above that obtained for the primary cultures. Subcultivation could be continued for at least 6 wk. These results, together with the reversible inhibition of Epo and CSA production by cycloheximide, demonstrate that these molecules are synthesized by the macrophage. The evidence supports the hypothesis that the macrophage is involved not only in extrarenal Epo production, but also in the possible short-range regulation of hemopoiesis.

Extrarenal erythropoietin production by macrophages

Murine bone marrow and adherence-separated spleen cells cultured on hydrophobic, gas-permeable Teflon foils (petriperm dishes) can be shown to synthesize and secrete erythropoietin (Epo) and colony-stimulating activity (CSA) simultaneously into the surrounding medium. The Epo activity in the supernatants of primary cultures as measured by the fetal liver erythroid colony-forming technique, from adherent and nonadherent spleen cells, increases over the first 7 days in culture, followed by a plateau until 14 days. Use of the macrophage-specific cytotoxic agent, crystalline silica, as a tool to release residual Epo contained in these cells produces a similar time-Epo activity curve to that found in the primary supernatants. This, together with functional and morphological examination of the cells, indicates that macrophages are responsible for this activity. The total Epo activity released from adherent and nonadherent spleen cells at plateau levels was estimated to be 25 mU/ml culture/day. Weekly subcultivation of bone marrow and adherence-separated spleen cells initiated from primary cultures demonstrated a massive increase in both Epo activity and CSA above that obtained for the primary cultures. Subcultivation could be continued for at least 6 wk. These results, together with the reversible inhibition of Epo and CSA production by cycloheximide, demonstrate that these molecules are synthesized by the macrophage. The evidence supports the hypothesis that the macrophage is involved not only in extrarenal Epo production, but also in the possible short-range regulation of hemopoiesis.

Modulation of antitumoral antibody-dependent cellular cytotoxicity and natural killer activity by Adriamycin and daunorubicin.

Mouse effector cells isolated from various anatomical sources failed to mediate antibody-dependent cellular cytotoxicity (ADCC) against alloantiserum-coated L1210 murine leukemia cell targets, whereas rat spleen cells appeared to be potent mediators of this activity. Following suppression of effector cell function 3 days after a single drug injection, the nylon wool non-adherent population of rat spleen cells from daunorubicin (DM)-treated rats demonstrated an increased ability to mediate ADCC compared to controls. Alternatively, although suppression occurred at day 7, no rebound enhancement was demonstrated by the same cell population isolated from Adriamycin (AM)-treated animals for as long as 12 days post-injection. Natural killer (NK) activity, measured as the ability of the nylon wool non-adherent rat spleen cell population to lyse uncoated L1210 cells, was modulated by drug treatment in a similar manner at each time point although the changes were not significant. In contrast to NK cells for which a substantial amount of activity remained adherent to nylon wool, all K cell activity was found in the non-adherent spleen cell population. The effector cell, in both cases, was not susceptible to antithymocyte serum plus complement treatment; however, NK activity appeared trypsin-sensitive whereas K cell activity did not. Therefore, AM and DM demonstrated different activities with regard to in vivo modulation of antitumoral ADCC.

A natural model of immunologic tolerance. Tolerance to murine C5 is mediated by T cells, and antigen is required to maintain unresponsiveness.

A unique experimental model is described, where natural immunologic tolerance to a well-defined soluble native antigen (murine C5) is examined in congenic strains of mice that differ only by the presence or the absence of C5. A highly sensitive hemolytic assay was developed to detect nanogram amounts of C5 as well as an assay of anti-C5 inhibition of C5 hemolytic activity. The latter was more sensitive than immunodiffusion. Two reciprocal approaches were used to study the cellular basis of tolerance in irradiated hosts of either strain. In the first, lymphoid cells from either strain were transferred to irradiated B10.D2OSN hosts that were lacking C5 and so would not hinder detection of anti-C5 antibody upon challenge with murine C5. Second, lymphoid cells from either strain were transferred to irradiated B10.D2NSN hosts, whose native C5 provided the antigenic stimulus. The immune response of whole nonadherent spleen cell suspension as well as mixtures of T and B cells (separated on the basis of surface immunoglobulin) from either strain were studied. In addition, the duration of tolerance and the antigen requirement to maintain it in irradiated C5-deficient hosts repopulated with C5-sufficient spleen cells was examined. The positive control of irradiated C5-deficient hosts repopulated with syngeneic spleen cells showed a primary and secondary response to immunization. In contrast, C5-sufficient spleen cells failed to respond both in the primary and the secondary response. Because the unresponsiveness was not caused by antigen carryover and was not antigen specific, it represents central tolerance. In C5-sufficient irradiated hosts (where immunization was not required and antigen was present in natural form and physiological concentration), transfer of C5-deficient cells mediated a drop in C5 levels to 10-20% of that noted in unreconstituted controls. T and B cell mixing experiments from the two strains into deficient or sufficient hosts demonstrated that tolerance is T cell dependent and that C5-sufficient or -deficient B cells could cooperate with nontolerant C5-sufficient T cells to produce significant anti-C5 antibody or mediate a significant drop in C5 levels. In addition, the presence of antigen was necessary to maintain tolerance. In conclusion, these results show that (a) natural tolerance to C5 is an active process that is T cell dependent and requires the presence of antigen; (b) in this natural model, clonal abortion does not seem to occur; and (c) both tolerant and nontolerant B cells retain the capacity to produce autoantibody.