Contact sensitivity in the mouse. IV. The role of lymphocytes and macrophages in passive transfer and the mechanism of their interaction.

Contact sensitivity skin reactions were produced in mice by immunization with 2-phenyl-4-ethoxymethylene oxazolone (oxazolone) and detected by the increase in ear thickness after challenging the ears with 2% oxazolone. These skin reactions can be transferred from immunized donors to irradiated recipients by peritoneal exudate cells induced by thioglycollate. The peritoneal exudate cells were separated into purified macrophage and purified lymphocyte populations. Both cell populations transferred skin reactions. However, their time course was different. The reactions produced by lymphocytes were greater at 24 hr than at 12 hr while the reactions produced by macrophages declined slightly between 12 and 24 hr. The working hypothesis was formed that the peritoneal lymphocytes conveyed a factor (presumptive cytophilic antibody) to peritoneal macrophages which enabled them to transfer ear reactions. Experiment showed that peritoneal and lymph node lymphocytes from sensitized donors within a Millipore chamber conveyed a factor to macrophages outside the chamber which enabled them to transfer ear reactions. In contrast, peritoneal macrophages (from sensitized donors) within the chamber and peritoneal lymphocytes outside the chamber were inactive. These findings suggested that there are three modes of immunological tissue damage: hypersensitivity mediated by lymphocytes (classical delayed hypersensitivity), hypersensitivity mediated by circulating antibody (classical immediate type hypersensitivity), and hypersensitivity mediated by macrophages which have passively acquired a factor (macrophage-mediated hypersensitivity).

Reaginic antibody produced in mice with contact sensitivity.

Mice produced reaginic antibody within 1 wk of painting with the contact sensitizing agent picryl chloride. The titers, measured by passive cutaneous anaphylaxis in rats, increased after repeated applications of picryl chloride. In contrast, serum agglutinins did not increase after two applications of picryl chloride. Reagin was also elicited by another contact sensitizing agent, oxazolone. Some strain variation of the response to picryl chloride was found, with CBA mice being good responders and BALB/c and C57BL/10 mice being poor responders.

Detection of radiolabelled bone marrow cells bearing surface immunoglobulins by combined autoradiography and immunoperoxidase.

A method is described for the simultaneous detection of radiolabelled bone marrow cells bearing surface immunoglobulins by combined autoradiography and immunoperoxidase. Bone marrow cells from normal CBA mice prelabelled in vivo with 125IUDR or exposed in vitro to [3H] thymidine were incubated with rabbit anti-mouse immunoglobulins under capping conditions, washed, cytocentrifuged and treated with methanol and hydrogen peroxide to destroy endogenous peroxidase. Cells were then covered with peroxidase-conjugated goat anti-rabbit immunoglobulins, washed, treated with diaminobenzidine and hydrogen peroxide and finally covered with autoradiographic stripped film and exposed for different times. Peroxidase-positive cells were typically capped and those radiolabelled had autoradiographic silver grains overlying the nucleus.

Regulation of B cell development in mouse bone marrow.

Cultured bone marrow cells, after in vitro treatment with hydroxyurea (HU) - a DNA synthesis inhibitor which kills cells in the S phase of the cell cycle - generated 40 to 70% more B cells than untreated control cells. This was shown by fluorescent-activated cell sorter analysis of labelled cells using FITC-F(ab')2 rabbit anti-mouse IgM and functional tests with LPS. The maximum increase was reached after 24 hr of incubation with HU while 6 or 2 hr of exposure had less effect. The effect of HU was dose dependent with a maximum at 4 mM. The same increase of B cells was observed with foetal liver cells but not with spleen or lymph node cells after 24 hr of in vitro HU treatment. Dialysed supernatants from HU treated bone marrow, spleen or foetal liver cells were themselves able to augment the B cell maturation in bone marrow cultures (test cells) as compared with supernatants from untreated cells, showing that soluble factors were involved. Preliminary data showed that inhibitory factors for B cell maturation were produced by normal bone marrow, spleen and thymus cells in vitro and their formation was prevented by HU pretreatment or irradiation (2500 R) whereas stimulatory factors were produced by lymph node cells. Cell separation experiments suggested that T cells and/or adherent cells may be involved in the production of these soluble factors. These data suggest that early B cell development may be under homeostatic control.

I-J revisited: is the I-J genetic restriction in downregulation due to an endogenous superantigen analogous to mammary tumour virus (Mtv)-encoded endogenous superantigen?

This article puts forward the hypothesis that the I-J genetic restriction observed between certain downregulatory (suppressor) T cells and antigen presenting cells is due to an endogenous superantigen analogous to the mouse mammary tumour virus (Mtv) products encoded by the open reading frames in the 3' long terminal repeat (LTR) of mtv's. In its weak form this hypothesis asserts that the I-J genetic restriction is due to an endogenous superantigen ligand on antigen presenting cells, which crosslinks the V beta and/or V alpha chains of certain T cell receptors (TCR) with major histocompatibility complex (MHC) class II, and that MHC together with this superantigen ligand causes positive selection of T cells bearing the appropriate I-J+ TCR in the thymus. In the periphery these T cells recognize peptide/MHC complex in the presence of the superantigen. In its strong form the hypothesis states that this superantigen ligand for TCR and MHC is encoded by integrated virus genome, e.g. Mtv. These possibilities can now be approached experimentally and their exploration may uncover one of the ways in which T cells are assigned to different functions, including downregulation.

Antigen-specific T-helper and -suppressor factors in the control of the immune response.

Antigen-specific T-helper and -suppressor molecules usually have a two-moiety, disulphide-bonded structure. One chain binds antigen, while the other chain bears I-A in the case of helper factor and I-J in the case of suppressor factor. Their genetic restriction, when it exists, parallels the 'MHC' determinants that they carry and probably reflects the fact that helper cells are activated by antigen in the context of I-A, while suppressor cells are activated by antigen in the context of I-J. T-helper factor probably augments the induction of contact sensitivity by approximating antigen with its own I-A determinants. Different factors act at the induction and the effector stage and they may be antigen or idiotype directed. The antigen-specific factors characteristically are part of complex circuits involving both antigen-specific and non-specific cells and factors and often have a mode of action through the macrophage.

Recombinant interleukin-2 limits the replication of Mycobacterium lepraemurium and Mycobacterium bovis BCG in mice.

BALB/c mice were infected with Mycobacterium lepraemurium in the footpad or with Mycobacterium bovis BCG intravenously with 5 x 10(7) bacilli. Recombinant interleukin-2 (IL-2) was injected intraperitoneally as a single dose (20,000 U), as a single course of five injections (400 U each), or as a 6-month course starting 3 days after the M. lepraemurium infection. BCG-infected mice received a single dose (1,000 U) or five daily injections of 100 or 1,000 U each. IL-2 significantly reduced the total bacterial counts in the footpad, lymph nodes, and liver of M. lepraemurium-infected mice (50 to 85%) by 6 months and viable counts in the spleen (30 to 50%) by 60 days after BCG infection. The courses of IL-2 started at 60 days were more effective than those started at 3 days after M. lepraemurium infection (P less than 0.05 to 0.001), and for BCG, 100 U of IL-2 was better than 1,000 U (P less than 0.05 to 0.01). These results indicate that IL-2 limits mycobacterial infections in mice and raise the question of its possible use in humans.

F1 mice make two species of antigen-specific, parental haplotype-restricted, T-helper factor whose restrictions correspond to the phenotype of the I-A determinants that they bear.

Antigen-specific T-helper factor (ThF) bears I-A determinants and is I-A restricted in its action. This I-A restriction may be explained by ThF binding, and hence approximating antigen to its own I-A determinants, thereby facilitating recognition by the T cell (I-A presentation theory), or by a recognition site for I-A on the ThF which approximates the antigen to I-A+ antigen-presenting cells (I-A recognition theory). When ThF is produced in F1 mice there may be a dissociation between the I-A phenotype of the ThF and the I-A restriction of any recognition site for I-A. In practice, ThF is made by spleen cells from (CBA x B10)F1 [(H-2k x H-2d)F1] mice pretreated with cyclophosphamide (100 mg/kg) and immunized with picrylated parental spleen cells intravenously (3 x 10(7)). This procedure produces haplotype-restricted contact sensitivity (corresponding to the parental cells) but the unfractionated F1 ThF does not show haplotype-specific restriction in its action. In fact, the F1 mice produces two species of ThF, each bearing determinants of the I-A molecule of one parental haplotype (k or d). When the two species were separated with monoclonal anti-I-Ak and anti-I-Ad antibodies, the genetic restriction in their action corresponded to the phenotype of the I-A determinants that they carried. In a further experiment, F1 ThF was split into its constituent antigen-binding (Ag+) and antigen non-binding (Ag-) chains by reduction, and the two species of Ag- chains separated with monoclonal anti-I-A antibodies. After complementation with the Ag+ chain, the two species of Ag- chains showed genetic restriction in their action that corresponded to the phenotype of the I-A determinants that they carried. These findings support the I-A presentation hypothesis that antigen-specific ThF acts by approximating antigen to its own I-A determinants, and hence facilitates recognition by I-A-restricted T cells.

Immune deviation in the mouse: transfer of selective depression of the contact sensitivity and interleukin-2 response with retention of interferon-gamma production requires CD8+ T cells.

Mice were injected intravenously (i.v.) with trinitrophenyl (TNP)-modified spleen cells. They were subsequently immunized by epicutaneous application of 2,4,6-trinitrochlorobenzene (TNCB, picryl chloride) or 'oxazolone'. The intravenous injection of antigen caused immune deviation (split tolerance) with selective loss of contact sensitivity (CS) and antigen-induced interleukin-2 (IL-2) production, and concomitant retention of antigen-induced interferon-gamma (IFN-gamma) production. This phenomenon was antigen specific as the response to oxazolone was unaffected. Moreover, lymph-node cells stimulated with antigen three times in vitro (from 'deviated' mice which had been injected with antigen i.v., and then sensitized with TNCB) showed limited proliferation. The per cent of IL-2R+ cells and the absolute number of V beta 8+ cells dropped. In contrast, lymph-node cells from 'undeviated' mice showed increased proliferation and IL-2 production on repeated stimulation with antigen in vitro and the per cent of IL-2R+ cells and the absolute number of V beta 8+ cells recovered increased. Spleen cells, taken from mice 3-7 days after the injection of antigen i.v., transferred immune deviation to normal recipients i.e. following epicutaneous immunization with TNCB, the recipients showed the same selective unresponsiveness as the donors. Thy-1+ CD4- CD8+ cells were required. These findings indicate that immune deviation can be demonstrated at the level of lymphokine production.

Recombinant interleukin-2 limits the replication of Mycobacterium lepraemurium and Mycobacterium bovis BCG in mice.

BALB/c mice were infected with Mycobacterium lepraemurium (MLM) in the foot pad or with M. bovis BCG intravenously with 5 x 10(7) bacilli. Recombinant interleukin-2 (IL-2) was injected intraperitoneally as a single dose (20,000 u), single course of 5 injections (400 u each) or 6 monthly courses starting 3 days or 60 days after the MLM infection. BCG infected mice received a single dose (1000 u) or 5 daily injections of 100 or 1000 u each. IL-2 significantly reduced the total bacterial counts in the footpad, lymph node and liver of MLM infected mice (50-85%) by 6 months and viable counts in the spleen (30-50%) by 60 days after BCG infection. The courses of IL-2 started at 60 days were more effective than at 3 days after MLM infection (P less than 0.05-0.001) and in the case of BCG, 100 u of IL-2 was better than 1000 u (P less than 0.05-0.01). These results indicate that IL-2 limits mycobacterial infections in mice, and raise the question of its possible use in humans.

T suppressor cells and suppressor factor which act at the efferent stage of the contact sensitivity skin reaction: their production by mice injected with water-soluble, chemically reactive derivatives of oxazolone and picryl chloride.

The water soluble, chemically reactive thioglycollic acid thioether derivatives of oxazolone and picryl chloride were synthesized and tested for their ability to prevent the development of contact sensitivity. Mice given two injections of these agents showed partial or complete unresponsiveness when subsequently sensitized and challenged with oxazolone and picryl chloride, respectively. This unresponsiveness was associated with T suppressor cells, Ts-eff(cs), which blocked the efferent stage of the contact sensitivity reaction, i.e. the passive transfer of contact sensitivity. These Ts-eff(cs) were entirely specific when tested with the corresponding antigen. However, the suppression which they caused had a non-specific final common pathway. Cell from mice injected with the oxazolone and picryl thioethers and painted with the corresponding contact sensitizer produced a suppressor factor in vitro. This factor specifically blocked passive transfer by immune cells incubated in it. It also armed macrophages which then caused suppression. These macrophages were most effective when injected intraperitoneally. The suppressor factor had a molecular weight between 30,000 and 100,000 and the alpha-oxazolone factor was absorbed by oxazolone-albumin Sepharose and could be eluted with oxazolone-epsilon-aminocaproic acid. It was also absorbed by concanavalin-A-sepharose and could be eluted with alpha-methylmannoside. It is proposed that the ability of water soluble, chemically reactive haptenes to evoke a Ts-eff(cs) population may be relevent to the rarity of severe drug reactions following the injection of chemically reactive drugs.

The effect of cyclophosphamide and irradiation on cells which suppress contact sensitivity in the mouse.

Contact sensitivity was produced in mice by painting the skin with picryl chloride and was assessed by the increase in ear thickness following local challenge. Contact sensitivity was passively transferred by immune lymph node and spleen cells taken at 4 days. The mice were then challenged immediately and the reactions read at 24 and 48 hr. Immune lymph node and spleen cells taken at day 8 virtually fail to transfer. Experiment showed that they contain cells which suppress passive transfer. These are demonstrated by mixing approximately equal numbers of 4-day cells, which transfer contact sensitivity, and cells taken at later times and injecting them intravenously into recipients. These 'suppressor cells' can be demonstrated by day 6 and are still present at day 11 after immunization. The precursors of the suppressor cells are sensitive to cyclophosphamide. Irradiation of immune mice 2 days before taking cells also selectively inactivates the suppressor cells. When mice are pretreated with cyclophosphamide before immunization or irradiated 2 days before transfer, the lymph node and spleen cells taken on day 9 after immunization transfer contact sensitivity. In contrast the same number of cells from untreated mice were inactive. This suggests that the cells which mediate passive transfer or their precursors may occur in an inhibited form in lymph nodes and spleen at later times after immunization. These suppressor cells in immune mice differ from the T suppressor cells produced by the injection of picryl sulphonic acid--an agent which causes unresponsiveness: (1) the precursors of the T suppressor cells resist cyclophosphamide; (2) the T suppressor cells are found in mice treated so as to produce unresponsiveness while the other type of suppressor cells occurs in mice immunized for contact sensitivity. However, both types of suppressor cells are selectively inactivated by irradiation as compared with the cells which mediate contact sensitivity and both are able to act on the effector stage of contact sensitivity.

Induction of cell-mediated immunity in the mouse: circumstantial evidence for highly immunogenic antigen in the regional lymph nodes following skin painting with contact sensitizing agents.

This paper describes an investigation of why contact sensitizing agents cause strong cell-mediated immunity. Contact sensitivity was induced in mice by painting the skin with 4-ethoxymethylene-2-phenyloxazolone (oxazolone), and measured by the increase of ear thickness following challenge six days later. Reactivity was transferred by taking the regional lymph node cells from mice 18 h after immunization and injecting them into the footpads of recipients. This "18-h transfer" has several characteristics. As few as 2 X 10(4) cells were effective. The donor lymph node cells were best taken one to three days after immunization, were less effective on day 4 and virtually inactive by day 7. The recipients developed contact sensitivity when challenged on day 4, but lacked sensitivity when challenged on days 1 and 2 after transfer. The transferred cells were still active after treatment with anti-theta serum and complement. They also resisted 2,000 R in vitro, mitomycin, vinblastine, and inhibitors of protein synthesis such as emetine, cycloheximide and puromycin. The transfer was prevented by treatment with trypsin, freeze-thawing, and heating at 56 C. Plasma membranes were also immunogenic. The evidence suggests that the "18-h transfer" is a special type of active immunization, not due to ordinary free oxazolone, and that the agent is present within the lymph node in a free oxazolone, and that the agent is present within the lymph node in a specially immunogenic location or form.

Effects of adult thymectomy on the contact sensitivity skin reaction and the unresponsiveness caused by feeding contact sensitizing agents.

Unresponsiveness was produced in mice by three feeds of high doses of oxazolone or picryl chloride. The mice were then sensitized and contact sensitivity assessed by ear swelling. Adult thymectomy prevented the unresponsiveness caused by feeding. In some but not all experiments adult thymectomy also increased the response to a single skin paint. This effect of thymectomy was apparent at 1 week and reached a plateau at 2-3 weeks. The effect of adult thymectomy, on unresponsiveness caused by feeding and its ability to increase the contact sensitivity skin reaction, was reversed by treatment with crude thymosin fraction V.