Enhanced transfer of experimental allergic encephalomyelitis with strain 13 guinea pig lymph node cells: requirement for culture with specific antigen and allogeneic peritoneal exudate cells

Previously, we reported that transfer of experimental allergic encephalomyelitis (EAE) with sensitized peritoneal exudate cells (PEC) in strain 13 guinea pigs is markedly enhanced if the cells are first cultured with specific antigen, myelin basic protein (BP). These cells also undergo considerable antigen-specific proliferation. In contrast, the data reported here show that lymph node cells (LNC) from sensitized animals display neither enhanced transfer nor antigen-specific proliferation after culture with BP. Enhanced transfer is obtained, however, if a second nonspecific signal is available. This second signal is provided by the presence of normal allogeneic strain 2 PEC in culture. After culture with BP and strain 2 PEC, 2.5 to 5 x 10(7) strain 13 LNC transfer disease reproducibly, in contrast with approximately 1 x 10(9) previously required for successful transfer. Addition of allogeneic or syngeneic PEC without antigen does not lead to enhanced transfer by LNC. Culture with normal syngeneic PEC plus BP oly infrequently enhances transfer by LNC. The intense mixed lymphocyte reaction (MLR) induced by addition of strain 2 PEC to strain 13 LNC precludes the use of 3H-TdR incorporation for detection of proliferation by EAE effector cells. However, inhibition of transfer with low doses of mitomycin C (2 to 5 micrograms/ml) pluse the fact that EAE effector cells are found almost exclusively in the light fraction of BSA gradients after (but not before) culture suggests that the latter are induced to proliferate in culture.     

Role of macrophage-myelin basic protein interaction in the induction of experimental allergic encephalomyelitis in Lewis rats

Stimulated peritoneal exudate cells (PEC) containing activated macrophages (M phi) of Lewis (Le) rats were exposed for 1 hr in vivo or in vitro to radioiodinated soluble myelin basic protein (MBP) or MBP incorporated into magnetic microspheres (MBP-microspheres). The uptake by M phi of the dose of microsphere-bound MBP averaged 6.2%, whereas the average uptake of soluble MBP was 0.17%. Naive rats were sensitized with M phi-associated MBP or M phi-associated MBP-microspheres via the hind footpads without the aid of conventional immunologic adjuvants. Draining lymph node cells (LNC) or spleen cells from sensitized rats were cultured for 3 days with guinea pig MBP (GPMBP) alone or in combination with concanavalin A (Con A), then injected i.v. into naive recipients. Clinical signs of experimental allergic encephalomyelitis (EAE) appeared 6 days after transfer of LNC or spleen cells, and typical CNS lesions were seen in recipients sacrificed 10 to 14 days after transfer. The challenge of MO-MBP-sensitized rats with MBP-CFA resulted in severe clinical signs of EAE marked by an accelerated onset of neurologic symptoms.     

Adoptive transfer of experimental allergic encephalomyelitis in mice with the aid of pertussigen from Bordetella pertussis

Adoptive transfer of experimental allergic encephalomyelitis (EAE) in (SJL X BALB/c)F1 mice was accomplished by an iv injection of 2.4 to 4.7 X 10(7) lymph node cells (LNC) from mice immunized with mouse spinal cord emulsified in complete Freund's adjuvant when both donors and recipients had been treated iv with 400 ng of pertussigen at the time of immunization for the donors and on transfer of cells for the recipients. Pertussigen was essential in both donors and recipients for development of frank EAE. Signs of EAE in recipients were delayed, appearing 21-23 days after cell transfer; the maximum response at about Day 27 is considerably delayed in comparison with other reported studies on passive transfer of EAE. Histologically, recipient mice with paralysis due to EAE had typical perivascular infiltrates of mononuclear cells in the brain and spinal cord. The mechanisms by which pertussigen promotes the development of EAE after adoptive transfer of sensitized LNC are uncertain.     

Micropolyspora faeni causes airway inflammation but not hyperresponsiveness in sensitized ponies

We assessed the effect of aerosol Micropolyspora faeni challenge in two groups of ponies by measuring lung function, airway reactivity to aerosol histamine, and bronchoalveolar lavage fluid cytology. One group of ponies was sensitized by subcutaneous injection of M. faeni in complete Freund's adjuvant, and the other group served as control. In both groups of ponies, measurements were made at base line and 5 h after aerosol administration of 30 ml of saline or 30 ml of 1% wt/vol particulate M. faeni antigen in saline. Saline challenge had no effect on any of the measured variables. M. faeni challenge had no effect on pulmonary mechanics or gas exchange in the control group but significantly increased respiratory frequency and minute ventilation and decreased arterial CO2 tension in the sensitized ponies. In both groups of ponies, aerosol M. faeni challenge significantly increased total white blood cell count and neutrophil numbers in bronchoalveolar lavage fluid while large mononuclear cell numbers decreased. Airway responsiveness was unaltered by saline or M. faeni challenge in both pony groups. We conclude that aerosol M. faeni challenge induces pulmonary neutrophilia and abnormalities of ventilation but is not accompanied by airway hyperresponsiveness in sensitized ponies.     

Encephalitogenic activity of guinea pig myelin basic protein in the SJL mouse

GPBP was shown to be encephalitogenic in SJL mice by direct challenge and in experiments in which an adoptive transfer system was employed. The three fragments obtained by treating GPBP with pepsin were assessed in the same manner. The encephalitogenic activity resided in the C terminal half of the molecule (residues 89-169). LNC also proliferated to the same fragment in vitro. Fragments 1-37, and, to a lesser extent, 44-48 stimulated sensitized LNC to proliferate but did not induce disease.     

Refractoriness to migration inhibitory factor of macrophages of LPS nonresponder mouse strains.

The responsiveness to macrophage migration inhibitory factor (MIF) of peritoneal exudate cells (PEC) from the LPS unresponsive C3H/HeJ and C57BL/10ScCR mice was assessed by the indirect agarose microdroplet macrophage migration inhibition assay. No migration inhibition with PEC from C3H/HeJ nor C57BL/10ScCR mice was detected, whereas PEC from both C3H/HeN and C57BL/10Sn mice were significantly inhibited by even a 1/32 dilution of MIF-containing supernatants. Responsiveness to MIF of C3H/HeJ PEC could, however, be induced. In vivo inoculations of Mycobacterium bovis, strain BCG, 7 days before in vitro assay rendered C3H/HeJ PEC responsive to MIF. The lack of responsiveness to MIF by C3H/HeJ PEC appeared related to some form of suppression, since a mixture of PEC from C3H/HeN mice with 10 to 15% PEC from C3H/HeJ mice resulted in undetectable migration inhibition at any MIF dilution. In contrast to the usual lack of responsiveness of their macrophage to MIF, C3H/HeJ mice were able to produce MIK in response to PPD as well as their counterpart C3H/HeN mice after BCG sensitization. These results demonstrate that macrophages from C3H/HeJ and C57BL/10ScCR mice are unable to be inhibited in their in vitro migration of MIF (possibly being directly or indirectly influenced by a suppressor cell), whereas lymphoid cells from at least one of these strains, the C3H/HeJ mice, can produce MIF in response to antigenic stimulation.     

LPS regulation of the immune response: separate mechanisms for murine B cell activation by lipid A (direct) and polysaccharide (macrophage-dependent) derived from Bacteroides LPS

Lipopolysaccharide (LPS) derived from Bacteroides fragilis has been reported to stimulate mitogenic responses in spleen cell cultures from the classical LPS-hyporesponsive C3H/HeJ mouse strain; however, we have shown that purified splenic B cells from C3H/HeJ mice are hyporesponsive to phenol-water extracted LPS from B. fragilis ATCC 25285 (B-LPS). In the present study, B-LPS and its purified lipid A and polysaccharide components were tested for their ability to induce mitogenic and polyclonal IgM synthesis in spleen cell and purified splenic B cell cultures from classical LPS-responsive and -hyporesponsive mice. Mitogenic responses to B-LPS and E. coli K235 LPS(Ph) of whole spleen cells (2 X 10(5) cells/culture) or purified B cells (5 X 10(5) cells/culture) from classical LPS-responsive mouse strains (C3H/HeN, BALB/c, C57BL/6J, C57BL/10Sn, and DBA/2), F1 mice (derived from crosses between LPS responsive and C3H/HeJ mice), and classical LPS-hyporesponsive mice (C3H/HeJ and C57BL/10ScN) were high, intermediate, and low, respectively. When a higher number of whole spleen cells (5 X 10(5) cells/well) were cultured, B-LPS induced high mitogenic responses in C3H/HeN, intermediate responses in F1, and lower but significant responses in C3H/HeJ cultures. Similar results were obtained when polyclonal IgM synthesis was assessed in cultures containing 1 X 10(6) cells/culture. In contrast, the purified lipid A component of B-LPS failed to induce mitogenic responses in either whole spleen or purified B cell cultures. The addition of purified splenic B cells from C3H/HeJ mice to C3H/HeN or C3H/HeJ splenic adherent cells resulted in mitogenic responses to B-LPS, implying that the hyporesponsiveness to B-LPS seen in whole spleen cell cultures from C3H/HeJ mice at the lower cell concentration was due to limiting numbers of M phi. When splenic B cells and M phi from either C3H/HeN or C3H/HeJ mice were incubated with the lipid A or the polysaccharide moiety of B-LPS, lipid A induced mitogenic responses only in C3H/HeN cultures, whereas the polysaccharide moiety induced similar responses in both C3H/HeN and C3H/HeJ cultures. These results suggest that Bacteroides lipid A does not stimulate B cells from the classical LPS-hyporesponsive C3H/HeJ mouse strain, whereas the polysaccharide moiety of B-LPS is biologically active and mediates B cell stimulation via M phi.     

Adoptively transferred experimental autoimmune encephalomyelitis in SJL/J, PL/J, and (SJL/J x PL/J)F1 mice. Influence of I-A haplotype on encephalitogenic epitope of myelin basic protein

Guinea pig basic protein (GPBP)-immune lymph node cells (LNC) from SJL, PL, and SJL x PL (F1) mice proliferated to whole GPBP and GPBP fragments 1-37, 43-88, and 89-169. All three strains of mice developed experimental allergic encephalomyelitis (EAE) by active immunization with whole GPBP or by passive transfer of LNC cultured with whole GPBP. SJL (H-2s) and PL (H-2u) mice developed EAE by active immunization with fragments 89-169 or 1-37, respectively, or by passive transfer of LNC cultured with the same Ag. F1 mice developed EAE by active immunization only with fragment 1-37 or by passive transfer of LNC cultured with either of the above fragments. Removal of macrophages (MO) from immune-F1 LNC resulted in the loss of a proliferative response and the ability to transfer EAE. Reconstitution of MO-depleted immune F1 T cells with either F1-, SJL-, or PL-MO restored the proliferative responses to whole GPBP and the three fragments. Cultures of immune F1 T cells reconstituted with any of the three MO populations and incubated with whole GPBP passively transferred EAE into naive F1 mice. Immune F1 T cells cultured with F1 MO in the presence of either fragment 1-37 or 89-169 transferred EAE. F1 T cells cultured with SJL MO were able to transfer EAE only if the Ag was fragment 89-169, whereas F1 T cells cultured with PL MO were able to transfer disease only if incubated in the presence of fragment 1-37. F1 mice are passively susceptible to EAE induced by adoptive transfer of cells reactive to either the N-terminal or C-terminal fragment and that the encephalitogenic determinant of GPBP is related to the genome of MO present in vitro.     

LPS regulation of the immune response: Bacteroides endotoxin induces mitogenic, polyclonal, and antibody responses in classical LPS responsive but not C3H/HeJ mice

Recent studies have suggested that lipopolysaccharide (LPS) derived from gram-negative organisms such as Bacteroides, which are not members of the Enterobacteriaceae, stimulate B cells from the classic LPS-hyporesponsive C3H/HeJ mouse. In the present study, purified, phenol-water-extracted LPS from Bacteroides fragilis ATCC 25285 (B-LPS) was tested for its ability to induce in vivo and in vitro responses in classic LPS-responsive C3H/HeN, LPS-hyporesponsive C3H/HeJ, and (C3H/HeN X C3H/HeJ)F1 hybrid mice. B-LPS induced mitogenic responses in both C3H/HeN and C3H/HeJ spleen cell cultures when cells were cultured under standard conditions, i.e., 8 X 10(5) cells/well. Interestingly, when lower spleen cell numbers were tested with B-LPS, a typical responsive-nonresponsive pattern developed in which good mitogenic responses were induced by B-LPS in C3H/HeN cultures and in which low responses in C3H/HeJ spleen cell cultures were evident. In vivo immunization of mice with B-LPS resulted in high antibody responses in C3H/HeN, intermediate responses in F1, and low responses in C3H/HeJ mice. When purified splenic B cells were incubated with B-LPS, both mitogenic responses and polyclonal immunoglobulin M (IgM) synthesis occurred in C3H/HeN cultures, whereas intermediate responses were noted in F1 cultures and no response was seen in B cell cultures from C3H/HeJ mice. Furthermore, in vitro TNP-B-LPS responses were induced in C3H/HeN spleen cells or purified B cell cultures, and intermediate anti-TNP PFC responses occurred in F1 spleen cells or purified B cell cultures. The toxicity of B-LPS was tested in galactosamine-sensitized mice. The LD50 values for B-LPS in classic LPS-responsive C3H/HeN and C57BL/6J mice were 0.6 microgram and 1.1 microgram, respectively; F1 hybrid mice were approximately 15-fold more resistant, whereas C3H/HeJ mice gave an LD50 of 1650 micrograms. This study shows that phenol-water preparations of B-LPS are biologically active and induce responses in the classic LPS-responsive but not in the LPS-hyporesponsive C3H/HeJ mouse strain.     

Cellular transfer of experimental allergic encephalomyelitis in Lewis rats: Effects of different sensitization regimens on in vitro reactivity of donor spleen cells to concanavalin A

Splenocytes from Lewis rats sensitized to guinea pig spinal cord (GPSC) and complete Freund's adjuvant (CFA) or to myelin basic protein (MBP)-CFA plus pertussis vaccine were less effective than spleen cells from MBP-CFA sensitized donors in transferring EAE to syngeneic recipients following culture with concanavalin A (Con A). Moreover, splenocytes from rats sensitized to GPSC-CFA plus pertussis vaccine showed no EAE transfer activity following culture with Con A. Diminished EAE transfer activity occurred in parallel with decreased proliferative responses of primed splenocytes to Con A. These effects were due, at least in part, to the use of pertussis vaccine and to Con A activation of a suppressive adherent cell subpopulation in sensitized donor spleens. Proliferative responses and EAE transfer activity were restored upon removal of plastic-adherent cells from splenocytes of rats sensitized to MBP-CFA plus pertussis vaccine prior to Con A activation of the non adherent lymphoid cells. Deletion of plastic-adherent cells from splenocytes of donors sensitized to GPSC-CFA plus pertussis vaccine prior to activation with Con A, however, had no effect on proliferative responses or EAE transfer activity. Furthermore, EAE transfer activity of Con A-activated splenocytes from MBP-CFA-sensitized donors was lost when such cells were cultured with splenocytes from donors sensitized to GPSC-CFA plus pertussis vaccine.     

Mechanisms of resistance to the intracellular protozoan Encephalitozoon cuniculi in mice

Mechanisms of resistance to the obligate intracellular protozoan Encephalitozoon cuniculi were studied in BALB/c mice. Resistance to lethal disease was T cell-dependent because transfer of T-enriched, but not T-depleted, spleen cells from sensitized BALB/c donors would protect infected BALB/c-nu mice. A modified focus-forming assay was utilized to measure effects on E. cuniculi infectivity in vitro. The results show that antibodies exert an opsonization effect and may block parasite entry into nonphagocytic cells. No cytotoxic T cells were demonstrated. Supernatants from E. cuniculi-sensitized spleen cells incubated with E. cuniculi in vitro could induce adherent PEC to kill E. cuniculi.     

Attempts to transfer immunity against Clonorchis sinensis in nude and DS mice.

The effects of peritoneal exudate cells (PEC) and sera of athymic nude and DS mice infected with Clonorchis sinensis metacercariae or sensitized by injection of metabolic products into footpad on transfer of immunity against the fluke to the syngeneic mice were studied. There was no significant difference in eggs per gram pattern between the sensitized and control groups, and between nude and DS mice. However, the worm burdens were slightly greater in nude mice than in DS mice. Also, a few plaque forming cells were found in only DS mice given PEC and serum from Group II DS mice. In the light of these results, it is likely that PEC and sera of nude or DS mice which are deficient, at least partially, in the cellular immune system are unable to transfer immunity against C. sinensis to syngeneic recipients.     

Variation among mouse strains in responsiveness to migration inhibitory factor.

Mineral oil-induced peritoneal exudate cells (PEC) from 10 different inbred mouse strains were tested for their responses to macrophage migration inhibitory factor (MIF). PEC from 5 out of 10 mouse strains responded to MIF, PEC from BALB/c mice showed an intermediate responsiveness, and PEC from A/J, C3H/HeJ, CBA/N, and C57BL/10ScCR mice were refractory to MIF. MIF responsiveness was not linked to the H-2 complex. However, a possible link between responsiveness to LPS and MIF was suggested, since the mouse strains not responding to MIF were previously reported to be deficient for responses to LPS.     

Opposing effects of cryostat sections of preneoplastic and neoplastic mouse mammary lesions on in vitro migration of peritoneal exudate cells.

Cryostat sections of normal mouse tissues and of preneoplastic HAN and neoplastic mammary tumors were used as "antigens" in MMI tests. Nonspecific inhibition of normal and sensitized PEC migration was induced by HAN and some normal tissues, including normal mammary gland. This inhibition did not require the presence of lymphocytes, was not species specific, and could be blocked by sera from HAN-bearing mice. Cryostat sections of mammary tumors did not inhibit, indeed occasionally enhanced PEC migration. Further, the presence of tumor cryostats and eluates interfered with inhibition induced by HAN cryostats and by PPD with PEC from donors sensitized to that antigen. Histologic examination of HAN and of mammary tumor tissue revealed inflammatory cells to be distributed diffusely in the former and localized peripherally around the latter type of lesion.     

Role of accessory cells in the in vitro growth of aggregate-derived murine T-cell colonies

C3H/HeJ lymph node cells (LNC) were seeded in 35-mm petri dishes containing 0.8% methylcellulose, 10% fetal calf serum, 2-mercapthoethanol, and supernatant from PHA or Con A-stimulated spleen cells. After 3–4 days incubation at 37 °C, colonies containing >50 cells appeared. The cells from individual colonies stained with a fluorescent anti-Thy-1 antiserum, and colony formation was prevented by treating the LNC with radiation or anti-T-cell serum + complement before culturing. When fewer than 1?2 × 10⁶ LNC were seeded, the number of colonies formed decreased exponentially; this observation suggested colony formation might require cell-cell interaction. Formation of cellular aggregates could be seen as early as 4–20 hr after plating. Colony formation of 2?5 × 10⁵ LNC was promoted by adding irradiated or anti-T serum + complement-treated LNC, and colony formation was inhibited by carbonyl iron treatment to remove adherent cells. Cell separation by velocity sedimentation showed colony promoting activity was associated with cells sedimenting at 4 mm/hr and also >6 mm/hr. These are properties similar to those of accessory cells that are required for immune responses in vitro and in vivo. Colony formation was also increased in LNC from tumor allograft immune mice, and in the uterine lymph nodes from mice bearing an allogeneic fetus. T-Cell colonies produced by direct plating of LNC in this system arise from proliferation of cellular aggregates, and are primarily a measure of accessory cell activity.     

Intratracheal priming with ovalbumin- and ovalbumin 323-339 peptide-pulsed dendritic cells induces airway hyperresponsiveness, lung eosinophilia, goblet cell hyperplasia, and inflammation

Dendritic cells (DC) are the primary APC responsible for the capture of allergens in the airways and the shuttling of processed allergens to the draining lymph nodes where Ag presentation and T cell activation take place. The mechanism of this Ag handling and presentation in asthma is poorly understood. In addition, the feasibility of asthma induction by DC priming has not been directly tested. In this report an asthma model using intratracheally (i.t.) injected splenic DC was used to address these issues. DC pulsed with a model Ag OVA or the major MHC class II-restricted OVA T epitope peptide OVA(323-339) and instilled i.t. primed mice to exhibit asthma-like diseases. With OVA as the Ag, mice exhibit airway hyperresponsiveness (AHR), lung eosinophilia and inflammation, and pulmonary goblet cell hyperplasia. In OVA(323-339)-immunized mice, AHR and goblet cell hyperplasia were noted, with little eosinophilia and parenchymal inflammation. The latter finding provides evidence for dissociating AHR from eosinophilia. In both cases mediastinal node hypertrophy occurred, and high levels of Th2 cytokines were produced by the lung and mediastinal lymph node cells (LNC). Interestingly, mediastinal LNC also produced high levels of Th1 cytokines. Lung cells produced much less Th1 cytokines than these LNC. These results demonstrate that DC when introduced i.t. are potent in inducing asthma-like diseases by recruiting lymphocytes to the lung-draining lymph nodes and by stimulating Th2 responses and also suggest that the lung environment strongly biases T cell responses to Th2.     

Lymphoid cell subpopulations. I. Synergy between lymph node cells and thymocytes in response to alloantigens and mitogens.

Mixtures of isogenic thymocytes (TC) and lymph node cells (LNC) were shown to exhibit synergistic responsiveness to M and H-2 alloantigens in the mixed lymphocyte interaction (MLI). With respect to the kinetics and magnitude of proliferation and effector cell generation, the response occurring in synergizing cultures closely resembled that of optimal numbers of LNC or spleen cells (SC). In addition, the antigen specificity of effector cells generated by synergizing cultures was similar to that of effectors derived from cultures containing optimal numbers of responding SC. LNC-TC mixtures also exhibited synergy in response to the phytomitogens concanavalin A and pokeweed mitogen but not to phytohemagglutinin. Weakly positive synergy was observed in response to bacterial lipopolysaccharide. It is proposed that the phenomenon of synergy is not restricted to cultures containing mixtures of LNC and TC but also occurs in cultures containing optimal numbers of LNC or SC as a result of interactions between subpopulations of lymphocytes contained within these tissues.     

Effects of antithymocyte serum on lymph node cells participating in the graft-vs-host reaction.

BALB/c mice were injected with 0.1 ml of antithymocyte serum (ATS) and tested at various times thereafter for graft-versus-host (GVH) reactivity of lymph node cells (LNC) and spleen cells (SC). Splenomegaly produced by LNC or SC injected alone was used as a measure of precursor T cell function and the capacity of such cells to produce synergy when combined with normal thymocytes was used to evaluate amplifier T cell activity. In lymph node, both precursor and amplifier function were strikingly depressed 2 days after ATS administration; by day 11, precursor function showed slight recovery while amplifier activity had recovered to supranormal levels. In spleen, precursor activity recovered two fold between 2 and 11 days after ATS inoculation but no amplifier activity could be detected at day 11. Since donors had not been deliberately stimulated with alloantigens prior to testing of LN and SC GVH acivity, these studies demonstrate (a) that precursors and amplifiers are disinct T cell populations that are committed to express unique functional activities before antigen exposure and (b) that, following depletion with ATS, these two populations recover independently at different rates in separate lymphoid compartments.     

Tolerance to nickel: oral nickel administration induces a high frequency of anergic T cells with persistent suppressor activity.

We adapted our mouse model of allergic contact hypersensitivity to nickel for the study of tolerance. Sensitization in this model is achieved by the administration of nickel ions with H(2)O(2); nickel ions alone are unable to prime naive T cells, but can restimulate primed ones. A 4-wk course of oral or i.p. administration of 10 mM NiCl(2) to naive mice induced tolerance, preventing the induction of hypersensitivity for at least 20 wk; long term desensitization of nickel-sensitized mice, however, required continuous NiCl(2) administration. When splenic T cells of orally tolerized donors, even after a treatment-free interval of 20 wk, were transferred to naive recipients, as with lymph node cells (LNC), they specifically prevented sensitization of the recipients. The LNC of such donors were anergic, because upon in vivo sensitization with NiCl(2) in H(2)O(2) and in vitro restimulation with NiCl(2), they failed to show the enhanced proliferation and IL-2 production as seen with LNC of mice not tolerized before sensitization. As few as 10(2) bulk T cells, consisting of both CD4(+) and CD8(+) cells, were able to specifically transfer tolerance to nickel. A hypothesis is provided to account for this extraordinarily high frequency of nickel-reactive, suppressive T cells; it takes into account that nickel ions fail to act as classical haptens, but form versatile, unstable metal-protein and metal-peptide complexes. Furthermore, a powerful amplification mechanism, such as infectious tolerance, must operate which allows but a few donor T cells to tolerize the recipient.