Lymph node localization and whole body distribution of radioiodinated encephalitogenic polypeptide in guinea-pigs

A bovine encephalitogenic polypeptide (BEP) labelled with radioiodide retained its capacity to induce experimental encephalomyelitis (EAE). Guinea-pigs were injected with ¹²⁵I BEP in Freund's complete adjuvant (FCA), to study changes in the architecture and the distribution of radioactivity in draining lymph nodes, and the amount of radioactivity in various organs.

After injection of BEP in FCA the lymph node rapidly enlarged. Within 48 hr there was depletion of lymphocytes, the enlarging lymphoid follicles had become confluent and there was proliferation of large `epithelioid' cells throughout the node. At 5 days the lymph node architecture was disorganized and lymph follicles with germinal centres could not be recognized; similar but less pronounced changes were present in regional nodes. By contrast, after injection of flagellin in FCA, there were numerous lymphocytes, plasmablasts and pyroninophilic cells, germinal centres were prominent, and the architecture was preserved.

From 0·5 to 0·8% of the total injected radioactivity was concentrated in the popliteal lymph node 2–5 days after injection of ¹²⁵I BEP in FCA. No radioactivity was concentrated in the node after injection of ¹²⁵I BEP without FCA, and animals thus immunized did not develop encephalomyelitis.

The popliteal lymph node was examined by autoradiography after injection of ¹²⁵I BEP in FCA. At 24 hr radioactive encephalitogen associated with droplets of adjuvant was present mainly in the peripheral sinus and at 48 hr encephalitogen–adjuvant droplets were deposited randomly throughout cortex and medulla. These droplets appeared to represent sites where lymphoid cells acquired their capacity for pathogenic reactivity with their target antigen in the central nervous system.

     

The cellular transfer of immunity to Nippostrongylus brasiliensis in inbred rats (Lewis strain)

Mesenteric lymph node cells obtained from highly inbred donor rats (Lewis strain), resistant to Nippostrongylus brasiliensis infection, were syngeneically transferred by intravenous injection into previously uninfected recipients. The adoptively immunized recipients were then challenged with either 1500 or 3000 third stage N. brasiliensis larvae on the day of cell transfer. The degree of resistance transferred was assessed by monitoring daily faecal egg output, differential worm burdens on days 6 and 10 of infection and the number of eggs per uterus in gravid worms.

The syngeneic transfer of 100 × 10⁶ immune mesenteric lymph node cells invariably resulted in suppression of egg production, a two- to four-fold reduction in the number of eggs per uterus in gravid females and rejection of at least 75 per cent of adult worms by days 6 and 10 of infection.

It was also noted that mesenteric lymph node cells obtained from donors on day 15 of a primary infection were more effective than those obtained from donors immunized by multiple infections.

Immune cells transferred from donors on day 4 of infection were equally effective with those transferred on day 0. However, immune cells transferred on or after day 10 of infection had little or no effect and this shows that the parasite is less susceptible to an attack mounted by the transferred cells during the later stages of infection.

     

Depression of delayed hypersensitivity by pretreatment with Freund-type adjuvants. II. Mechanism of the phenomenon

Recipient guinea-pigs pretreated with Freund's complete adjuvant alone show depressed 4 hr (Arthus) reactions following passive transfer of antiserum to bovine γ-globulin and human serum albumin. Recipient outbred guinea-pigs and inbred rats also show depressed 24 hr delayed reactions following passive transfer of immune peritoneal exudate cells and serum. This indicates that Freund's complete adjuvant depresses certain inflammatory responses.

Donor guinea-pigs and inbred rats pretreated with FCA alone and then immunized with BGG in FCA transfer smaller 24 hr reactions to normal recipients than comparable donor animals which have not been pretreated with FCA. This suggests that pretreatment with FCA depresses the central state of delayed hypersensitivity which normally follows immunization with BGG in FCA.

These two findings may explain why pretreatment with FCA depresses the 4 and 24 hr skin reactions which otherwise follow immunization with antigen in FCA.

     

Suppression of experimental allergic encephalomyelitis with thoracic duct lymphocytes

Thoracic duct lymphocytes (TDL) from Lewis rats immunized 9-10 days previously with basic protein in complete Freund's adjuvant (BP-CFA) failed to induce experimental allergic encephalomyelitis (EAE) in syngeneic recipients. This contrasts with the successful transfer of EAE by lymph node cell suspensions from donors immunized 9 days previously with BP-CFA. Only minor EAE was induced passively by TDL from rats immunized 11-12 days before with BP-CFA. TDL collected 9-20 days after BP-CFA immunization, however, were successful in transferring specific suppression of EAE tested by the lack of disease in the recipients immunized actively with BP-CFA 1 week after the TDL transfer. The data indicate that the thoracic duct contains specific suppressor cells shortly before, during and after the development of clinical EAE.     

Alterations in granulation tissue growth induced in vivo by lymphocytes from adjuvant-diseased rats.

Lymph node cells from Lewis and Wistar rats, treated 9 or 11 days previously with Freund's complete (FCA) or incomplete adjuvant (FIA), were transferred into polyether sponges implanted subcutaneously into syngeneic, recipient rats. FCA-treated lymphocytes enhanced or reduced granuloma formation (measured after 8 days), when compared with FIA-treated controls, depending on the strain of mycobacterium present in the FCA. The stimulatory effects of lymphocytes from FCA-treated, Lewis rats were abolished by pre-incubation with mitomycin C (25 microgram/ml). Whole serum and isolated serum immunoglobulin from adjuvant-diseased rats had no effect on the sponge granulomas. These data confirm that cell-mediated immunity is involved in the articular granuloma formation of adjuvant arthritis.     

Contact and delayed hypersensitivity in the mouse: II. The role of different cell populations

Mice sensitized to oxazolone show contact sensitivity which can be assessed by painting oxazolone on the ear and measuring the increase in ear thickness at 24 hours. Contact sensitivity can be transferred passively by peritoneal exudate, lymph node and bone marrow cells. When CBA mice are challenged immediately after transfer, at a 3:1 donor recipient ratio, the peritoneal exudate cells give bigger transferred reactions than lymph node cells. The converse is true on challenge at 6 days after transfer, at which time the lymph node cells give bigger transferred reactions than the peritoneal exudate cells. These results suggest that different types or functional states of cells are involved in these two types of transfer.

The possibility that the successful transfer with delayed challenge by lymph node cells was due to active sensitization by antigen transferred with the cells, was made unlikely by passive transfer into `tolerant' recipients.

Mice first show contact sensitivity when challenged at 3 days (about 72 hours) after sensitization. The lymph nodes develop the ability to transfer contact sensitivity with delayed challenge at the same time but the bone marrow only acquires this activity at 5 days.

     

Dendritic cells and T cells transfer sensitization for delayed-type hypersensitivity after skin painting with contact sensitizer

The cells involved in the development of delayed-type hypersensitivity (DTH) to the contact sensitizer fluorescein isothiocyanate (FITC) were examined. Cells used for transferring sensitization were obtained from donor mice up to 5 days following skin painting with FITC. Recipient mice were sensitized by footpad injections of dendritic cells (DC) obtained from donor lymph nodes up to 3 days following skin painting, when DC expressed high levels of antigen. DTH, assessed by ear swelling 24 hr following ear challenge with FITC, was detected when recipient mice were challenged 5 days after transfer of DC, but not when ear challenged immediately after transfer. Removal of donor DC using the cytotoxic antibody 33D1, plus complement, either from the DC-enriched population or from whole lymph node cells 24 hr after skin painting, abolished the capacity to transfer DTH. Purified T lymphocytes obtained from donor mice between Days 3 and 5 after skin painting, transferred DTH when recipients were ear challenged with FITC 5 days after footpad injections. DTH also occurred in mice ear challenged with antigen immediately after receiving footpad injections of either normal or irradiated T cells obtained from donors 4 days after skin painting. B cells and macrophages did not transfer sensitization for DTH throughout the time-course. Therefore an early stage in the immune response, where antigen-bearing DC initiated DTH, was distinguished from a later stage, where T cells transferred sensitization.     

Potentiation of nephrotoxic serum nephritis in Lewis rats by Freund's complete adjuvant--possible role for cellular immune mechanisms

Lewis rats receiving subnephritic doses of nephrotoxic serum (NTS) showed increased albuminuria and glomerular histopathologic alterations during the autologous phase of nephrotoxic nephritis (NTN) when they received simultaneous footpad injections of Freund's complete adjuvant (FCA). Lymph nodal lymphocytes from such experimental rats showed increased in vitro cellular sensitization to the nephrotoxic IgG as measured by [3H]thymidine incorporation. Such a lymphocyte blastogenesis response was not detected in rats receiving the same doses of FCA or NTS alone. Antibody titers to the nephrotoxic rabbit IgG were not different in the two groups of rats as measured by enzyme-linked immunosorbent assay. The transfer of lymph nodal mononuclear cells from rats with NTN potentiated by FCA, was able to induce albuminuria and glomerular histopathologic alterations in recipients treated with NTS. In the above experimental model FCA appears to potentiate the autologous phase of NTN by cellular immune mechanisms.     

Depression of delayed hypersensitivity by pretreatment with Freund-type adjuvants. III. Depressed arrival of lymphoid cells at recently immunized lymph nodes in mice pretreated with adjuvants

Pretreatment of mice with Freund's complete adjuvant (FCA) or Corynebacterium parvum depresses the contact sensitivity which otherwise follows immunization with picryl chloride in FCA (PCL/FCA). In contrast, Bordetella pertussis is inactive. The hypothesis that this depression was partly due to the failure of lymph nodes immunized with PCL/FCA to collect lymphoid cells was tested. Mice were pretreated with FCA, C. parvum or B. pertussis, subsequently immunized with PCL/FCA, and injected with ⁵¹Cr-labelled normal lymph node cells.

Pretreatment with FCA 10 days before immunization depressed the arrival of labelled cells at draining nodes immunized with PCL/FCA by 10–19%.

Pretreatment intravenously with C. parvum 4 days before immunization virtually abolished the increased arrival at draining lymph nodes which is otherwise seen 2 and 4 days after immunization, but had little effect on the increased arrival seen 1 day after immunization. In contrast, B. pertussis caused a slight but significant depression at day 1, but had no effect 2 and 4 days after immunization.

The depression of contact sensitivity by these three bacterial adjuvants was paralleled by their depression of the arrival of labelled lymph node cells at recently immunized nodes, and supported the hypothesis that failure of recently immunized lymph nodes to collect lymphoid cells is part of the mechanism of depression of delayed hypersensitivity by bacterial adjuvants, and in particular, by C. parvum.

     

Requirement of B Lymphocytes in Local Adoptive Transfer of Experimental Allergic Orchitis (EAO) by Lymph Node Cells

ABSTRACT: The necessity of T lymphocytes in local adoptive transfer of experimental allergic orchitis (EAO) is an established fact. The present study was undertaken to elucidate the participation of B lymphocytes in the local transfer system of EAO. The capacity of purified lymph node T and B cells to transfer EAO was compared. Donor strain 13 guinea pigs were sensitized with testicular antigen and complete Freund's adjuvant. We then injected 1 × 10⁸ cells just under the tunica albuginea of the testis of a syngeneic recipient. The recipients were killed 5 days after cell transfer. Nylon-wool nonadherent T cells alone produced only minimal lesions, but could induce more severe mononuclear lesions with the addition of syngeneic, normal macrophages. In contrast, B-cell enriched populations either alone or combined with a small number of macrophages, were able to transfer extensive lesions. In the combined transfer of immune T and immune B cells the severity of transferred lesions was dependent upon the number of B cells. These results suggest that cooperative interactions among T cells, B cells, and macrophages are involved in the generation of transferred testicular lesions, with emphasis on the participation of B cells.     

The suppression of rejection of Nippostrongylus brasiliensis in lactating rats: the nature of the immunological defect

Lactating female rats infected with 3000 third-stage larvae of Nippostrongylus brasiliensis showed significant increases in worm fecundity and total worm burdens when compared with infected nulliparous controls. Statistically significant differences were recorded for each of the three periods of infection, although these differences were of greatest magnitude during Period 3 (16–30 days of infection).

Immune mesenteric lymph node cells (100 × 10⁶), obtained from nulliparous female donors on Day 15 of a primary infection, were transferred syngeneically to lactating female recipients. The transferred cells invariably caused suppression of worm fecundity, reduction in the number of eggs per uterus in gravid female worms and rejection of a substantial proportion of worms by Day 10 of a challenge infection in the lactating recipients. The results of this study showed that immune cells were functional in lactating female recipients and that transfer of immune cells repaired the deficit in the rejection mechanism.

Mesenteric lymph node cells (100 × 10⁶), obtained from lactating female donors on Day 15 of a primary infection, were transferred syngeneically to nulliparous female recipients. The transferred cells caused suppression of worm fecundity, reduction in the number of eggs per uterus in gravid female worms and rejection of the majority of parasites by Day 10 of a challenge infection in the nulliparous recipients. Clearly, potentially immune lymphoid cells were present in the mesenteric nodes of lactating females at the time that the rejection mechanism was severely impaired.

Mesenteric lymph node cells obtained from infected lactating donors were substantially less effective in lactating recipients than in nulliparous recipients. These cells caused the expulsion of 51 per cent of worms by Day 10 in lactating recipients, whereas they caused expulsion of 99 per cent of worms in nulliparous recipients.

These observations suggest that the inductive processes of the immune response occur normally, but that differentiation of induced cells to effector cells is impaired in lactating animals.

     

Activation of cytotoxic T cells and effector cells in experimental autoimmune thyroiditis by shared determinants of mouse and human thyroglobulins

Previous studies have shown that T cells from genetically susceptible mice developing experimental autoimmune thyroiditis (EAT) proliferate in response to restimulation with mouse thyroglobulin (MTg) in vitro and differentiate into cells cytotoxic for syngeneic thyroid monolayers. To examine further the effector cells involved in pathogenesis and the determinants on MTg responsible for their activation, spleen cells (SC) and lymph node cells (LNC) from mice immunized with MTg or human (H) Tg, and adjuvant (complete Freund's adjuvant (CFA) or lipopolysaccharide (LPS] were cultured in vitro with MTg or HTg. Control cultures were incubated with concanavalin A (Con A) or purified protein derivative (PPD). The in vitro-activated cells which proliferated in response to MTg, HTg, or Con A adoptively transferred thyroiditis to normal recipients, whereas cells transferred directly without in vitro culture were very ineffective. The capacity to transfer EAT was abrogated by irradiation (1500 R), and SC from CFA-immunized control mice which responded in vitro to PPD stimulation did not transfer thyroiditis. The serum titers of MTg autoantibodies were uniformly low and were not correlated with severity of disease. The localization of EAT-effector (precursor) cells depended upon the site of immunization; they were found in the spleens after inguinal (subcutaneous) or systemic (intravenous) immunizations, but were present in the popliteal lymph nodes after hind footpad injections. Both homologous MTg and heterologous HTg functioned as in vivo sensitizing antigen and in vitro activating antigen for each other; such cultured cells transferred thyroiditis in vivo and became cytotoxic for thyroid monolayers in vitro. These findings show that shared determinants are autoantigenic and thyroiditogenic, and support the hypothesis that EAT-effector cells responsible for initiating thyroid damage include cytotoxic cells.     

Permanent hapten-specific tolerance in B lymphocytes.

Tolerance to the hapten TNP was induced in mice congenic with CBA but bearing the Ig-b allotype (Ig-b mice). To induce a high degree of tolerance it was necessary to give five injections of TNP-sulphonate followed by an immunogenic challenge (alum precipitate of TNP-BSA with pertussis adjuvant). Lymph node or spleen cells from these mice were transferred, with or without an equal number of non-tolerant CBA spleen cells, to irradiated CBA recipients and these were challenged with a different TNP-protein conjugate. Anti-TNP antibody bearing the Ig-b allotype was then assayed separately from total anti-TNP, as a measure of the contributions made by tolerant and non-tolerant B-cell populations respectively. Tolerant lymph node cell did not depress the response of normal cells, nor did the normal cells 'break' the tolerance of the Ig-b population even when the latter had been treated with anti-T-cell serum and complement. No response was obtained from tolerant lymph node cells when the recipients were challenged at different time up to 12 weeks after transfer. By this time the control non-tolerant lymph node cells had also lost the capacity to respond. It is concluded that: (1) effectively permanent tolerance, which is not maintained by afferent mechanisms, can be induced in lymph node B cells; (2) B-cell tolerance can be greatly enhanced by immunogenic challenge; (3) spleen may contain a distinct population of B cells which is less susceptible to tolerance; and (4) the life-span of virgin lymph node B cells is probably less than 12 weeks.     

The cellular transfer of immunity to Trichostrongylus colubriformis in an isogenic strain of guinea-pig: III. The localization and functional activity of immune lymph node cells following syngeneic and allogeneic transfer

Mesenteric lymph node cells obtained from guinea-pigs immunized with Trichostrongylus colubriformis have been transferred allogeneically (McMaster outbred to Heston inbred) and syngeneically (Heston to Heston inbred) by intravenous injection. The immune cells were transferred on days 6, 7 and 8 of infection when the parasite was in the susceptible fourth stage of development. The syngeneically transferred cells caused rejection of the parasite but the allogeneically transferred cells were not effective.

Immune mesenteric lymph node cells were labelled in vitro with ⁵¹Cr before transfer to infected recipients. The percentage of the intravenously injected cells which localized in infected small intestine has been calculated from γ-counts recorded at 1, 6, 16, 24 and 48 hours after injection of the labelled cells. The mean percentage of cell dose per 12 in. of small intestine recorded following syngeneic transfer was 1·29 and following allogeneic transfer 0·68. A significant difference in the localization of allogeneic and syngeneic cells in the small intestine was already apparent at 6 hours following intravenous injection.

     

Effects of heterologous anti-lymphocyte serum on the distribution of 51Cr-labelled lymph node cells in mice

The effects of heterologous anti-lymphocyte serum (ALS) were studied in a syngeneic cell transfer system, in which lymph node cells from donor CBA mice were labelled in vitro with ⁵¹Cr and transferred intravenously into syngeneic recipients. Labelled cells treated in vitro with ALS were unable to migrate to lymph nodes or spleens of recipients, as did normal cells, but instead distributed themselves very similarly to cells which had been killed by exposure to heat. It is thus likely that cells treated in vitro with ALS are killed after transfer by the cytotoxic action of ALS mediated by the complement of the recipient.

ALS administered directly to the recipients of labelled lymphocytes could also reduce their uptake into lymphoid tissue; however, the magnitude of this effect appeared to be critically dependent upon the timing of the antiserum dose with respect to the labelled cell dose. ALS given immediately prior to labelled cells showed the greatest effect, while treatment given either 24 hours before or after the labelled cells was much less effective. While with prior treatment the reduced effect could be due to a fall off in antibody titre during the interval between the dose of antiserum and cells, in the latter situation no drop in titre would have occurred. It thus seems that lymphocytes that have already established themselves in lymphoid tissue may be less susceptible to the action of ALS.

ALS given chronically to lymphoid cell donors resulted in a population of cells which upon transfer to normal recipients were distributed differently from either normal or NRS-treated donor cells.

These data support the hypothesis that the effects of ALS may be exerted preferentially on circulating lymphocytes, and that ALS may act to selectively reduce the representation of this cell type in lymphoid tissue.

     

Studies on the pathogenesis of experimental anti-tubular basement membrane nephritis in the guinea pig.

Using the model of renal disease induced in guinea pigs by immunization with bovine TBM preparations in adjuvant, the following observations were made. Animals with activity induced disease show bright staining for IgG along the TBM and only faint, inconstant staining along the GBM. Following transfer of serum animals with anti-TBM disease to normal recipients, accumulation of IgG was found predominantly in glomeruli at 4 hours, but at Days 3 and 5, IgG was seen predominantly along the TBM. There was no appreciable accumulation of neutrophils in the kidneys of recipients of anti-TBM serum, even at early intervals (4 and 24 hours) after transfer. However, within 2 days, small numbers of mononuclear cells were found. By Day 3, mononuclear cells were numerous, and multinucleate giant cells and tubular cell damage were present. After that, the lesions increased in severity and by 10 days were indistinguishable from those found in actively immunized animals at 14 to 21 days. Study of frozen section of kidneys obtained from animals with active disease at 14 days, employing sheep cells coated with rabbit antibody (IgG EA) revealed rosettes around many of the mononuclear cells in the infiltrate, indicating that they are mononuclear phagocytes (monocytes or macrophages). IgM complexed with sheep cells and complement (EAC) did not react and thus failed to provide evidence for the presence of B lymphocytes. Transfer of 7 X 10(8) lymph node cells from the TBM-immunized Strain 13 donors to normal Strain 13 recipients failed to result in renal lesions. The findings are interpreted as indicating that anti-TBM antibodies mediate the renal disease without the participation of cell-mediated immunity and further that these antibodies bring about an influx of ciculating mononuclear cells, predominantly monocytes, without attracting appreciable numbers of neutrophils.     

Intracerebral injection of myelin basic protein (MBP) induces inflammation in brain and causes paraplegia in MBP-sensitized B6 mice

Brain inflammation and paraplegia can be induced by an additional intraperitoneal (i.p.) and intracerebral (i.c.) restimulation in B6 mice after standard immunization with MBP in Freund's complete adjuvant (FCA) and Bordetella pertussis coadjuvant. Only the combination of i.p. MBP/FCA and i.c. MBP injection could induce clinical paraplegia; either one alone was not effective. Clinical symptoms would develop 2 days after the i.c. injection. The induction of paraplegia was MBP-specific, as irrelevant bovine serum albumin with the same protocol could not induce it. The i.p. restimulation was requisite and needed the MBP in FCA, as MBP in PBS was ineffective. Histopathological observation manifested cellular infiltration by leucocytes in perivascular spaces and cerebral cortex. Neutrophils were prominent at 12 h after i.c. injection, then were replaced by mononuclear cells 24 h later. There were dynamic changes in cell number and immunophenotype of VLA-4⁺ expression in cervical lymph node cells after i.c. injection. The cells derived from cervical lymph nodes had higher MBP-stimulated proliferation than that of distal lymph nodes. This additional i.p. and i.c. stimulation provides a new manipulation to study brain inflammation.     

Suppression of experimental allergic encephalomyelitis by transfer of lymph node cells from Lewis rats pretreated with complete Freund's adjuvant

Experimental allergic encephalomyelitis (EAE) in Lewis rats was suppressed by pretreatment with complete Freund's adjuvant (CFA) 14-16 days before immunization with basic protein (BP) in CFA. Suppression of active EAE was also accomplished clinically and even histologically if lymph node cells from CFA-treated donors were passively transferred. Obviously this depends first on a certain amount of transferred lymph node cells (about 3 X 108) and secondly on the interval between CFA application and cell harvest (more than 14 days). No suppression was obtained by transfer of serum from CFA-treated donors. This indicates that there is a suppressor cell population which can be raised or stimulated by CFA and which is capable of suppressing the immune response EAE.     

Cellular transfer of autoimmune aspermogenic orchiepididymitis (AIAO) by the i.v. route in the guinea pig.

In this study, AIAO was adoptively transferred with a high proportion of success to syngeneic recipient guinea pigs. Donors of strains 2 and 13 were sensitized with a series of spermatozoal autoantigens (whole spermatozoa and three autoantigens isolated therefrom: S, P and T). Syngeneic (experimental) and allogeneic (control) recipients were all transferred by strictly i.v. injections of lymphoid cells. The damage observed in testis and epididymis (mainly in the latter) was identical to, but milder than, that seen in active forms of AIAO. The incidence and severity of the disease were dependent on: the type of inducing antigen, S, T, P in order of decreasing efficiency; the length of immunization in donors, with increasingly serious lesions as periods ranged from 1 to 4 weeks; the presence or not of a complementary treatment of recipients with bacterial adjuvant enhancing the disease. Other parameters were less important, such as the strain 2 or 13 specificities, the amount of immunogen or the addition of peritoneal cells to lymph node cells. Skin hypersensitivity was concomitantly transferred to a large majority of isogenic recipients. But the incidence and severity of the disease showed only a partial correlation with Arthus type or delayed type responses to autoantigens. Thus guinea pig AIAO, already known to be transferable by immune sera (mainly anti-P and also anti-T) may also be transferred in physiological conditions by sensitized lymphoid cells (mainly anti-S and also anti-T).     

Adoptive Transfer of Allergic Encephalohyelitis by Inoculation of Cells Into Lymph Nodes

Experimental allergic encephalomyelitis can be transferred passively by lymph node cells from actively immunized donor rats to immunologically naive syngeneic recipients. In the present work, passive transfer was accomplished by direct inoculation of donor cells into the recipient's lymph node, and this route was more effective than the conventional intravenous or intraperitoneal routes.