Cellular Reaction in the Footpad and Draining Lymph Nodes of Mice Induced by Mycobacterial Fractions and BCG Bacilli

Ten micrograms of trehalose-6, 6'-dimycolate (cord factor), injected into the footpad of mice, induced histological changes similar to those following injection of living BCG bacilli. Both materials induced in the draining lymph nodes the formation of granulomas composed of epitheloid cells, macrophages, and small numbers of lymphocytes. Apart from the granulomatous inflammatory process, marked hyperplasia of the lymphoid tissue in the paracortical zone of the nodes and accumulations of macrophages were evident. In some cases, the macrophages were very numerous and replaced part of the lymphoid tissue. Compared to cord factor, wax D showed weak granulomagenic activity. Only slight and transient inflammation was found in the footpads as well as transient slight lymphoid hyperplasia. Wax D also induced small accumulations of macrophages. Complete Freund's adjuvant induced, under the same experimental conditions, large accumulations of macrophages in the draining lymphnode and lymphoid hyperplasia in the paracortical zone. No cellular reaction was seen in the liver, spleen, and lungs after injection of cord factor and BCG into the footpads of the animals. The results and implications are discussed.     

Adjuvant and mitogenic properties of a supernatant fraction of sonically treated Myobacterium bovis (BCG).

A water-soluble, oil-free supernatant fraction of sonically treated BCG (BCG-SS) was shown to be an immunological adjuvant and a mitogen. When BCG-SS and sheep erythrocytes (SRBC) were injected intravenously into mice, the plaque-forming cell (PFC) response was 10 times greater than that induced by injection of SRBC alone. Circulating antibody responses to SRBC and to bovine serum albumin were also enhanced by BCG-SS. The in vitro enhancement of PFC and circulating antibody responses did not require mineral oil or exogenous lipids. In vitro PFC responses by normal mouse spleen cells were also greatly increased in the presence of BCG-SS. Anti-theta serum-treated spleen cells from mice that had been lethally irradiated and reconstituted with normal bone marrow cells also gave a higher PFC response to SRBC in the presence of BCG-SS. This suggests that BCG-SS can stimulate B lymphocytes to develop an immune response when T lymphocytes are severely depleted or absent. BCG-SS also stimulated the uptake of 125IUdR by normal spleen cell cultures, indicating that it is a mitogen.     

Systemic or local co-administration of lactoferrin with sensitizing dose of antigen enhances delayed type hypersensitivity in mice

Lactoferrin (LF), a major defense protein synthesized and stored in granulocytes has been implicated in maintaining immune homeostasis during an insult-induced metabolic imbalance. In this study, we demonstrated that lactoferrin augments the delayed type hypersensitivity (DTH) response to specific antigens in mice. Lactoferrin (LF) was given to mice orally or intraperitoneally (i.p. ) at the time of immunization, or subcutaneously (s.c.) in a mixture with the immunizing doses of the following antigens, sheep red blood cells (SRBC), Calmette-Guerin bacillus (BCG) or ovalbumin (OVA). A DTH reaction was determined 24 h after administration of an eliciting dose of antigen as a specific increase in foot pad swelling. Lactoferrin enhanced DTH reaction to all studied antigens in a dose-dependent manner. Lactoferrin (LF) given to mice in conjunction with antigen administered in an incomplete Freund's adjuvant induced the DTH response at the level of control mice given antigen in a complete Freund's adjuvant. In addition, LF remarkably increased DTH response to a very small, otherwise non-immunogenic SRBC dose. The increase in DTH response was less pronounced for orally administered LF than for any other routes of administration, however, statistically significant augmentation was demonstrated for each antigen studied. Although the costimulatory action of LF was accompanied by the appearance of bovine lactoferrin-specific cellular responses in mice, it is very unlikely that such responses will be generated in humans, since bovine lactoferrin is a dietary antigen to which a tolerance has been acquired. Considering the involvement of LF in generation of stimulatory signals during the induction phase of an antigen specific immune responses, we suggest that LF may be useful for development of safer and more efficacious vaccination protocols.     

Immunological Properties of Antigen 60 of BCG Induction of Humoral and Cellular Immune Reactions

Antigen 60 (A60), a member of the thermostable macromolecular antigen family (TMA) and main component of old tuberculin and purified protein derivative (PPD), has been purified from the cytoplasm of Mycobacterium bovis BCG; its structure and metabolism have already been described. In the present paper, the action of A60 on humoral immunity has been analysed by an ELISA type immunoassay, and that on cellular immunity by the mouse footpad swelling test. Injection of very low A60 doses into unprimed mice produced an undetectable level of anti-A60 antibodies; the effect of a booster inoculation was not appreciable in the absence of incomplete Freund's adjuvant, but was evident when the latter was added. Higher doses of the antigen produced an appreciable primary response, and a sharp and long-lasting secondary response, which had a 10-fold higher intensity in the presence of incomplete adjuvant. No detectable delayed hypersensitivity reactions were observed in unprimed mice after footpad injection of A60, whereas clear responses were elicited in primed mice. This effect was more pronounced when the footpad was injected after a secondary response than after a primary response, and it was invariably magnified by incomplete adjuvant. It is concluded that A60 is a powerful immunogen, which is able to induce primary and secondary responses and delayed hypersensitivity reactions, effects that are adjuvant-modulated and develop concurrently.     

Route-dependent immunomodulation: local stimulation by a surfactant and systemic stimulation by a polyanion

Immunomodulatory activity of the two synthetic adjuvants dimethyldioctadecylammonium bromide (DDA) and dextran sulfate (DXS) in relation to route and time of injection was investigated in mice. Humoral responses to sheep red blood cells (SRBC) were measured as the number of direct anti-SRBC plaque-forming cells (PFC) in the spleen 5 days after immunization. Both adjuvants stimulated the anti-SRBC response if adjuvant and antigen were injected simultaneously via the same route (either intraperitoneally or intravenously). Administration of adjuvant and antigen via different routes (intraperitoneally or intravenously, respectively or vice versa) resulted in enhanced humoral responses after DXS, but not after DDA. Intraperitoneal immunization of mice which were injected intraperitoneally with either adjuvant 4 days earlier resulted in diminished humoral responses. Immune responses in pretreated mice were not suppressed when the antigen was injected intravenously instead of intraperitoneally. In conclusion, DDA and DXS differ in immunostimulating properties as DDA enhanced only a response to antigen injected via the same route whereas DXS induced a systemic state of increased immunoresponsiveness. The immunosuppressive state induced by intraperitoneal injection of either adjuvant prior to immunization is restricted to the peritoneal compartment. Mechanisms underlying differences between both adjuvants and aspects of systemic immunopotentiation are discussed.     

Water-soluble adjuvant obtained from Bacterionema matruchotii.

The adjuvant effect of a butanol-extracted water-soluble adjuvant (bu-WSA) obtained from Bacterionemia matruchotii, a gram-positive oral bacteria, was studied on the antibody response at the plaque-forming cell (PFC) level in murine spleens. Intraperitoneal injection of Bu-WSA caused significant increase in direct PFC numbers in spleens 1 to 3 days after the antigenic stimulation with sheep erythrocytes (SRBC). Injection of 100 to 800 microgram of Bu-WSA was effective, and 400 microgram of Bu-WSA seemed to be the optimum for induction of the adjuvant effect. The adjuvant effect was strongest when Bu-WSA was injected at the same time as the SRBC, but some effect was still observed when Bu-WSA was injected 7 days before or 1 day after the immunization. The adjuvant effect of Bu-WSA was greatest at high dose of antigen. The mice injected with Bu-WSA at the time of priming SRBC and then immunized with trinitrophenylated SRBC showed greater anti-trinitrophenyl PFC response than controls without the injection of Bu-WSA. These findings suggest that a part of the adjuvant effect of Bu-WSA depends on thymic cell function and another part does not.     

Relationships among differentiated T-cell subpopulations. I. Dissociated development of tuberculin type hypersensitivity, Jones-Mote type hypersensitivity and activation of helper function.

Relationships among tuberculin type hypersensitivity, Jones-Mote type hypersensitivity and activation of helper T cells were studied in AKR mice by means of footpad reaction, migration inhibition test and antibody production against the trinitrophenyl group. (1) Immunization with SRBC in saline, Freund's incomplete adjuvant (FIA) or complete adjuvant (FCA) and fixed-SRBC (FRBC) in FIA- or FCA-induced delayed hypersensitivity as demonstrated by footpad swelling. (2) Migration inhibition was positive in the groups immunized with SRBC or FRBC in FCA, but negative in those immunized with SRBC in saline or FIA or FRBC in FIA. This may suggest that the former has to be assigned to tuberculin type and the latter to Jones-Mote type. (3) Both pre-treatment with BCG and with cyclophosphamide (CY) augmented delayed footpad reaction in the mice immunized with SRBC in saline. However, migration inhibition was positive only in the group pre-treated with BCG. BCG may convert the reaction from Jones-Mote type to tuberculin type, while CY may augment the reaction of Jones-Mote type. (4) FRBC in saline scarcely induced delayed footpad reaction, whereas they activated helper function efficiently. Thus, three types of immunological phenomena attributable to the functions of T cells may depend upon distinct subpopulations of differentiated T cells which are raised by different methods of immunization.     

Influences of anti-mouse interleukin-6 receptor antibody on immune responses in mice

In the present study, we examined the effect of anti-IL-6 receptor antibody (MR16-1) on humoral and cellular immune responses in mice. MR16-1 did not affect antigen-specific antibody production in either the primary or secondary response in mice immunized with dinitro-phenyl (DNP)-keyhole limpet haemocyanin (KLH) in saline. DNP-KLH immunization with complete Freund's adjuvant (CFA) markedly augmented anti-DNP antibody production and induced interleukin 6 (IL-6) production in serum. In this case, MR16-1 significantly suppressed antibody production and further increased serum IL-6 levels. Regarding the cellular response, we studied the effect on the delayed-type hypersensitivity (DTH) response. DTH response was induced in mice by the immunization with Mycobacterium butyricum with incomplete Freund's adjuvant and following antigen challenge into the footpad 14 days after immunization. When MR16-1 was injected immediately after immunization, the DTH response was significantly suppressed and enlargement of the spleen was also suppressed. This suppressive effect was observed, when MR16-1 was administered on day 0, but not on days 5 and 10. Again, serum IL-6 levels were much higher in MR16-1-treated mice compared with controls. Furthermore, spleen cells from control mice released IL-2 and INFgamma by the stimulation of antigen in vitro. In contrast, spleen cells from MR16-1-treated mice produced these cytokines at a marginal level. In contrast, MR16-1 did not suppress the DTH response, when it was injected immediately after antigen challenge. Our results suggest that IL-6 does not always involve antibody production, although IL-6 augments antibody production, and that IL-6 is essential for the induction of Th1 cells.     

The detection and production of macrophage cytophilic antibody to different antigens in different laboratory animals

An in vitro assay for macrophage cytophilic antibody is described, based on the measurement of the adherence of ¹²⁵I-labelled antigen to peritoneal exudate cells pretreated with antiserum. Using this assay, the production of cytophilic antibody to SRBC, BSA, polymerized flagellin, flagellin and a CNBr digest of flagellin was investigated in guinea-pigs, rats, rabbits and mice. Antigens injected in either Freund's complete or incomplete adjuvant were almost equally efficient at inducing cytophilic antibody production. In contrast, antigens injected in saline failed to induce detectable cytophilic antibody. The ratio of cytophilic antibody titre/passive haemagglutinating antibody titre was calculated and it was found that the ratio for rabbit>guinea-pig>mouse>rat. Furthermore, in those sera which were examined, cytophilic antibody was only detected in the 7S fractions.     

Local adoptive transfer to mice of human delayed hypersensitivity: reactions by the radioisotopic footpad assay.

Human delayed hypersensitivity to living BCG organisms and/or Varidase was adoptively transferred to mice and assayed in mice by a radioisotope footpad assay (FPA). A mixture of the antigen and peripheral blood lymphocytes from patients (positive skin reactions to PPD and/or Varidase) or healthy volunteers was inoculated into the footpads of lethally X-irradiated mice. A positive footpad reaction was accompanied by an increased leakage of the radiolabelled serum protein from the blood stream into the intercellular space at the site of inoculation, and was measured by the 'foot-count ratio', or radioactivity in the test foot divided by radioactivity in the contralateral foot. The intensity of the footpad reaction correlated directly with the skin test response of the human lymphocyte donors.     

Characterization of antibody responses to endogenous and exogenous antigen in the nonobese diabetic mouse

It is suggested that a T-helper cell 2 (Th2) shift and Th2 spreading of autoimmunity following immunization with beta-cell antigen causes diabetes protection. To address this, antibody titer and subclass to insulin, glutamic acid decarboxylase (GAD)65, IA-2, and IA-2beta proteins were measured by radiobinding assays in untreated or immunized female nonobese diabetic mice. Untreated nonobese diabetic mice developed autoantibodies to insulin (IAA), but not GAD or IA-2/IA-2beta, and IAA-positive mice had increased diabetes risk (P < 0.001). IAA were IgG1 and IgG2b. In immunized mice, IgG1 and lesser IgG2b insulin antibodies were promoted by subcutaneous injection of insulin plus incomplete Freund's adjuvant, insulin plus Montanide ISA 720, and glucagon plus incomplete Freund's adjuvant, but not by incomplete Freund's adjuvant plus GAD65, IA-2beta, or phenylethanolamine N-methyltransferase, or adjuvant alone. Diabetes incidence was significantly reduced in immunized groups with elevated insulin antibody (IA) responses. Spreading of antibody responses to GAD or IA-2/IA-2beta following immunization was rare, and antibody epitope spreading was only detected in IA-2beta immunized mice. Humoral autoimmunity in nonobese diabetic mice is, therefore, limited to IAA with Th2 subclass phenotype and is associated with increased diabetes risk. This contrasts the diabetes protection provided by immunization protocols that promote this response and suggests that Th2 immunity may not be the principal regulator of beta-cell destruction in autoimmune diabetes.     

Antibody affinity maturation in selectively bred high and low-affinity mice

The affinity of serum antibodies produced by selectively bred lines of mice [high affinity, low affinity, low nonmaturing (N/M)] injected with T-dependent [human serum albumin (HSA), dinitrophenylated bovine gamma-globulin (DNP-BGG)] and T-independent (DNP-Ficoll) antigens in saline and adjuvant has been determined. The lines of mice differ significantly in the affinity of antibody produced to T-dependent antigens injected in saline but not to the T-independent antigen. Unlike mice of the high and low-affinity lines, low-affinity N/M mice failed to show affinity maturation to HSA and DNP-BGG injected in Freund's incomplete adjuvant. However, low-N/M mice responded to DNP-Ficoll injected in adjuvant by the production of antibody of affinity comparable to that produced in the other lines and with a similar maturation in affinity. Carrier priming resulted in the suppression of anti-hapten antibody affinity in all lines but low-N/M mice showed significantly greater suppression late in the response to challenge. Low doses of cyclophosphamide produced a significant increase in affinity in low-N/M mice. These results suggest that the failure of low-N/M mice to show affinity maturation results from increased suppressor T cell activity. The availability of the selectively bred mice provides a useful model for the detailed study of the cellular basis of the control of antibody affinity maturation.     

A limited role of IL-1 in immune-enhancement by adjuvants.

Generation of interleukin-1 (IL-1) in the draining lymph nodes after injection of complete Freund's adjuvant (CFA), incomplete Freund's adjuvant (IFA) and alum was studied in line with IL-1 mRNA expression (cytoplasmic slot blot analysis) and IL-1 beta antigen detection (ELISA and immunohistochemistry) in rabbits. The expression of IL-1 beta mRNA was marked from 6 to 96 hr, with a maximum at around 24 hr post-injection of CFA, while injection of the other two adjuvants elicited only a moderate or negligible response. On the other hand, IL-1 alpha mRNA expression was almost negligible during the entire 8-week observation period after injection of the above three adjuvants. Generation of IL-1 beta antigen in the draining lymph nodes after CFA injection paralleled the expression of IL-1 beta mRNA. Immunohistochemistry revealed that cells containing IL-1 beta resided in the medullary sinuses, marginal sinuses and para-cortical area, but not in the follicles. Despite marked generation of IL-1 beta in CFA-treated draining lymph nodes, the primary antibody response (IgG) to ovalbumin differed only slightly between the three animal groups that were immunized with the antigen incorporated in CFA, IFA and alum. Further, rIL-1 beta did not significantly enhance the immune response when it was entrapped together with the antigen in IFA and alum. IL-1 beta enhanced the immune response only when it was injected with antigen without adjuvant. Thus, IL-1 seemed to play only a limited role, if any, in the augmentation of the primary immune response by the above-mentioned adjuvants.     

FCA和FICA可增强淋巴滤泡捕捉抗原的能力

目的　探讨向体内注入非抗原类物质能否引起反应性淋巴滤泡形成 ,观察体内注入FCA(完全弗式佐剂 )、FICA(不完全弗式佐剂 )对滤泡树突状细胞 (FDC)捕捉抗原的能力有无影响。方法　将FCA、FI CA分别注入小鼠足底 ,数日后取出腘淋巴结 ,应用PNA B法及PAP免疫组化法染色 ,采取三维重塑方法 ,统计淋巴滤泡、FDC细胞群数量。结果　注入FCA、FICA后第 5日出现初级淋巴滤泡 ,第 3 5日数量达到高峰 ,之后逐渐减退。同时 ,滤泡树突状细胞 (FDC)与B细胞聚集同步出现。结论　动物体内注入有丝分裂物质FCA、FICA引起引流淋巴结内次级淋巴滤泡形成 ,初级滤泡形成过程中B细胞聚集与FDC形成是同步发生的     

Comparability of delayed hypersensitivity in various rodents. II. Jones-Mote type hypersensitivity in guinea-pigs immunized with sheep erythrocytes and its modification by cyclophosphamide or BCG pre-treatment.

Guinea-pigs were immunized via footpads with sheep red blood cells (SRBC) in saline. Histological examination of erythematous skin reaction was performed and effects of cyclophosphamide (CY) or BCG pre-treatment on the skin reaction were examined. Delayed-in-onset erythematous skin reaction accompanied by substantial basophil infiltration was elicited in guinea-pigs immunized with SRBC in saline. The erythema was augmented in size by CY which was injected 2 days before immunization. The reaction may be comparable to Jones-Mote type. In BCG pre-treated guinea-pigs, basophil infiltration at the skin reaction sites was reduced in number, but significant inhibition of macrophage migration was not detected in the presence of SRBC antigen. The reaction may be intermediate between Jones-Mote and the tuberculin type. Comparability of delayed skin reactions in guinea-pigs and delayed footpad reactions in mice or hamsters against SRBC is discussed.     

Separate application of adjuvant and antigen: the effect of a water-in-oil emulsion on the splenic plaque-forming cell response to sheep red blood cells in mice

The effect of a non-immunogenic adjuvant on the murine splenic plaque-forming cell (PFC) response against sheep red blood cells (SRBC) was studied. The adjuvant, a stable water-in-oil (W/O) emulsion, was injected intraperitoneally at the same time as or prior to the intravenous (i.v.) injection of SRBC. Enhancement of the SRBC-specific IgM-, but not IgG- and IgA-responses was observed. The stimulatory effect depended on the dose of both adjuvant and antigen and on the interval between their application. The minimal dose of adjuvant needed to induce maximal stimulation increased with the interval between the injections. Administration of an optimal adjuvant dose one week before antigen application still resulted in a clear stimulation of the response to the antigen. In adjuvant-treated animals, the primary PFC response did not exceed the maximum level reached after i.v. injection of a high dose of SRBC. Adjuvant therapy also resulted in polyclonal B cell-activation, since the number of spontaneous Ig-secreting cells in the spleen was increased. The kinetics and isotype distribution of the SRBC-specific and polyclonal responses, however, were different. Therefore, the observed stimulatory effect on the SRBC-specific PFC-response cannot be explained by the polyclonal activation of the immune system. From this study it appears that injection of a W/O emulsion provokes an active stimulation of the immune system, which demonstrates that the adjuvant effect of W/O emulsions is not only passively obtained by prolonged antigen presentation by depot formation.     

Comparative studies on antibody and antibody production to poly(ADP-ribose) in mice

Antibodies to poly(ADP-ribose) were produced in C3H/He mice by injection of poly(ADP-ribose) in Freund's incomplete adjuvant or its complexes with methylated bovine serum albumin in Freund's incomplete adjuvant. Titres of the antibody obtained from the latter were about 50-fold higher than those from the former. Thus, the effect of methylated bovine serum albumin on the antibody production to poly(ADP-ribose) resembled the case of poly(I).poly(C) and was different from the case of single stranded DNA. The class of the antibodies obtained from these two different procedures mainly consists of 7S antibodies, and the specificity of these antibodies was independent of their titres. It was also found that antibodies to poly(ADP-ribose) were most reactive to poly(ADP-ribose) with 20 repeating ADP-ribose units. The reactivity of the antibody was dependent on the chain length of the polymer. Neither the complex in Freund's incomplete adjuvant nor poly(ADP-ribose) alone in Freund's imcomplete adjuvant could induce an immune response to poly(ADP-ribose) in athymic nude mice. Therefore, the immune response to poly(ADP-ribose) may occur through a thymus function.     

Monoclonal antibody production by hybridoma growth in Freund's adjuvant primed mice

Incomplete Freund's adjuvant was used as a priming agent prior to the injection of hybridoma cells in mice to expand monoclonal antibody production. Two hybridoma cell lines, FDO28B (IgG1) and FDO31C (IgM), which produce monoclonal antibodies reactive towards human placenta, were used. Monoclonal antibody was detected in the ascites fluids by agarose gel electrophoresis. It was found that the time interval between adjuvant priming and cell injection could be reduced to 1 day, allowing collection of ascites fluid containing monoclonal antibody within 2 weeks of priming. In addition, as few as 1 X 10(5) hybridoma cells were needed to collect approximately 5-7 ml of ascites fluid containing antibody detectable by gel electrophoresis. Thus priming with incomplete Freund's adjuvant enables production of large amounts of monoclonal antibody in a short time using a low number of hybridoma cells.     

Dependence of the antibody response of guinea-pigs to collagen on the type of adjuvant and on the conformation of the antigen.

The immunological response to collagen of guinea-pigs is strongly dependent on the conformation of the antigen and on the type of adjuvant. Freund's complete adjuvant facilitated excellent delayed hypersensitivity skin reactions to native (triple helical conformation) as well as denatured (random coil conformation) collagen. Immunization of guinea-pigs with collagen in this adjuvant gave rise to very low levels of antibody to native collagen and failed to induce antibodies to denatured collagen. Use of Freund's incomplete adjuvant resulted in excellent antibody responses to native collagen, but it did not induce antibodies to denatured collagen. Animals injected with collagen and Freund's incomplete adjuvant were not sensitized for cell-mediated immunological reactions. The antibodies to collagen were specific with regard to collagen from various species but displayed different degrees of cross-reactivities depending on the species of collagen used for immunization. They were specific for the triple helical conformation of the collagen molecule.     

Interactions between the immunological responses of a thymus-independent antigen (Salmonella adelaide O antigen) with a thymus-dependent antigen (sheep erythrocytes) in the adult bird.

The bird's antibody response to a thymus-dependent antigen (sheep erythrocytes) (SRBC) and a thymus-independent antigen (SALMONELLA ADELAIDE O antigen) were characterized: whereas the former proceeded through a brief 19S response to a declining 7S response, the latter failed to switch from 19S TO 7S for several weeks and consisted in repeated excursions of 19S antibodies. When injected intravenously and simultaneously an injection of S. adelaide-killed organisms and SRBC interact, so that the response to the latter fails to switch from 19S TO 7S and consists of repeated excursions of 19S antibodies. The changed character of the SRBC response is interpreted to be due to the relative lack of 7S antibody: passive 7S antibody to S. adelaide O antigen or 7S anti-SRBC produces a negative feedback inhibition of their respective responses, so that only one excursion of 19S antibody is observed. The effect is not, however, symmetrical; the thymus-independent antigen is dominant. Thus, whereas 7S antibody to S. adelaide produces the same negative feedback inhibition on the response to S. adelaide and the response to SRBC (when injected with adlaide), 7S antibody to SRBC inhibits only the response to SRBC and not the response to S. adelaide. These results are discussed relation to current hypotheses of antibody biosynthesis and mechanisms of adjuvant action. They are also discussed in relation to the function of the germinal centres of the spleen which may function to mediate the negative feedback of 7S antibody on the antibody response.