Antigen in tissues: IV. The effect of antibody on the retention and localization of antigen in rat lymph nodes*

A study has been made of the role of specific antibody in causing increased retention and specific localization of a weak antigen, human serum albumin (HSA), in the popliteal and aortic lymph nodes of rats. The antigen was labelled with ¹²⁵I prior to mixing with antibody. HSA mixed with excess homologous antibody was trapped to the greatest extent in these nodes after footpad injection of the antigen. Injection of HSA with antibody caused increased uptake of HSA into the medulla but retention was poor as autoradiographs showed the area to be essentially free of antigen 4–5 days after injection. By contrast, antigen injected with antibody localized strongly in lymphoid follicles and persisted at this site. Both IgM and IgG antibody were found to cause follicular localization of HSA in rats. Heterologous, isologous and homologous antibody also caused follicular localization of the antigen. Purified homologous γ-globulin was found to localize in the follicles. A moderate increase in the net negative charge of the γ-globulin by acetylation did not appreciably affect the ability of the globulin to localize in the follicles. Detectable formation of antibody did not occur in the rats after injection of antigen—antibody complexes, owing possibly to the inhibitory effect of free antibody on the primary response.     

A study of the effects of salt and of pH on precipitation of antigen—antibody compounds

Tobacco mosaic virus (TMV) combines with its homologous antibody to much the same extent irrespective of whether or not salt is present, but without salt the complex not only fails to precipitate, but the virus particles do not aggregate. TMV—antibody precipitate formed in the presence of salt, like that formed between human serum albumin (HSA) and its homologous antibody, dissolves when suspended in distilled water to form stable and transparent solutions, although the precipitate may not disaggregate completely.

To dissolve HSA—antibody complex in distilled water, the pH of the water must be raised to about 7.0. At pH near 6.0, HSA—antibody complex precipitates even in the absence of salt, but the precipitate dissolves immediately when the pH is raised to 7.0.

All these facts are incompatible with the theory of precipitation based on the `lattice hypothesis', and argue strongly in favour of the theory that antigen—antibody complexes are hydrophobic and, as such, flocculate when sufficiently discharged either by salt or by suitably adjusting the pH of the medium.

     

Antigen in tissues: II. State of antigen in lymph node of rats given isotopically-labelled flagellin, haemocyanin or serum albumin

1. Three antigens, flagellin from Salmonella adelaide, haemocyanin from Maia squinado blood, and human serum albumin (HSA), each labelled with radioactive iodide, have been injected singly into rats to give a primary or a secondary response. After hind footpad injection, the popliteal and aortic lymph nodes were removed and fractionated in sucrose medium into a nuclear debris fraction, a large granule fraction and a cell supernatant fraction. The large granule fraction was divided into an extract and residue after nine cycles of freezing and thawing.

2. At intervals after injection of antigen, determinations were made of the radioactivity in the node which was: (a) bound to sub-cellular components: (b) macromolecular but not bound to sub-cellular components, or (c) associated with low molecular weight components. Except as mentioned below, the patterns of behaviour of antigen injected into normal or primed rats were essentially similar. Notable features were: flagellin to a large extent became bound to sub-cellular components; after HSA injection, most radioactivity was in a macromolecular but unbound form; whereas nodes from haemocyanin injected rats had the highest proportion of label which was attached to low molecular weight components. Injection of the same dose of HSA with rat anti-HSA serum had a profound effect; more than 100 times more label was recovered in the large granule fraction and the overall distribution of isotope resembled that exemplified by flagellin.

3. With each antigen the time at which the highest proportion of total radioactivity in the node was found associated with low molecular weight component was 2–3 days after injection. In each case, the large granule extract showed the highest proportion of radioactivity in this state.

4. The extent to which the isotope remained associated with specific antigen in the tissues was studied by testing the ability of any fraction to react with specific immune serum to the antigen. With flagellin a large proportion (up to 80 per cent) of radioactive substances associated with the large granule residue reacted with antiserum. In the case of haemocyanin, a very small proportion only of the radioactivity in this fraction reacted with antiserum but this proportion increased as the serum antibody titre rose. The greatest contrast in the behaviour of the fraction occurred with HSA. After injection of HSA alone there was very little specific reaction (about 12 per cent) shown by this fraction. After injection of HSA with antiserum, the proportion reacting was five-fold higher. This increased ability of the radioactive fraction in the case of HSA to react specifically with antiserum could be correlated with the increased localization of the antigen in the lymphoid follicles of the node, as revealed by radioautography.

     

The influence of adjuvants on the immunological response of the chicken: I. Effects on primary and secondary responses of various adjuvants in the primary stimulus

The effect of various adjuvant procedures on the antibody response (first 21 days) for human serum albumin (HSA) has been studied in the chicken. The lack of effect on the circulating antibody level contrasts with their action in some mammals.

The administration of depôt-type adjuvants failed to increase the peak circulating antibody levels (8–12 days after injection of antigen) by comparison with control birds. However, the circulating antibody level declined more slowly in birds given HSA in a water-in-oil emulsion than in birds given HSA in saline.

The administration of endotoxin and `surface active' adjuvants also failed to increase the peak circulating antibody levels over that of control birds. In three experiments there was significant depression of peak antibody levels in birds given endotoxin adjuvant in comparison to control birds.

The administration of HSA in Freund's complete adjuvants containing Mycobacterium tuberculosis or Mycobacterium avium did not result in elevation of peak antibody levels compared to those of control birds given HSA in saline or HSA in a water-in-oil emulsion.

Experiments to determine the effect of adjuvants from each of the main groups on the establishment of immunological memory were performed. Chickens were given adjuvant with the primary injection of HSA. A second injection of HSA without adjuvant was given 56 days later. None of the adjuvants used produced an increase in the peak antibody level attained during the secondary response compared to control birds.

     

The localization of non-microbial antigens in the draining lymph nodes of tolerant, normal and primed rabbits

Soluble haemocyanin (HCy) or human serum albumin (HSA) labelled with ¹²⁵I at 8 and 0·7 μc/μg, respectively, were injected into the footpads of rabbits in doses just sufficient to elicit a primary response in normal animals, and the distribution of radioactivity in the popliteal lymph nodes between 6 hours and 21 days later was studied by autoradiography. The recipient rabbits had either been primed by a single prior injection of unlabelled antigen, or made putatively tolerant by repeated neonatal administration of antigen, or were previously untreated. Localization of antigen in germinal centres, in a typical dendritic pattern, was marked in the primed animals throughout the period of observation; in those tolerant animals which had no detectable serum antibody initially and made no detectable antibody response such localization was not seen at any time; in the animals that had no previous contact with antigen there was no localization in germinal centres until about the time when antibody became detectable in the serum. Localization of radioactivity in medullary sinus macrophages did not differ significantly between the three groups.

It is concluded that localization of these soluble antigens on the dendritic web in lymphoid follicles occurs as a consequence of the presence of circulating antibody. Uptake of the antigens by medullary macrophages, however, can occur in the absence of antibody. Although the degree of labelling of medullary macrophages was not evidently affected by the presence of antibody in these experiments, it is emphasized that the antibody levels, even in the primed animals, were low, and that this finding is unlikely to apply when the amount of antibody present is relatively much greater than the amount of antigen injected.

     

Antibody production in mice: II. The mechanism of antigenic stimulation in the secondary immune response

To study the mechanism of antigenic stimulation in the secondary immune response, primed lymphoid cells were stimulated with antigen in various ways in vitro. The effect of antigenic stimulation was assessed by the antibody titres obtained after in vivo culture of primed cells in X-irradiated recipients.

Primed cells were much more effectively stimulated by antigen—antibody (Ag—Ab) complex than by free antigen. Primed cells could also be stimulated by spleen or lymph node cells from normal mice which had been exposed to free antigen or Ag—Ab complex in vitro or in vivo and thoroughly washed. Under these conditions, Ag—Ab complex was again much more effective than free antigen. When the cells were incubated with Ag—Ab complex, the dose of antigen bound to the cells was somewhat increased. But this increased binding of antigen could not solely account for the increase in immunogenicity.

It is suggested that the ingestion of antigen by macrophages is facilitated by the presence of antibody and that the macrophages mediate the effective immune stimulus to memory cells.

The effect of antibody in increasing the immunogenicity of antigen was lost completely when antibody was digested with pepsin. Thus, the Fc portion of antibody seemed to be important for this effect. However, it was demonstrated that antibody does not operate by becoming attached to macrophages as cytophilic antibody, and that complement is not involved in this process. The augmenting mechanism of antibody on the antigenic stimulation mediated by macrophages was discussed.

     

The influence of amount and avidity of persisting primary antibodies on the secondary response to human serum albumin in rabbits

The primary antibody response in rabbits to human serum albumin (HSA) has been analysed. Doses of 0.04–25 mg were given subcutaneously with Freund's complete adjuvant. Maximum antibody response about 60 days after primary stimulation was independent of the antigen dose given.

After 175 days the amount of persisting antibody was lowest in the animals stimulated with the lowest dose of antigen. The avidity of the antibody was inversely proportional to the antibody titre. Secondary stimulation of all rabbits with 1 mg of HSA intravenously caused secondary antibody responses which were insignificantly different from each other.

It is suggested that the magnitude of the secondary antibody response is determined by the regulatory activity of the circulating antibodies following primary stimulation.

     

Antigens in immunity: VIII. Localization of 125I-labelled antigens in the secondary response

Two flagellar antigens, intact flagella and monomeric flagellin from Salmonella adelaide were labelled with ¹²⁵I by the Chloramine-T method. They were injected into the hind foot-pads of rats, and the localization in the draining lymph nodes was studied by scintillation counting and autoradiography. Injected rats belonged to one of four groups: normal, unimmunized adult rats (NI); rats that had been given an unrelated antigen 6 weeks previously (HI); rats that had been given a priming dose of the same antigen (though unlabelled) 6 weeks previously (AI); and rats that had been given passive antibody by intraperitoneal injection (PI).

Follicular localization was more rapid in AI and PI groups than in NI or HI rats. With flagellin, but not with flagella, the final follicular concentration reached was also greatly increased. No differences were observed between NI and HI rats, or between AI and PI rats.

In primary lymphoid follicles, the antigen was distributed throughout the follicle in a diffuse network, presumably of macrophage fibrils. In secondary follicles, the antigen localized in a crescentic cap occupying the superficial aspect of the follicle.

The study stressed the importance of antibody acting as an opsonin in determining details of antigen localization.

     

Antigen in tissues: III. The separation of antigen-containing components from lymphoid tissues

Particulate and soluble antigens were labelled with ¹²⁵I, injected into rats and the lymphoid organs examined. As determined by autoradiography of tissue sections, one antigen used localized exclusively in vacuoles of medullary macrophages of the lymph nodes and others on the surfaces of reticular cells in the lymphoid follicles of nodes or the white pulp of spleen. The remaining antigens studied localized in both medulla and lymphoid follicles of nodes.

Tissues containing antigen were homogenized in a sucrose medium and most radioactivity was recovered in a large granule fraction. This fraction was submitted to equilibrium centrifugation. The preparations were not resolved in gradients of sucrose or dextran but in gradients of Urografin the preparations were resolved into two or more peaks of radioactivity. Medullary localized antigen banded in a region of the gradient rich in lysosomal enzymes and was considered to be present in vesicles. Antigen was not found in a region of the gradient rich in mitochondria. Antigen from lymphoid follicles of nodes or from spleen white pulp banded at high density values and was considered to be present as an antigen—antibody complex, possibly associated with membrane.

Equilibrium density centrifugation in Urografin gradients provides a means of separating and examining the properties of antigen in lymphoid tissues.

     

The tolerance inducing properties in rats of bacterial flagellin cleaved at the methionine residues

Flagellin (molecular weight 40,000) from Salmonella adelaide was reacted with CNBr to give mainly four peptide fragments, A, B, C and D, the largest of which (fragment A, molecular weight 18,000) retained all the serological activity of flagellin itself. Mixtures of the fragments or individual fragments were injected into neonatal or adult rats, either as a single dose in saline or in adjuvant, or in multiple doses in saline. Antibody titres, measured by bacterial immobilization, were compared with those given after an injection of flagellin. Fragment A and the complete CNBr digest of flagellin (the `digest') were both immunologically active whereas fragments B, C and D were inactive.

When given as a single injection in saline into adult rats, the `digest' or fragment A was much less efficient than flagellin at inducing either a primary antibody response or the production of primed cells for a secondary antibody response. In contrast, the `digest' or fragment A was effective at triggering primed cells to give a secondary antibody response. These findings were consistent with an interpretation given in detail elsewhere relating to `in vivo' localization of labelled flagellin and fragment A in rat lymph nodes to the immune response. Fragment A and flagellin were equally immunogenic when injected in Freund's complete adjuvant.

Rats given a course of injection of the `digest' starting on the day of birth became almost completely tolerant to either flagellin or polymerized flagellin. Daily injections of the `digest' or fragment A for 4 weeks or longer into adult rats resulted in a significant degree of tolerance to flagellin and to polymerized flagellin. Adult rats made tolerant in this way responded normally to BSA injected in Freund's complete adjuvant and to a slightly decreased extent to sheep RBC.

It was concluded that by a process of partial degradation, a highly immunogenic substance, polymerized flagellin, had been converted into a preparation with strong tolerance-inducing properties. The relevance of this approach to transplantation antigens was discussed.

     

The relationship between circulating soluble antigen—antibody complexes and the production of chronic glomerulonephritis in the rabbit

Twenty-three rabbits were injected over long periods of time with isotopically labelled bovine serum albumin. Varying injection schedules and primary and secondary responses were studied, but all schedules were designed to preclude the presence of free circulating antibody at any time. All animals became tolerant of injected antigen after a period of 60–80 days. During the intervening period (from 10 to 60 days) large amounts of antigen—antibody complex were shown to be circulating.

Proteinuria was intermittent in all animals. Histological examination of the kidneys from 50 days on showed only very minor evidence of renal damage; no animal developed chronic renal disease.

     

The immune response to sheep erythrocytes in the mouse: II. A study of the cytological events in the draining lymph node utilizing cellular imprints

The cytological events of the primary and secondary immune response in the popliteal lymph node of Swiss White mice were studied following administration of sheep erythrocytes into the hind footpad. Four morphological features of cellular activity of immunologically competent cells—basophilia, synthesis of RNA, mitotic activity and distinctive cellular morphology—were analysed, and correlated with previous studies of 19S and 7S antibody forming cellular activity employing plaque assays performed on the residual lymphoid tissue remaining after production of node imprints.

The findings support the view that 19S and 7S antibody forming cells in the primary immune response are derived from two populations of cellular precursors. It is suggested that the lymphoid cell producing 19S immunoglobulin arises by transformation from the reticular cell following activation by antigen, while the 7S antibody forming cell arises from the small lymphocyte following some degree of initial transformation and subsequent cellular proliferation. The possibility that the 7S antibody forming cells had passed through a transient period of biosynthesis of 19S antibody was suggested in the present studies. Finally, evidence was provided for the presence of two morphological types of plasma cells, which, by virtue of their appearance at different stages of the primary immune response, could represent cells producing different immunoglobulins at varying rates of protein biosynthesis.

     

The immune response of mice to antigen in macrophages

Peritoneal macrophages obtained following an injection of proteose peptone, and after uptake of Maia squinado haemocyanin were transferred to syngeneic hosts. Immunogenicity was tested by the capacity of macrophages containing the antigen to prime normal or irradiated (660–700 r) recipients for a secondary immune challenge. The immunogenicity of macrophages containing antigen depended on interaction with immunocompetent lymphoid cells since irradiated hosts were unresponsive unless normal lymphoid cells were also supplied. For optimal immune response the live macrophages had to gain access to lymphoid organs. Depending on the amount of antigen transferred with the macrophages, the recipient mice synthesized on secondary challenge 7S and/or 19S antibody. The kinetics of response to the antigen in macrophages were similar to those seen when using free soluble material except for some quantitative differences. Although the immune response was dependent on the total dose of antigen transferred with the macrophages, somewhat higher antibody titres were obtained with macrophages having a high antigen—cell ratio. Antigen in macrophages could elicit a secondary response in primed mice. The immunogenicity of macrophage-held haemocyanin was not impaired by X-irradiation of macrophage donors.     

Antibody response to Escherichia coli O antigens and influence of the protein moiety of the endotoxin

Exposure of mice to different serotypes of E. coli bacteria, either O4 or O6, resulted in an enhanced indirect IgG-PFC response to the alternate bacteria. This effect seemed to be mediated by a protein connected with the endotoxin structure. This protein moiety had some weak adjuvant activities and increased the antibody response against sheep red blood cell about two-fold. This effect was not likely to be due to any contamination with the B-cell mitogen lipid A, a constituent of the endotoxin from which the protein was isolated.

In addition, experiments were performed in which irradiated spleen cells (0–400 R) from mice injected with E. coli O4 bacteria were transferred to irradiated (800 R) recipients together with E. coli O6 bacteria. Decreasing numbers of antibody forming cells with increasing irradiation dose were found. The parallel experiment employing E. coli O6 bacteria for both primary and secondary antigen injections revealed an increased immune response for an irradiation dose of 50 R, showing that suppressor cells are more irradiation sensitive than the other cells involved in this immune response, but that the effect of such cells is possibly overcome by the influence of the protein residue isolated from endotoxin.

A secondary response to E. coli O6 bacteria was also noted in agreement with previous results. It was found that this immune response could be reduced drastically by injecting primed thymocytes, simultaneously with the second injection.

     

Phylogenetic studies on the immune response: I. Localization of antigens and immune response in the toad, Bufo marinus

Toads of the species Bufo marinus were injected subcutaneously with ¹²⁵I-labelled flagella from Salmonella adelaide. Observations were made on the ensuing serum antibody response, the antigen localization pattern and sequential cellular changes in lymphatic and other tissues.

The serum antibody findings confirmed the work of previous investigators in showing a good primary response, prolonged synthesis of mercaptoethanol sensitive antibody and little or no evidence of secondary responsiveness.

Antigen became localized in the jugular bodies and spleen where proliferation of pyroninophilic cells could be observed after 5 days. Both the antigen-trapping cells and the first pyroninophilic blasts were scattered randomly throughout the jugular bodies. There was no clear-cut separation into cortex and medulla. Nothing resembling the antigen-trapping web of rat lymph node follicles was observed, nor were there any germinal centres. In the spleen, antigen was trapped in the red pulp and some degree of concentration around the islands of white pulp could be noted 1 day later. However, unlike in the rat, entry of antigen into the white pulp did not occur.

Both focal and diffuse collections of lymphoid and pyroninophilic cells were found in the kidney after antigenic stimulation. It seems likely that the kidney is a major antibody-forming organ in the toad.

The hypothesis is advanced that the absence of immunological memory may be due to the absence of the follicular antigen-trapping web and of resultant germinal centres.

     

Antigen—antibody complexes in the immune response: I. A analysis of the effectiveness of complexes on the primary antibody response

Soluble crystalline bacterial α-amylase (BαA)-mouse anti-BαA antibody complexes (Ag—Ab complexes) elicited a primary antibody response in mice with a single intravenous injection, while free BαA could not. The response was dose dependent. Ag—Ab complexes were not only phagocytosed but also degraded more rapidly than free BαA in vivo and in vitro but these characteristics themselves were not important for immunogencity of the complexes.

The Ag—Ab complexes phagocytosed by cells in normal spleen and lymph node elicited a primary antibody response when injected into non-irradiated mice but the response was suppressed when anti-BαA antibody was simultaneously injected. On the other hand, free BαA phagocytosed by cells could not elicit the response.

The degraded products of complexes phagocytosed by normal spleen and lymph node cells were highly immunogenic and probably retain antigenic fragments. They elicited an even higher primary antibody response than the original complexes and were also more effective in eliciting a secondary response from primed cells than the original complexes or free BαA. The degraded products of free BαA, however, were ineffective not only for the primary response but also for primed cells.

Ag—Ab complexes prepared with heterologous rabbit antibody were ineffective for the primary antibody response.

     

Studies on antigenic competition: Effect of antigen dose on the immune response of mice injected simultaneously with human serum albumin and ferritin

Human serum albumin (HSA) and ferritin in Freund's incomplete adjuvant (FIA) were injected simultaneously, intraperitoneally into mice in various dose/ratios. The degree of suppression of the primary and secondary immune responses to a small dose of HSA was dependent on the dose of ferritin injected simultaneously. A totally suppressed primary response was associated with a secondary response which only attained the level of a primary response to HSA in FIA.

Similar results were obtained in experiments in which a constant dose of ferritin and varying amounts of HSA were injected simultaneously in FIA into mice.

Suppression of the immune response to alum-precipitated HSA in the presence of soluble ferritin was not as striking as when the antigens were injected with FIA. Nevertheless, suppression of the primary response was again associated with a reduced secondary response.

The results are discussed in the context of the possible mechanism(s) of antigenic competition.

     

Mechanism of induction of immunological tolerance: VI. Tolerance induction following thoracic duct drainage or treatment with anti-lymphocyte serum*

Adult Wistar rats were injected with flagellin from Salmonella adelaide three times weekly for different periods of time. With the range of doses 100 fg to 100 μg (fg, femtograms), varying levels of immune responsiveness were demonstrable following challenge, but not one of the doses induced complete immunological tolerance, although a reduced antibody response was elicited in rats which received injections of 100 μg of flagellin.

In contrast to this finding, complete tolerance could be demonstrated following challenge if rats, previously drained of lymph and lymphocytes from the thoracic duct for 5 days, were injected with either 100 μg of flagellin three times weekly or with 1 μg/g body weight/day for 6 weeks. Similarly, anti-lymphocyte serum treatment prior to the injection of antigen resulted in partial tolerance in adult rats and nearly complete tolerance in adult C57BL/Brad mice. The primary response to flagellin of C57BL mice was abrogated if ALS was administered prior to but not after the injection of antigen. The implications of these findings are discussed.

     

Differentiation of lymphocytes in the mouse bone marrow: III. The adoptive response of bone marrow cells to a thymus cell-independent antigen*

The response of mouse spleen cells to the T cell-independent antigen dinitrophenylated polymer of flagellin (DNP—POL), has been studied using an adoptive transfer system, and compared with the response of bone marrow cells. Spleen cells showed a complex cell dose—response relationship, with a markedly discontinuous curve, for assays performed before day 9 after transfer and antigen challenge. This discontinuity could be explained by a delay in attainment of the peak response for lower cell inocula. The curve became linear on a log—log scale when spleens were harvested on days 9 and 10 post-transfer.

Bone marrow cells gave a lower response than would be expected from their lymphocyte content. This response increased progressively with a delay before antigen challenge in the irradiated recipient or in tissue culture prior to cell transfer, suggesting a functional maturation in this cell population, whereas the performance of spleen cells fell off under similar circumstances. The findings were consistent with, but could not prove, the hypothesis that the immediate precursors of anti-DNP antibody-forming cells in bone marrow were high surface immunoglobulin density small lymphocytes that had arisen locally from precursors lacking detectable surface immunoglobulin, by a non-mitotic maturation.

     

On the existence of a live immunologically unresponsive lymphoid cell

Immunological unresponsiveness to human IgG in rabbits was terminated with a single intravenous injection of 3 mg of purified rheumatoid factor. Anti-Fc fragment antibodies were produced within 3–6 days after this injection. These were all IgG antibodies as in a secondary immune response.

Irradiated rabbits injected with lymphoid cells from a donor unresponsive to human IgG produced anti-IgG antibodies upon stimulation with rheumatoid factor.

On the basis of these findings, it is postulated that `blocked' cells (live lymphoid cells with antigen on their surface) are the common substrate of unresponsiveness and of the proliferative phase of the immune response. If all cells are blocked, unresponsiveness ensures; whereas if only some are blocked, these are stimulated to multiply by antibody produced in others.