7S Antibody Research Articles

THE MECHANISM OF TOLERANCE PRODUCED IN RATS TO SHEEP ERYTHROCYTES. I. PLAQUE-FORMING CELL AND ANTIBODY RESPONSE TO SINGLE AND MULTIPLE INJECTIONS OF ANTIGEN.

Previous studies suggested that an active immune response was partially responsible for maintaining immunological unresponsiveness to sheep erythrocytes. Measurement of the plaque-forming (antibody-releasing) cell response proved to be a sensitive indicator of an immune response to sheep erythrocytes in the absence of detectable circulating antibody to the antigen. The present studies were undertaken to determine whether an active immune process, measured by the plaque-forming cell response, was partially responsible for induction and maintenance of tolerance. Rats injected intraperitoneally with large doses of sheep erythrocytes beginning at the day of birth develop tolerance to the antigen. In this paper, the plaque-forming cell and antibody response to sheep erythrocytes was characterized for rats receiving a single antigen injection at various ages and for rats which received repeated antigen injections as adults. The dose of antigen was the same as that used to produce tolerance; the injection schedule for repeated immunizations was also the same as that used to produce tolerance. Rats receiving a single antigen injection on the day of birth or at age 7 days had no measurable response to the antigen. Rats receiving a single antigen injection at age 17 days and sacrificed 4 days later had an unequivocal response to the antigen. The spleens had about one-tenth as many plaque-forming cells as spleens of adult animals immunized similarly, but the antibody titers were as high as titers for adult animals. Presumably the high titers of these young animals resulted from the high ratio of plaque-forming cells to body weight and blood volume. Adult animals receiving a single antigen injection had a peak or near peak plaque-forming cell response 4 days after immunization; at this time, sera contained high titers of 19S antibody and the numbers of plaque-forming cells in spleens correlated reasonably well with circulating antibody titers. 7S antibody appeared in serum 5 or 6 days after immunization. The numbers of plaque-forming cells declined progressively 2 and 3 weeks after immunization. Repeated twice weekly, injections of the antigen in adult rats produced a marked decline and then stabilization of numbers of plaque-forming cells in spleens. Although the numbers of plaque-forming cells were fewer, titers of 19S and 7S antibody stabilized at high levels. A progressive recovery of the plaque-forming cell response and a rise in antibody titer occurred when the interval between the last 2 injections was increased from 3 to 10, 17, or 32 days. These findings suggested that repeated closely spaced antigen injections interfered with either cell division or maturation of antibody-forming cells. As the interval between injections was increased, additional antibody-forming cells matured or were formed through cell division. Thus, relatively constant antigenic stimulation provided a mechanism for controlling or limiting the response of antibody-forming cells. In the following paper, it will be shown that this mechanism operating together with a homeostatic mechanism which prevents induction of new antibody-forming cells serves to regulate the immune response to repeated injections of antigen.

CELLULAR SITES OF SYNTHESIS OF RABBIT IMMUNOGLOBULINS DURING PRIMARY RESPONSE TO DIPTHERIA TOXOID-FREUND'S ADJUVANT.

The present studies are based on previous observations that the intravenous injection of diphtheria toxoid and complete Freund's adjuvant into rabbits resulted in an increased proliferation of cells associated with antibody synthesis; an accelerated, enhanced, and prolonged synthesis of antibody; and a lengthened interval between the appearance of gammaM- and gammaG-hemagglutinating antibodies in the circulation. The molecular species of antibodies that were synthesized by fragments of the spleens were determined by the incorporation of labeled amino acid into antibody and by binding of radioactive antigen by antibody. These studies were paralleled by determination of the presence and type of antibody within the cell by immunofluorescence. Evidence was obtained that non-phagocytic mononuclear cells in the walls of the sinusoids of the red pulp of the spleen are a major source of 19S gammaM-antibody and plasma cells in the non-follicular white pulp are a major source of gammaG-antibody. The data did not exclude the synthesis of gammaG-antibodies by the mononuclear cells, the synthesis of gammaM-antibodies by the plasma cells, or the synthesis of both antibodies by an occasional cell of either morphology. It was hypothesized that the 19S and 7S antibody responses evolved independently with the development of at least two different cell types, a mononuclear cell with capacity for 19S immunoglobulin synthesis and a plasma cell with capacity for 7S immunoglobulin synthesis.

SUPPRESSION OF MEASLES 19S ANTIBODY FORMATION AS EVIDENCE OF IMMUNITY. PRIMARY IMMUNE RESPONSE MASKED BY MATERIAL ANTIBODY.

Secondary immune responses to measles virus are characterized by a rise in 7S measles antibodies with little or no 19S antibody production. Sera collected a month after challenge with live measles vaccine of 9-month-old infants who had received inactivated measles vaccine before they were 7 months of age revealed 19S antibodies in 50% of postchallenge sera tested. Suppression of 19S antibody formation in the other 50% was well correlated with resistance to the clinical effects of measles and provided a more sensitive measure of immunization during the maternal antibody period than seroconversion or incidence of clinical reactions to challenge.

Immune Globulins in Human Viral Infections

EXTENSIVE investigations in animals, including man1–3, have shown that the first immune globulins which appear after a primary antigenic stimulus are of high molecular weight and can be readily inactivated by sulphydryl reducing compounds such as 2-mercaptoethanol (2-ME). In most systems these antibodies, which will be referred to as 19S, are replaced within a few weeks by 7S γ2-globulins which resist reduction. The 19S antibodies can be demonstrated in early post-immunization sera by sedimentation characteristics or more easily by titration of antibody activity before and after treatment with 2-ME. In late sera or following a secondary stimulus, 19S globulins make up no more than a small portion of total antibody so that 2-ME treatment does not result in over-all titre reduction.

ALTERATIONS IN SERUM PROTEINS AND 19S ANTIBODY PRODUCTION DURING THE COURSE OF INDUCED MALARIAL INFECTIONS IN MAN.

SummaryIncreases in β2M macroglobulins have been detected by means of immunoelectrophoresis during the course of induced malarial infections in man. The increase in this serum globulin coincides closely with the appearance of antibodies to malaria as detected by fluorescent antibody studies. Evidence is presented that 19S β2M macroglobulin antibodies as well as 7S gamma globulin antibodies are formed during the course of primary malarial infections in human volunteers.

Depression of the Primary Immune Response by dl-Penicillamine.

SummaryTreatment of mice with dipenicillamine for 3 weeks before antigen injection resulted in an overall reduction in antibody formation while treatment immediately after antigen injection for 8 consecutive days had no effect. In those groups showing depression of antibody response, the formation of both the 19S and 7S antibody was reduced, the latter type also showing a delay in appearance but subsequently reaching levels comparable to those seen in normal animals. Administration of penicillamine intravenously to animals having only 19S antibody in the serum resulted in an immediate but transient reduction of antibody titer. This observation, along with that showing failure of penicillamine to affect the formation of both 19S and 7S antibody when given after antigen injection, suggests that the depressive effect is primarily at the cellular level of the immunologic process rather than through an intravascular dissociation process.

Adult brain antigens demonstrated in chick embryos by fractionated antisera

ABSTRACT Antisera produced in rabbits against homogenates of adult chicken brains and absorbed with liver and yolk were used to detect, via complement fixation, the appearance of brain antigens during development of the embryonic chick brain. Reacting antigens are found at the earliest stages studied (stages 14–16, ca. 2 days) but remain at a low level (relative to protein) until after stage 28 ( days), when a rapid increase begins, reaching the adult level by stage 38 (12 days). Antisera were fractionated on DEAE cellulose and the anti-brain activity found in two fractions, identified as 7S and 19S respectively. The 19S antibody reacted with a chloroform-methanol extract of adult brain homogenate but not with the residue, while the 7S antibody reacted only with the chloroform-methanol insoluble residue. When tested against brains of embryos at different developmental stages, the 19S antibody reacted at a low and essentially unchanging level from the earliest stages studied to the adult. The 7S antibody gave no reaction with embryonic brain until after stage 28 at which time a reacting antigen was found which reached the adult level by stage 38. Based on the appearance in detectable quantity, and the rapid accumulation, of a brain-specific protein antigen, the period between stages 28 and 38 is considered to be critical for neural differentiation.

ANTIBODY ACTIVITY IN DIFFERENT CHICKEN GLOBULINS.

Hemagglutination is of questionable validity for following the synthesis of 19S to 7S chicken anti-BSA antibodies, since only small amounts of the major antibody population (7S) are measured in early primary antisera. The 7S antibodies in primary antisera were demonstrated by antigen-binding methods (ammonium sulfate salting out procedure and RIE). Radioimmunoelec-trophoretic patterns localized anti-BSA and anti-HSA primary antibody activities in the 7S γ−, γ1A−, and γ1M−globulins; however, only a few of these sera had γ1A− activity. No γ1M− antibody was detected by RIE in hyperim-mune antisera, All anti-azobenzoate antisera had γ− and γ1M−globulin antibodies, but no activity in the γ1M−globulin. The species of chicken antibody synthesized seems to be related to the nature of the immunizing antigen.

Effect of prior administration of 7S and 19S antibody on active antibody formation

Effect of prior administration of 7S and 19S antibody on active antibody formation

ANTIBODY FORMATION IN VITRO BY SEPARATED SPLEEN CELLS: INHIBITION BY ACTINOMYCIN OR CHLORAMPHENICOL.

The formation of 19S antibody to poliovirus by rabbit spleen cells in vitro was interrupted by adding actinomycin D (1 to 10 micrograms per milliliter) for 30 minutes or longer. Shorter treatment (5 to 15 minutes) caused partial suppression. Antibody formation was slowly renewed upon removal of the drug. These results suggest that 19S antibody formation is contingent upon DNA-dependent RNA synthesis and that the genetic messenger has an average lifetime of one-half day or less. Treatment with chloramphenicol (20 to 50 micrograms per milliliter) for 3 days caused the cessation of 19S antibody formation.

SPECIFIC INHIBITION OF ANTIBODY FORMATION BY PASSIVELY ADMINISTERED 19S AND 7S ANTIBODY.

Intravenously administered 7S antibody is more effective than 19S antibody in inhibiting the formation of antibody to bacteriophage X174. Since considerable amounts of 7S antibody are needed for inhibition, serum antibody formation may act as a "feedback" mechanism to prevent hyperimmunization.

In Vitro Passive Sensitization of Chopped Guinea Pig Lung by Guinea Pig 7S Antibodies

Summary Guinea pig 7S γ-1, but not γ-2, antibodies directed against the same antigenic determinant sensitized chopped guinea pig lung tissue for antigen-induced release of histamine. Thus, in vitro experiments confirm previous in vivo findings that guinea pig 7S γ-1, but not γ-2, antibodies sensitize the guinea pig for passive cutaneous and systemic anaphylaxis. Succinate or maleate markedly enhanced antigen-induced release of histamine from tissue passively sensitized in vitro with γ-1 antibodies. Maximum enhancement was achieved by simultaneous addition of antigen and succinate. Succinate did not enhance hemolysis produced by γ-2 anti-hapten antibodies and complement, nor did it enhance lysis of sheep erythrocytes sensitized with guinea pig anti-sheep erythrocyte antisera.

THE ROLE OF NONSPECIFIC SERUM FACTORS IN THE ANTIBODY RESPONSE TO THE PHI-CHI 174 BACTERIOPHAGE.

The role of the “antibody cofactor” and of other heat-labile serum components (complement) in the neutralization of the ϕX 174 bacteriophage by means of specific antibodies was studied. Sera of white mice, guinea-pigs and rabbits obtained mainly early after phage administration were investigated. The character of antibodies was estimated from their sensitivity to 2-mercaptoethanol or else by chromatography on Sephadex G-200. Sera from the first days after the administration of the phage containing mostly type 19S antibodies, and sera from later periods after the administration containing mostly type 7S antibodies, were tested. (Some evidence was also obtained about the formation of slowly sedimenting antibodies sensitive to 2-mercaptoethanol in the rabbit.) With a single exception the tested sera showed no significant decrease of the neutralization activity after 30-mins. heating at 56°C or at 60°C and no increase of the neutralization power could be observed after the application of homologous or normal mouse serum. It is concluded that the heat-labile components of normal sera including the complement and the “antibody cofactor” play no role in specific phage neutralization.

COMBINATION WITH RED CELLS OF IMMUNE RABBIT 7S ANTIBODY AND ITS SUB-UNITS.

COMBINATION WITH RED CELLS OF IMMUNE RABBIT 7S ANTIBODY AND ITS SUB-UNITS.

ENHANCEMENT OF THE INFECTIVITY OF ARBOVIRUSES BY SPECIFIC ANTISERA PRODUCED IN DOMESTIC FOWLS.

SummaryThe apparent infectivity of certain arboviruses is increased by the action of specific antisera prepared in domestic fowls when assayed on chick embryo fibroblast (CEF) monolayers or the chorioallantoic membrane of the developing chick embryo. Of the viruses tested, Murray Valley encephalitis (MVE), West Nile (WNV) and Japanese encephalitis viruses from Casals group B, and Getah virus from group A demonstrate this enhancement, but Sindbis and SFV from group A do not. The virus plaque count is increased by up to 12 times the control plaque count, depending on the potency of the antiserum.Enhancement could not be demonstrated with rabbit or mouse antisera to MVE on the CEF monolayer, nor with fowl antiserum on PK‐2a cells or in suckling mice.Cross enhancing activity could be demonstrated between viruses within group B (MVE and WNV), but not between viruses from different groups (MVE and Getah).Preliminary work indicates that 19S antibody is involved, as well as antibody not susceptible to depolymerisation with mercaptoethanol.

INHIBITION OF 19S ANTIBODY SYNTHESIS BY 7S ANTIBODY.

A brief course of treatment with 6-mercaptopurine prevented conversion of 19S to 7S antibodies during the immune response of rabbits to bovine gamma globulin. Specific reactive homologous 7S antibodies given intravenously to animals with such incompletely developed humoral reactions led to an abrupt fall in 19S antibodies. After a lag period the synthesis of 7S antibodies was apparently normal. These results suggest that the concentration of 7S antibodies controls the synthesis of 19S antibodies.

ANTIGENS IN IMMUNITY

SummaryAn investigation of the immunogenicity of three flagellar preparations, namely, intact flagella, polymerized flagellin and the soluble, low molecular weight protein flagellin, was performed in rats. Despite the similarity of in vitro serological behaviour of these three antigens, important differences in their in vivo immunological effectiveness were noted. While flagella and polymerized flagellin caused first 19S antibody formation and later prolonged 7S antibody formation, flagellin appeared to be quite incapable of inducing a 19S primary response. Further, the peak titres achieved with Hagellin were lower than those with the other antigens. Polymerized flagellin was somewhat less immunogenic than were flageila.The study covered a dosage range of 10 logs.10, i.e., from 10–12 g. to 10–2 g. Clear‐cut dose‐response relationships could be observed for the shortlived 19S response caused by flagella or polymerized flagellin, over the dose range 10 ng. (0·01 μg.) to 10 μg. For the 7S response, particularly with flagellin, dose‐response relationships were not established. Remarkably small doses, namely, 100 ng. (0·1 μg.) of flagella, 1 μg. of polymerized flagellin and 10 μg. (0·01 μg.) of flagellin caused peak 7S titres to appear. Doses as small as 100 pg. (0·0001 μg.) occasionally caused antibody production.Doses of antigen too small to cause a detectable 19S response were followed by a lag period of about two weeks during which serum antibody levels remained below the threshold of our test. Then antibody litres rose steadily until six or eight weeks after injection, Peak levels were maintained for over six weeks, and fell slowly. At 34 weeks, when the experiments were stopped, antibody litres were still quite high, The lag period with doses which caused 19S antibody production was much shorter, generally less than four days. In such animals, 19S antibody was gradually replaced by 7S antibody during the second week, and again it took several weeks for peak 7S litres to be leached.These results emphasized the need to use small doses in any meaningful study of antigen distribution, localization, behaviour and fate.

ANTIGENS IN IMMUNITY

SummaryA wide range of soluble and particulate substances were iodinated and injected into rats. Some preparations were classical antigens; others were heterologous proteins of minimal antigenicity; and others were homologous substances believed to be non‐antigenic.It was found that all antigens showed a tendency to concentrate in lymphoid follicles, as well as in medullary macrophages. Non‐antigens were taken up to a varying extent by cells in the medulla, but did not localize in lymphoid follicles. The only exception to this rule was rat gamma globulin. In view of the role of globulins in tbe opsonization of particulars antigens, this exception seemed especially significant, The study suggested that the phagocytic cells in lymphoid follicles possessed the capacity to “recognize foreignness”, and the possibility exists that this is mediated by opsonic factors, rather than being an inherent property of the cells.The localization pattern of iodinated flagellin, and the cellular changes induced by it, were carefully compared with those of equivalent doses nf flagella. The differences in localization were relatively slight, yet the cellular changes typical of the immune response occurred 10–14 days later with flagellin than with flagella. It is unlikely that this delay was due to the chemical differences between the antigens, as polymerized flagellin behaved more like flagella. It appears that a particulate antigen entering a follicular macrophage may cause the more dramatic appearance of plasma cell precursors around it, and the earlier formation of 19S antibodies, but the reasons for this are not clear.

SINGLE CELL STUDIES ON 19S ANTIBODY PRODUCTION.

Rats were immunized with Salmonella adelaide flagella. By zone centrifugation of serum samples in sucrose gradients, it was shown that, as in many other systems of antibody formation, the first response was the formation of 19S, mercaptoethanol (ME)-sensitive antibody. This was quickly replaced by 7S, ME-insensitive antibody. Popliteal lymph node cell suspensions were prepared, and cells with antibody on their surface were identified by the method of bacterial adherence. By micromanipulation such cells were washed, placed into microdroplets, examined under high-power phase contrast and broken to release intracellular antibody. These droplets were then studied in either of two ways. In the first method, each droplet was halved and one half treated with ME. Then both halves were titrated for immobilizing antibody through serial twofold dilution of the half microdroplets. Droplets showing destruction of antibody by ME were classified as 19S; those showing no reduction in titer as 7S; and those showing significant (>1 log(2)) reduction as double producers; i.e., cells containing both 7S and 19S antibodies. In the second method, droplets were divided into 4 equal quarters, for testing after treatment with either ME, or a specific rabbit anti-rat 7S globulin serum, or both. In these experiments, cells showing some remaining antibody after treatment with either reagent, but not after treatment with both reagents, were classified as double producers. Of 144 cells tested, 123 contained readily detectable amounts of antibody. These comprised 42 19S cells, 64 7S cells, and 17 double producers. The double producers were frequent at times when the switchover from 19S to 7S antibody production was occurring. All except 4 of the cells in the study could clearly be identified as members of the plasma cell series. Though 7S cells became more frequent as the cell population matured, no clear-cut correlation between cell immaturity and 19S production could be obtained. In the primary response many fully mature plasma cells contained only 19S antibody; conversely, in the secondary response many blasts contained 7S antibody. No morphological difference between 19S and 7S cells could be found. The results suggested that many cells or cell clones go through a sequence whereby each forms first 19S and later 7S antibody with identical combining sites.

PRODUCTION OF 7S AND 19S ANTIBODIES TO THE SOMATIC ANTIGENS OF SALMONELLA TYPHOSA IN RABBITS.

SummarySeven rabbits immunized with acetone dried Salmonella typhosa produced both 19S and 7S agglutinins for the typhoid somatic antigens although the 19S antibodies predominated in most serum samples even after repeated injections of vaccine. The results suggest that the route of injection and amount of antigen injected may influence the relative amounts of 19S and 7S antibodies appearing in the serum.