Immune responses to thyroglobulin in experimental allergic thyroiditis

Guinea-pigs immunized with homologous thyroglobulin in complete Freund's adjuvant were observed for lesions in the thyroid gland, and for development of specific delayed type skin hypersensitivity. Their blood leucocytes were examined for specific sensitivity in vitro by determining the inhibition of migration in the presence of thyroglobulin. Serum was tested for humoral antibodies by the passive haemagglutination technique. There was a significant correlation between the development of thyroiditis and the intensity of the skin reactions. No relationship was observed between the presence of thyroiditis and leucocyte sensitivity nor between thyroiditis and circulating antibodies. On the other hand serum antibody titres were correlated to the degree of leucocyte sensitivity. Thyroglobulin inhibited the migration of cells from normal animals in the presence of plasma from thyroglobulin-sensitized animals. The activity of sensitized plasma disappeared when diluted 1:10 in normal plasma irrespective of the serum antibody titre. Guinea-pigs immunized with thyroglobulin or kidney antigen in complete Freund's adjuvant and tested for skin or leucocyte hypersensitivity showed specific but no cross reactivity to the two preparations.     

Immunological unresponsiveness during induction of experimental autoimmune orchitis in guinea-pigs: studies in vivo and in vitro.

Groups of male and female guinea-pigs were immunized with homologous sperm derived from testis (TS) or epididyimis (ES) in Freund's complete adjuvant (FCA). In vivo investigations included skin tests at 2 weeks and development of aspermatogenesis (testis weight) at 4 weeks; in vitro assays were inhibition of migration of peritoneal exudate cells (PEC) and culture of blood leucocytes (lymphocyte transformation) at weekly intervals after immunization. Antigens used were heat-treated extracts of sperm used for immunization (BTS, BES); cells were also cultured simultaneously with PPD. Skin tests revealed anergy in males as compared with females: a larger quantity of antigen which caused partial unresponsiveness in females, caused profound unresponsiveness in males although the aspermatogenesis was less severe. In vitro tests also showed anergy during the active stages of the orchitis. This was non-specific for PEC (specific unresponsiveness was not excluded), but blood leucocytes showed only specific unresponsiveness (to BES). These and previous studies suggest that the unresponsiveness results from a desensitisation by sperm antigens released during the development of aspermatogenesis.     

In vivo macrophage suppression of delayed hypersensitivity in the guinea-pig.

The nature of the suppressive activity in the peritoneal exudate cells (PEC) of guinea-pigs immunized with dinitrophenyl bovine gamma globulin (DNP50-BGG) was investigated. A method was developed to isolate from the peritoneal exudate large numbers of macrophages. Using density gradient centrifugation on Percoll it was possible to obtain a population of cells which contained over 90% macrophages. This macrophage preparation was found to respond to lymphokine but to be incapable of passively transferring delayed hypersensitivity reactions. When these immune macrophages were transferred into antigen immunized animals, which had been pretreated with cyclophosphamide (CY), the skin reactions were suppressed to the same extent as when the total PEC was transferred. PEC from guinea-pigs immunized with ovalbumin in Freund's incomplete adjuvant did not suppress the skin reactions in CY-pretreated DNP50BGG immunized animals. However, in contrast, macrophages from these animals did suppress the skin reactions in the recipient guinea-pigs indicating that the macrophage suppression was not antigen specific.     

Antibodies to gluten and reticulin in gastrointestinal diseases

Ribonucleic acid extracts (RNA) obtained from the lymph nodes and spleens of guinea pigs, which were immunized with testicular antigen emulsified in Freund's complete adjuvant (FCA), were injected intraperitoneally into normal guinea pigs. The transferred guinea pigs developed a delayed hypersensitivity to sperm antigens and testicular lesions which resembled the lesions obtained in the donor RNA guinea pigs. When the transfer was performed with RNA extracted from guinea pigs immunized with FCA alone or with 'immune' RNA treated with Ribonuclease, neither cellular immunity nor testicular lesions were observed.     

Nifurtimox-induced alterations in the cell-mediated immune response to PPD tin guinea-pigs.

Positive skin reactions to PPD in guinea-pigs immunized with Freund's complete adjuvant (FCA) were reversed after treatment with 10 mg/kg/day nifurtimox for 12 days. The in vitro migration of peripheral blood leucocytes from FCA-immunized guinea-pigs was inhibited with PPD, but it returned to normal values after nifurtimox treatment. Furthermore, the cell-free supernatant from PPD-stimulated lymphocytes from FCA-immunized nifurtimox-treated guinea-pigs did not inhibit the migration of normal cells. Thus the administration of nifurtimox impaired the specific cell-mediated immune response to PPD both in vivo and in vitro.     

Delayed Hypersensitivity, Macrophage Migration, and Antibodies in Guinea-Pigs Immunized with Diphtheria Toxoid

Guinea-pigs were immunized with diphtheria toxoid (DT) or with DT-anti-toxin precipitate, both in Freund's complete adjuvant (FCA). DT-induced inhibition of peritoneal cell migration was equally strong in both groups and increased with time after immunization. Some guinea-pigs immunized with DT-antitoxin precipitate were boosted with DT or DT-antitoxin precipitate, both in FCA. This increased the antibody titre but did not affect 24-hour skin reactivity or migration inhibition. Migration inhibition did not correlate with anti-DT passive haemagglutinins, haemolysins, or cytophilic antibody on peritoneal cells, but did correlate with 24-hour skin reactivity.     

Migration inhibition of lymph node lymphocytes as an in vitro assay for cell-mediated immunity in the draining lymph nodes of parenterally immunized mice

This paper describes a method for in vitro measurement of specific cell-mediated immunity in the mouse. Animals were immunized parenterally with ovalbumin in Freund's incomplete or complete adjuvant, and a direct migration inhibition assay was performed, using lymphoid cells from the draining lymph nodes. Migration inhibition was found to be antigen specific, correlated with systemic delayed-type hypersensitivity measured in vivo by skin testing, and had a high degree of sensitivity for ovalbumin. The migrating cells were identified as lymphocytes. Lymph node lymphocyte migration inhibition provides a reliable in vitro assay for regional CMI in the mouse.     

The mechanism of action of soluble lymphocytic mediators: I. A pulse exposure test for the measurement of macrophage migration inhibitory factor

Culture supernatants containing macrophage migration inhibitory factor (MIF) were obtained by incubating lymphocytes of guinea-pigs, immunized with Freund's complete adjuvant (FCA), with tuberculin PPD in vitro. Exposure of normal peritoneal macrophages to MIF-containing supernatants for 2 hours at 37° (pulse exposure), followed by suspension in culture medium and transfer to capillaries, resulted in inhibition of migration in vitro for the next 24 hours. No inhibition was seen when macrophages were incubated with MIF at 4°. On the other hand when exposure to MIF at 4° was followed by incubation of the cells for 2 hours at 37° in culture medium, in the absence of MIF, inhibition of migration was obtained. These results indicate that: (a) macrophages possess a specific receptor able to bind MIF at either 4° or 37°, and (b) inhibition of migration by receptor bound MIF requires a temperature-dependent active process, the nature of which remains unknown.

Passage of lymphocytes through columns of glass beads resulted in a population of cells with intact or heightened MIF-forming ability, as assessed by both conventional and pulse exposure techniques.

     

Inhibition of Macrophage Migration by Nucleotide-Containing Streptococcal Preparations

Certain extracts of streptococcal cell walls are known to inhibit macrophage migration in vitro. In this study, we attempted to identify the streptococcal components responsible for this phenomenon. Trypsinized cell walls and cytoplasm from groups A and B streptococci were extracted with hot formamide followed by acetone precipitation. Subsequent gel filtration in aqueous solutions yielded a fraction devoid of C-carbohydrate and containing mostly oligonucleotides, apparently derived from streptococcal cytoplasm. This fraction significantly inhibited the migration of peritoneal exudate cells from rats sensitized to groups A and B streptococci. It was noteworthy that no inhibition of migration was observed with cells from nonsensitized animals or control rats injected with BCG or complete Freund adjuvant. Similarly, no inhibition was obtained with formamide extracts of calf thymus RNA. Although the inhibition does not show specificity for streptococcal groups, it seems to have immunological specificity since prior sensitization with streptococci is required for migration inhibition.     

Immune deviation: demonstration of split tolerance in vitro by inhibition of macrophage migration

Migration of cells, taken from animals immunized with ovalbumin in Freund's complete adjuvant which gave normal delayed hypersensitivity skin responses, was found to be significantly inhibited in the presence of antigen. Migration of peritoneal exudate cells from guinea-pigs in which immune deviation had been induced by immunization with antigen in Freund's incomplete adjuvant was not inhibited.     

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.     

The effect of respiratory immunization on cell-mediated immune effector cells of the lung.

In order to understand the mechanisms of cellular immune injury in hypersensitivity pneumonitis, the effect of type of antigen on cell-mediated immunity in guinea-pigs receiving respiratory immunization was studied. Lymphocytes obtained by pulmonary lavage were compared with those from peritoneal exudate following immunization with either a soluble protein, human serum albumin, or a particulate suspension of Thermoactinomyces vulgaris. Assays were obtained without mixing cells from these two sources. Statistically significant increases (13-22%) in the number of alveolar rosette-forming cells (RFC) were found in the animals immunized with either antigen, but only the particulate T. vulgaris was also capable of inducing a systemic increase of such cells. That this increase in RFC could be due to specifically reactive lymphocytes was demonstrated by the production of antigen-stimulated macrophage migration inhibition. Some evidence was obtained that indicated that T. vulgaris could act both as a non-specific B-cell stimulant and a specific T-cell activator. The concept of a hypothetical pulmonary 'barrier' is discussed which must be overcome to induce systemic immune responses following respiratory immunization. T. vulgaris must be added to the list of known agents or means for overcoming this 'barrier'.     

Relationship Between Tuberculin Hypersensitivity and Cellular Immunity to Infection in Mice Vaccinated with Viable Attenuated Mycobacterial Cells or with Mycobacterial Ribonucleic Acid Preparations

The migration inhibition technique has been used to study delayed hypersensitivity in vitro by using peritoneal exudate cells and splenic lymphocytes from mice vaccinated with viable cells of the attenuated H37Ra strain of Mycobacterium tuberculosis and from mice vaccinated with ribonucleic acid (myc RNA) preparations obtained from viable mycobacterial cells of the same strain. Inhibition of macrophage migration was noted when purified protein derivative (PPD) or viable H37Ra cells were added to peritoneal exudate cells obtained from mice immunized with viable H37Ra cells and not from mice immunized with myc RNA. Splenic lymphocyte cultures were exposed to the same antigens in vitro. Filtered supernatant fluids from these lymphocyte cultures, when added to peritoneal exudate cells obtained from nonimmunized mice, inhibited migration only when they were obtained from lymphocytes which came from mice immunized with viable H37Ra cells. Injection of PPD intravenously into vaccinated mice resulted in inhibitory supernatant fluids from splenic lymphocyte cultures only when the lymphocytes came from mice immunized with viable H37Ra cells. However, intravenous injection of either viable H37Ra cells or of myc RNA preparations into mice vaccinated with myc RNA occasionally produced inhibitory supernatant fluids when lymphocytes were obtained from these mice. On the other hand, mice vaccinated with myc RNA or viable H37Ra cell preparations were consistently and equally protected against intravenous challenge with the virulent H37Rv strain. Thus, although some evidence was obtained for a delayed type hypersensitivity in mice vaccinated with H37Ra cells or with myc RNA to ribosomal proteins or other proteins associated with the RNA preparation, no evidence of tuberculin hypersensitivity could be detected in any mice vaccinated with the myc RNA. These results argue against a role for tuberculin hypersensitivity in immunity to tuberculous infection.     

Kinetics of cellular and humoral response of rabbits immunized with mycoplasma pneumoniae.

The cellular and humoral responses of rabbits immunized with Mycoplasms pneumoniae antigen in saline or incorporated in Freund's complete adjuvant were examined. Peripheral blood leukocytes were used for the leukocyte migration inhibition (LMI) test. Both groups of animals showed significant LMI activity in the presence of M. pneumoniae as well as cross-reacting M. salivarium antigens but response to M. pneumoniae antigen was more pronounced. In the humoral response no such cross-reactivity was observed. Although some of the animals (3/8) demonstrated antibodies to M. salivarium prior to immunization the titers were not influenced by the immunization with M. pneumoniae antigen. Both groups of animals produced antibodies to M. pneumoniae antigen only, but significantly higher titers were observed in the adjuvant group. Cold hemagglutinins in both groups appeared earlier than the specific antibodies to mycoplasma. The adjuvant had no effect on the production of the cold agglutinins.     

Selective and specific inhibition of 24 hour skin reactions in the guinea-pig. I. Immune deviation: description of the phenomenon and the effect of splenectomy

Strong 24 hour skin reactions occur in guinea-pigs immunized with antigen in Freund''s complete adjuvant. These reactions were reduced by the injection of 1 mg of the same antigen, in either the soluble or alum precipitated form, 14 days before immunization with antigen in Freund''s complete adjuvant. There was also a reduction in the corneal reaction, which has been regarded as an index of delayed hypersensitivity. This phenomenon was called immune deviation.The phenomenon was demonstrated for bovine γ-globulin, human serum albumin, bovine serum albumin, egg albumin, diptheria toxoid, haemocyanin, purified protein derivative (PPD) and dinitrophenylated proteins. Ten mg of soluble bovine γ-globulin caused considerable reduction of the circulating antibody level and of the 24 hour skin reaction. Alum precipitated bovine γ-globulin and smaller doses of soluble antigen reduced the skin reaction but had less effect on the antibody level. Similar results were obtained with human serum albumin. This suggested that the conditions for eliciting immune paralysis and immune deviation were different.Immune deviation was obtained with either footpad or intravenous injections and with as little as 100 μg of antigen. Alum precipitated bovine γ-globulin caused immune deviation when given 14, 7 or 1 day before or 1 day after immunization with antigen in Freund''s complete adjuvant. It was inactive when given 6 days after immunization. Splenectomy had no effect on the production or deviation of 24 hour skin reactions.Alum precipitated antigen had a variable and usually slight effect on the level of antibody following immunization with the same antigen in Freund''s complete adjuvant. There was, however, a qualitative alteration in the antibody. The sera of guinea-pigs, which had been deviated by a prior injection of bovine serum albumin or bovine γ-globulin, showed only a γ₁ line of antibody on immunoelectrophoresis, while the sera of control guinea-pigs also showed the γ₂ line characteristically seen in guinea-pigs immunized with antigen in Freund''s complete adjuvant.It was concluded that immune deviation was distinct from classical immune paralysis and that the immunologically specific reduction of the 24 hour skin reactions might be due, at least in part, to a selective loss of delayed hypersensitivity.     

The immune response to oxidized ferredoxin. I. Specificity of the response to the amino terminal determinant.

Several synthetic peptide analogues of the amino terminal antigenic determinant (ala-tyr-lysile-ala-asp-ser) of oxidized ferredoxin (O-Fd) were tested for their ability to inhibit the complement fixation reaction between O-Fd and homologous rabbit antiserum, and to inhibit the migration of spleen cells from guinea-pigs immunized to O-Fd or to a conjugate of the amino terminal heptapeptide (N7) and bovine serum albumin (N7-BSA). The results of the migration inhibition assay suggest that the tetrapeptide and longer peptides of the native sequence were all recognized and stimulated the production of migration inhibition factor. Peptides modified at the aspartic residue were partially active while the serine modified peptide was not. Modification at the amino end of the heptapeptide had no effect on migration inhibition. As specificity controls, it was shown that the N7-BSA conjugate inhibited migration in O-Fd immunized animals, while O-Fd inhibited migration in N7-BSA immunized animals. The hexa-, hepta-, aspartic-deleted and serine-modified peptides were able to inhibit the complement fixation reaction with O-Fd and specific rabbit antiserum. Inhibition found with the serine-modified peptide and the lack of inhibition with the amino-modified peptide or the di-, tri-, tetra- and pentapeptides indicates the determinant recognized by the rabbit antibodies is either larger or is located nearer the middle of the heptapeptide than the determinant which induced the production of MIF.     

Specificity of the macrophage spreading test with reference to Leishmania antigens and correlation with delayed hypersensitivity.

The inhibition of the macrophage spreading test, claimed to be an in vitro correlate of delayed hypersensitivity, was examined in guinea-pigs immunized with L. enriettii and L. tropica soluble antigens. Cells from peritoneal washings of the guinea-pigs were tested in presence of the homologous and heterologous antigens and also without antigen. Inhibition of macrophage spreading compared to control preparations was noted only in the presence of the homologous antigen when the skin test response of the donor animal was relatively small. The degree of inhibition decreased as the skin test volume increased and when skin test volumes were large there was actual stimulation of macrophage spreading, rather than inhibition. The addition of heterologous antigen to the peritoneal cell preparation always resulted in the augmentation of macrophage spreading above control levels. The possible mechanisms of this in vitro technique and its use as a taxonomic or diagnostic tool are discussed.     

Lithium potentiates antigen-dependent stimulation of lymphocytes only under suboptimal conditions

Immunization of hamsters with DNP-BSA in either Freund's complete or incomplete adjuvant led to the induction of antigen reactive lymph node cells. As assessed by in vitro lymphocyte stimulation assays, antigen in complete adjuvant was more effective than antigen in incomplete adjuvant in inducing immunity. Supplementing antigen-stimulated cultures from animals 14 days post-immunization with LiCl led to no enhancement of tritiated thymidine incorporation into cells from animals immunized with antigen in complete adjuvant, but did enhance antigen-dependent stimulation of cells from animals immunized with antigen + incomplete adjuvant. LiCl was, however, able to enhance stimulation of cells from animals immunized with antigen + complete adjuvant at 22 and 29 days post-immunization, when in vitro responsiveness was declining. Lymph node cells from animals optimally immunized antigen + complete adjuvant were fractionated by passage over Sephadex G-10 columns. Sephadex G-10 non-adherent cells, deficient in cells such as macrophages, exhibited a depressed responsiveness to antigen, compared to unfractionated cells, and responsiveness was not restored by LiCl. Stimulation of cells by antigen was found to be inhibited by supplementing the cultures with theophylline or dibutyryl cyclic AMP and this inhibition could be reversed by LiCl. Lithium would, therefore, appear to be able to influence lymphocyte adenylate cyclase. Thus, LiCl can exert an immunopharmacologic effect on in vitro antigen stimulation primarily when conditions are suboptimal, possibly through an influence on cyclic AMP metabolism.     

The effect of lymphokines on the development of delayed hypersensitivity to an unrelated antigen

The development of delayed hypersensitivity to sheep red cells in rabbits was assessed by measuring the inhibition of migration of cells from spleen fragments in the presence of sheep red cell antigen. Supernatants containing migration inhibition factor were prepared by incubating lymph node cells from rabbits immunized with Freund's complete adjuvant with PPD. Sheep red cells injected together with these supernatants intravenously gave rise to delayed hypersensitivity. In contrast the injection of sheep red cells alone or with control supernatants did not give rise to delayed hypersensitivity.     

Lymphocyte transformation and macrophage migration in guinea-pigs immunized with Freund''s complete adjuvant

In vitro peripheral blood lymphocyte transformation and peritoneal macrophage migration in the presence of purified protein derivative have been studied at weekly intervals in Freund's complete adjuvant sensitized guinea-pigs. Peripheral blood lymphocytes from half of the sensitized animals when cultured with purified protein derivative 1 week after immunization manifested more than double the thymidine-2-¹⁴C incorporation of the unstimulated cells. By the 3rd week after immunization the response of the lymphocytes to antigen was maximal. The average of peritoneal cell migration inhibition was statistically significant only in the group of animals examined 3 weeks after immunization. The in vitro peripheral blood lymphocyte transformation was closely related with the appearance of the delayed cutaneous reactivity while the migration of peritoneal cells was markedly inhibited only when there was much evidence of in vivo delayed hypersensitivity and of in vitro peripheral blood lymphocyte response to antigen. No relationship has been demonstrated between these in vitro phenomena and the presence in the sera of anti-purified protein derivative haemagglutinating antibodies.