Further study of the problem of specific cellular receptors in delayed hypersensitivity]

The work presents the materials obtained as a result of the further study of specific T lymphocyte receptors with the use of so-called receptor antisera, i. e. antisera against the lymphoid cells of mice sensitized with one of the two antigens (Macobacterium tuberculosis or bovine gamma globulin); thus these differed from control antisera against the lymphoid cells of intact mice. These mouse antisera reacted with the lymphoid cells of guinea pigs in experiments of delayed-type hypersensitivity transfer. The cells of sensitized guinea pigs lost their ability to transfer hypersensitivity if, prior to their injection into the recipient guinea pigs, these cells were treated with the above-mentioned mouse antisera, i. e. antisera against the lymphoid cells of mice had a blocking effect on the lymphoid cells of guinea pigs. The blocking action of the antisera proved to be specific: antisera against the lymphoid cells of mice sensitized to bovine gamma gloublin blocked the cells of guinea pigs, also sensitized to bovine gamma globulins, but did not block the cells sensitized to Mycobacterium tuberculosis. The control antisera, taken in the same doses as the factor antisera, did not show a blocking effect on the specific activity of lymphoid cells.     

Passive transfer of cell-mediated immunity in xenogeneic animals

Cell-mediated immunity (delayed hypersensitivity) to 1-chloro-2,4-dinitrobenzene (DNCB) was passively transferred from sensitized guinea pigs to mice. Transfer was accomplished by subcutaneous injection of peritoneal exudate cells of sensitized guinea pigs.     

Specific depression of delayed hypersensitivity to purified proteins, with relation to production of circulating antibody

In guinea pigs sensitized in the footpads with a purified protein, such as hen egg albumin or diphtheria toxoid, in incomplete Freund's adjuvant, delayed hypersensitivity precedes the appearance of circulating antibody. This expression of delayed hypersensitivity by skin-test declines sharply the day before circulating antibody is detected. Adoptive transfer of spleen and peritoneal exudate cells from guinea pigs showing this decline suppresses the expression of delayed hypersensitivity in already sensitized recipients. This suppression of delayed hypersensitivity is immunologically specific. The intensity of this suppression does not correlate directly with the dose of sensitizing antigen, nor does it depend directly on the amount of circulating antibody.     

The role of immunoglobulin receptors on lymphocytes in production of MIF in guinea pigs

The effect of anti-guinea pig IgG sera and anti-rabbit light kappa chain serum on the capacity of sensitized lymphocytes of guinea pigs to production of migration inhibitor factor (MIF) was investigated. The lymph node cells, thymocytes and circulating lymphocytes taken from dinitrophenyl- (DNP) sensitized guinea pigs were preincubated with antisera against gamma₁ + gamma₂ globulins, gamma₁ globulins, gamma₂ globulin, light kappa chains or normal rabbit serum as control and stimulated with antigen in vitro to production of MIF. The inhibitory effect of lymphocyte culture supernates on the migration of guinea pig normal macrophages was determined by capillary tube test. It was found that all the anti-immunoglobulin sera used suppressed, in varied degree, the release of MIF by sensitized lymphocytes. It is suggested that the suppressive influence of anti-IgG sera reflects their blocking effect on surface receptors specific for antigen.     

Blocking of delayed hypersensitivity by humoral antibody in an in vivo mouse transfer assay using sensitized guinea pig cells

Humoral antibody was shown to interfere specifically with the expression of cell-mediated immunity (delayed hypersensitivity) in an in vivo system. Mice that received peritoneal exudate cells obtained from guinea pigs sensitized to 1-chloro-2,4dinitrobenzene (DNCB) exhibited delayed hypersensitivity reactions after challenge with the sensitizing agent. While control groups that received either normal sera, saline, or anti-BSA (bovine serum albumin) in addition to peritoneal exudate cells from sensitized guinea pigs exhibited positive delayed reactons to challenge with DNCB, mice that received anti-DNP (dinitrophenyl group) in addition to the senstized cells were prevented from exhibiting a delayed reaction to DNCB.     

Role of mediators of cellular hypersensitivity in the stimulation of the antibody formation to unrelated antigen

The effect of a concurrent delayed hypersensitivity reaction on the antibody response to sheep red cells was assessed by a plaque assay. Guinea pigs with delayed hypersensitivity to tuberculin purified protein derivative (PPD) or egg albumin showed an increased antibody response to sheep red cells when the cells were injected intravenously at the same time as PPD or egg albumin. This effect was transferred to normal guinea pigs by serum from guinea pigs with delayed hypersensitivity to PPD or egg albumin taken 24 hr after injecting the corresponding antigen. Supernatants containing migratory inhibitory factor were prepared by incubating lymphocytes from sensitized rabbits with antigen. These supernatants were injected with sheep red cells and gave rise to an enhanced plaque response. Similar results were obtained with supernatants from normal rabbit thymus cells. The role of mediators of delayed hypersensitivity in enhancing antibody formation and in T cell/B cell cooperation is discussed.     

Experimental allergic orchitis and aspermatogenesis. VI. Transfer of allergic orchitis with immune cells.

Typical experimental allergic orchitis (EAO) and aspermatogenesis were successfully transferred to strain 13 guinea pigs with peritoneal exudate and lymph node cells from male and female donor guinea pigs (lacking detectable antibody) previously sensitized with 9 mug of highly purified GP1 glucoprotein isolated from the sperm acrosome. Attempts to transfer the disease with circulating antibody from hyperimmunized animals were not successful. These studies support a cell-mediated basis for the immunopathologic events in EAO.     

Studies on the mechanism of suppression of delayed hypersensitivity by the antischistosomal compund niridazole.

Niridazole given in a single oral dose of 100 mg/kg to guinea pigs sensitized to ortho-chlorobenzoyl chloride-bovine gamma-globulin (OCB-BGG) regularly abolished delayed cutaneous reactivity. Little effect was observed, however, when cells from these animals were tested in vitro with either direct or indirect assays for migration inhibitory factor (MIF). On the other hand, sera taken from nonsensitized guinea pigs after they had received 100 mg/kg of niridazole markedly diminished antigen-induced inhibition of migration of sensitized peritoneal exudate cells in vitro. The immunosuppressive effects of such sera could not be produced by niridazole itself, thereby suggesting an effect of niridazole metabolites. This suppressive activity was readily removed from the serum by dialysis. The active serum blocked the production of MIF by sensitized lymph node cells but did not affect the action of preformed MIF on macrophages. The effect of this serum was reversible; lymph node cells incubated for 24 hr with active serum, then washed and reincubated with antigen in normal serum, produced normal amounts of MIF. These studies suggest that metabolites of niridazole, but not the parent compound itslef, suppress delayed hypersensitivity in guinea pigs and prevent MIF production by lymphocytes without affecting the macrophage response to MIF.     

A heterophile antigen associated with experimental toxoplasmosis

The biological characteristics of a heterophile protein (HP) in peritoneal exudate from mice, hamsters, rats, and guinea pigs infected with Toxoplasma gondii were studied by immunofluorescence, immunoelectrophoresis and immunodiffusion techniques using specific antisera raised in rabbits. HP of mice had the highest antigenicity, HP of hamsters and rats had intermediate antigenicity and HP of guinea pigs had the lowest antigenicity. HP was found in normal peritoneal exudates from mice, hamsters and rats inoculated with paraffin oil instead of T. gondii and in normal guinea pig serum. HP was detected by the fluorescent antibody technique on the surface of T. gondii in peritoneal exudates of mice, but not on mouse peritoneal cells, and by the indirect fluorescent antibody technique on L cells infected with T. gondii and on free Toxoplasma derived from them, but not on uninfected L cells. T. gondii could make host cells produce HP to cover its surface for protection. The relation between HP from host cells and T. gondii is discussed.     

Effect of Corynebacterium parvum on the immune response in guinea pigs. II. Passive transfer of enhanced anamnestic response and delayed hypersensitivity observed after treatment with Corynebacterium parvum]

After intradermal immunization with a mixture of Corynebacterium parvum (C. parvum) and ovalbumin guinea pigs show a markedly increased anamnestic response to an intradermal booster of ovalbumin as compared to controls treated with ovalbumin only. At the same time a reaction of delayed type hypersensitivity is observed in the treated animals, but not in controls. The enhanced anamnestic response as well as the posivitive skin reaction were transferred to strain 2 histocompatible guinea pigs by peripheral blood leukocytes as well as by peritoneal exudate cells. Passive transfer was not obtained after prior irradiation of donor animals.     

RNA extracts with polyadenylic acid sequences transfer specific sensitivity for a low molecular weight antigen (MW 486).

RNA prepared from the lymphoid tissues of guinea pigs specifically sensitized to mono(p-azobenzene-arsonate)-N-chloroacetyl-l-tyrosine (ARSNAT) (MW = 486) was fractionated by oligo(dT)-cellulose affinity chromatography. Two fractions designated as I and II were eluted from the column. Fraction II, binding to the column and containing polyadenylic acid sequences, transferred ARSNAT or keyhole-limpet-hemocyanin (KLH) sensitivity to nonsensitized guinea pig peritoneal exudate cells (GP-PEC) in 14 experiments. Fraction I was unable to transfer KLH or ARSNAT sensitivity to GP-PEC. The amount of Fraction II needed to transfer ARSNAT sensitivity was 10 times less than previously reported. Synthetic nonlymphoid cell poly(A) tested in this system failed to transfer ARSNAT or KLH sensitivity to GP-PEC. Both Fractions I and II were inactivated by ribonuclease. The results suggest a possible messenger function for the poly (A)-containing RNA fractions.     

Amblyomma americanum: requirement for host Fc receptors in antibody-mediated acquired immune resistance to ticks

Guinea pig recipients of anti-tick immune serum or immune peritoneal exudate cells expressed 25 and 30% tick rejection, respectively, when challenged with Amblyomma americanum larval ticks. Previous studies have shown that IgG1 antibodies are responsible for the ability of immune serum to transfer resistance to ticks and to mediate the accompanying, and required, cutaneous basophil response. Since IgG1 antibodies induce mast cell-mediated passive cutaneous anaphylaxis and cutaneous basophil responses by interaction with cell surface Fc receptors, we investigated whether host Fc receptors were involved in the mechanism of antibody-mediated immune resistance to ticks. Recipients of immune serum pretreated intravenously with rabbit IgG failed to express resistance when challenged. In contrast, recipients of immune peritoneal exudate cells similarly pretreated expressed normal resistance. Sheep IgG had no inhibitory effect on the transfer of resistance by either immune serum or peritoneal exudate cells. Furthermore, recipients of immune serum pretreated with the Fc fragment from papain digestion of rabbit IgG failed to express resistance when challenged with ticks. Rabbit Fab and sheep Fc and Fab had no effect on the transfer of resistance by immune serum. Purity of rabbit Fc preparations was verified by the ability to inhibit mast cell-mediated passive cutaneous anaphylaxis due to high-titered IgG1 antiovalbumin antibodies. Rabbit Fab and sheep Fc fractions did not inhibit passive cutaneous anaphylaxis reactions. These findings suggest that immunoglobulin Fc receptors on host cells, such as mast cells and basophils, are required for antibody-mediated immune rejection of ticks from guinea pigs.     

Killing effect of normal peritoneal exudate cells in guinea pigs on microfilariae of Dirofilaria immitis evoked by passive transfer of immune humoral factor.

Parasiticidal activity of normal peritoneal exudate cells on microfilariae of Dirofilaria immitis in diffusion chambers implanted into normal guinea pigs was evoked by intraperitoneal passive transfer of anti-D. immitis serum. The immune serum was fractionated into supernatant and sediment by ammonium sulfate precipitation. The parasiticidal effect was reproduced with the supernatant and, to a less extent, with the sediment. Anti-D. immitis serum from the animals sensitized 5 days before was also effective in this respect. From these results it can be concluded that the factor responsible for the phenomenon is some other factor(s) than the antibody. In addition, the in vitro cytotoxicity test demonstrated an enhanced Mf-killing activity of sensitized peritoneal exudate cells by adding with anti-D. immitis serum.     

BN大鼠和豚鼠对卵清白蛋白致主动过敏反应的免疫学特性比较

目的 比较BN大鼠和豚鼠对卵清白蛋白(OVA)致敏前后机体免疫学特性的变化.方法 BN大鼠和豚鼠分别用OVA(每只1 mg)隔日致敏(i.p.),共5次;于末次致敏第10天以OVA(每只2 mg)激发致敏(i.v.);分别设正常对照组和OVA致敏组.于激发致敏后1h处死动物,分离腹腔肥大细胞、脾脏和骨髓,并制备脾脏和骨髓淋巴细胞.以annexin-V作为标志检测肥大细胞活性,同时以Fluo-3/AM标记胞内钙离子,检测钙离子水平;以PHA和LPS作为有丝分裂原,分别检测脾脏和骨髓T、B淋巴细胞活性.结果 ①致敏BN大鼠和豚鼠脾脏及骨髓T、B淋巴细胞活性均升高,其中骨髓淋巴细胞活性BN大鼠显著高于豚鼠,脾脏淋巴细胞活性两种属间差异无显著性;②致敏后,腹腔肥大细胞活性两种属间差异无显著性,但BN大鼠致敏后是致敏前的6倍,豚鼠是3倍;③肥大细胞内钙离子水平两种属致敏后均升高,豚鼠致敏前后钙离子水平具有统计学意义.结论 OVA致敏后,BN大鼠骨髓淋巴细胞活性明显高于豚鼠,豚鼠肥大细胞内钙离子较BN大鼠升高明显,肥大细胞活性两者无明显差异.因此,在实验中可以根据两种属在过敏反应中的特点以及具体的实验要求选择动物模型.     

Passive immunization protects guinea pigs from lethal toxoplasma infection

Abstract The cellular and humoral interactions that contribute to protective immunity in toxoplasmosis were studied by adoptive transfer of selective cell populations or immune serum and its fractions into normal syngeneic strain 2 guinea pigs. The results of this study with the RH strain of Toxoplasma gondii confirm and extend the findings of previous studies by showing that the passive transfer of parasite-sensitized T cells or of immune serum from previously infected donors protected recipient guinea pigs against lethal toxoplasmosis. An additional key finding was that similar levels of complete protection against lethal infection occurred in guinea pigs receiving partially purified anti- Toxoplasma immunoglobulins or immune cells that had been enriched for B cells prior to transfer. Cells residing in the spleen, lymph nodes and peritoneal cavity, but not the thymus, were equally effective in conferring immunity to challenged recipients. In addition, cell titration experiments revealed that guinea pigs could survive T. gondii infection by infusing them with as little as 2 × 10⁷ sensitized T cells or B cells. Unlike protection mediated by T cells, protection against lethal disease occurring in the B cell recipients was associated with the formation of Toxoplasma antibodies. These findings illustrate the major role of both humoral and cell-mediated immunity in affording protection against toxoplasmosis based on a guinea pig model of the human disease.     

Detection of guinea pig macrophages by a new CD68 monoclonal antibody, PM-1K

A new monoclonal antibody, PM-1K, was raised against 24-h cultured human peritoneal macrophages. In immunohistochemical assays, PM-1K recognized freshly isolated blood monocytes and most tissue macrophages as well as myeloid dendritic cells such as Langerhans cells and interdigitating cells. The molecular size of the antigen recognized by PM-1K was determined to be 110 kD by means of immunoaffinity purification. Because this affinity-purified antigen recognized by PM-1K was also recognized by anti-CD68 antibodies, it is believed to be one of the heterogeneous molecules of the CD68 antigen. Analysis showed interspecies reactivity of PM-1K with macrophages from guinea pigs, pigs, bovine species, and monkeys. Among these macrophages, those of the guinea pig reacted strongly with PM-1K. Patterns of PM-1K immunostaining in guinea pig tissues were similar to those found in human tissues. Studies with the immunoelectron microscope revealed reaction products of PM-1K in the cytoplasm, especially around endosomes. Since only a few antibodies are available to label guinea pig macrophages, PM-1K is considered to be one of the most suitable antibodies to examine macrophages in experimental guinea pig models.     

Species heterogeneity of hepatic alpha 1-adrenoceptors: alpha 1A-, alpha 1B- and alpha 1C-subtypes.

alpha 1-Adrenergic activation stimulated phosphorylase and phosphoinositide turnover in hepatocytes from guinea pigs, rats and rabbits. Chlorethylclonidine inhibited these effects in rat and rabbit cells but not in guinea pig hepatocytes; low concentrations of 5-methyl urapidil blocked the alpha 1 actions in guinea pig and rabbit liver cells, but not in rat hepatocytes. Binding competition experiments also showed high affinity for 5-methyl urapidil in liver membranes from guinea pigs and rabbits and low affinity in those from rats. The data indicated that guinea pig hepatocytes express alpha 1A-, rat hepatocytes alpha 1B- and rabbit hepatocytes alpha 1C- adrenoceptors. This was confirmed by Northern analysis using receptor subtype-selective probes.     

Antibodies to guinea pig lymphokines. II. Suppression of delayed hypersensitivity reactions by a "second generation" goat antibody against guinea pig lymphokines.

A "second generation" antibody to a highly purified lymphocyte product was raised in a goat against material eluted from a rabbit anti-guinea pig lymphokine immunoadsorbent column. This anti-lymphokine serum, in constrast to anti-lymphocyte serum (ALS) did not appear to contain cytotoxic antibodies directed against membrane antigens on guinea pig lymph node lymphocytes. Furthermore, the anti-lymphokine serum did not inhibit the formation of spontaneous T rosettes nor significantly depress lymphocyte response to mitogens. The anti-lymphokine serum totally suppressed the delayed skin reactivity to PPD and contact sensitivity to DNCB when injected intradermally around the site of antigen challenge. By contrast, intradermally injected ALS did not appear to suppress the PPD response in sensitized guinea pigs. Intravenously and i.p. administered anti-lymphokine serum was somewhat less effective in suppressing the delayed skin response to PPD. The intradermal injection of the antiserum had no effect on nonspecific inflammation evoked by turpentine-olive oil or on the extravasation of circulating Evans blue evoked by intradermally injected histamine. Histologic examination of 24-hr DNCB-induced skin lesions from sensitized guinea pigs treated with intradermally injected anti-lymphokine serum showed marked reduction of mononuclear infiltration of the dermis and of epidermal lesions, as compared with skin sites taken from sensitized animals pretreated with normal goat serum. The anti-lymphokine serum injected i.v. also markedly reduced the perivascular infiltration of the dermis and subcutis in skin reaction sites from sensitized animals challenged with PPD. Intravenous treatment with ALS for 3 consecutive days caused extensive depletion of the paracortical areas of peripheral lymph nodes whereas treatment with normal serum and anti-lymphokine serum caused no such depletion. It is proposed that the anti-lymphokine serum is directed against activated lymphocyte products, one of them being MIF. These products are involved in the mediation of delayed hypersensitivity reactions. This is in marked contrast to ALS, the suppressive action of which appears to be central rather than peripheral.     

Modulation of adoptive cell transfers in experimental allergic encephalomyelitis by antigen treatment of donors

Experimental allergic encephalomyelitis has been adoptively transferred using lymph node cells from Strain 13 guinea pig donors sensitized with purified encephalitogenic myelin basic protein. Adoptive cell transfer was used to examine the immunocompetence of lymph node cells obtained from guinea pigs protected from disease development by treatment with MBP. Lymph node cells from guinea pigs unresponsive to EAE challenge do not adoptively transfer disease. Cells obtained from guinea pigs treated with MBP following encephalitogenic challenge are competent in adoptive transfer with respect to pathologic lesions, but not clinical disease. The clinical and pathologic responses of recipients of the histocompatible lymphocyte populations are similar to those seen in the treatment-matched donor controls, suggesting that under these circumstances lymphoid cells, rather than circulating soluble factors, are responsible for disease induction and suppression.     

In vitro studies on transplantation immunity. II. The migration inhibition in homograft reactions in guinea pigs

The migration of peritoneal exudate cells from guinea pigs exhibiting transplantation immunity is inhibited in the presence of donor antigens. This inhibition of migration is demonstrable whether the donor transplantation antigens are presented in the form of viable cells (peritoneal exudate cells) or as particulate subcellular antigens (spleen microsomes). A greater degree of inhibition was observed when transplantation immunity was induced with lymphoid cells in Freud's adjuvant compared to sensitization with orthotopic skin grafts. There was no inhibition of migration in mixtures of normal allogeneic cells or when peritoneal cells from guinea pigs exhibiting tuberculin hypersensitivity were mixed with similar cells from normal animals. Finally, supernatants from cultures of sensitive lymphocytes plus donor antigens inhibited the migration of normal peritoneal cells indicating the presence of migration inhibitory factor (MIF) activity.