Antibody-mediated and delayed-type hypersensitivity reactions to Brucella skin test antigens in guinea pigs.

Cutaneous hypersensitivity responses to brucella antigens of different composition were studied in guinea pigs sensitized by infection with smooth brucella or immunization with killed rough brucella in adjuvant. These animals had circulating antibodies to smooth lipopolysaccharide or protein antigens, respectively. Intradermal skin tests, active cutaneous anaphylaxis, passive cutaneous anaphylaxis, and immunodiffusion tests were performed. Delayed-type hypersensitivity reactions uncomplicated by accompanying antibody-mediated reactions were seen only in infected guinea pigs with protein antigen that was entirely free of lipopolysaccharide. In the adjuvant-immunized animals, the protein antigen evoked overlapping antibody-mediated and delayed-type reactions. Lipopolysaccharide and polysaccharide preparations contained varying amounts of protein components. In infected animals, reactions of these antigens were clearly antibody mediated, but participation of delayed-type hypersensitivity could not be excluded. In adjuvant-immunized animals, the antibody-mediated reaction to the lipopolysaccharide preparation was caused by its protein component.     

Relationship between the tuberculin-type and Jones-Mote-type hypersensitivities: suppression of basophil infiltration by mycobacterial adjuvant

Guinea-pigs immunized with bovine gammaglobulin (BGG) in incomplete Freund's adjuvant (IFA) showed the typical Jones-Mote-type hypersensitivity (JMH) reaction when tested 5 days later. This is characterized by prominent basophil infiltration. After pretreatment with complete Freund's adjuvant (CFA) 16 days before immunization with BGG in IFA, the lesions resembled the JMH reaction macroscopically in their evolution over time and in the absence of a positive macrophage migration inhibition (MIT) test. However, histologically, the lesions resembled classical tuberculin-type hypersensitivity with prominent mononuclear cell infiltration without any basophils. The pretreated animals, which failed to show basophil infiltration, were able to transfer JMH reactions with basophil infiltration into normal animals. In contrast, pretreatment of recipients with CFA or Corynebacterium parvum prevented the passive transfer of the characteristic effect on the JMH reaction when given shortly before skin testing. We postulate that macrophages activated by CFA may play an important role in regulating basophil infiltration in the effector phase of the delayed hypersensitivity reaction.     

Basophil hypersensitivity response in rabbits.

A cutaneous basophil hypersensitivity response has been observed in rabbits immunized with bovine serum albumin and challenged intradermally with this antigen 7 days later. The cellular response appears to be similar to cutaneous basophil hypersensitivity reported in guinea pigs and humans. A basophil response was also observed in rabbits immunized with Staphylococcus aureus and challenged with viable staphylococcal cells 7 days later. A method of observing basophil infiltration in rabbits by means of connective tissue spreads obtained from the subcutaneous connective tissue is described. The rabbit should serve as an excellent model for the study of basophil responses as these animals have a significant basophil component with few if any tissue mast cells which may be confused both morphologically and functionally with the basophil.     

Impaired delayed hypersensitivity in germ-free guinea-pigs

The delayed-type hypersensitivity response of germ-free guinea-pigs was found to be defective. Whereas almost all conventionally reared guinea-pigs became hypersensitive to an allergenic hapten (picryl chloride), most germ-free guinea-pigs did not. When injected with a fully antigenic substance, bovine γ-globulin (BGG), none of the germ-free animals acquired BGG-specific delayed hypersensitivity. Further, none of the germ-free guinea-pigs developed spontaneous iso-hypersensitivity for a beta globulin as do conventional guinea-pigs. In addition, germ-free guinea-pigs given Freund's complete adjuvant did not develop the characteristic induration or erythema normally seen at injection sites and most animals died within 21 days.

Germ-free guinea-pigs given competent lymphoid cells from highly sensitized conventional guinea-pigs were unable to translate adoptive hypersensitivity into delayed dermal reactions.

A permeability factor in aged guinea-pig sera, injected into the skin of germ-free and conventional animals to determine whether the skin of germ-free guinea-pigs was able to support reactions, initiated immediate dermal reactions of equal intensity in both sets of animals.

     

A Histological Study of the Early Stages of the Development of the Tuberculin Reaction after Passive Transfer of Cells Labelled with [3H]Thymidine

A comparison has been made of the histology of the lesions developing as a result of the injection of PPD (protein purified derivative of tuberculin) or HGG (human γ globulin) into the skin of normal guinea-pigs and those passively sensitized with lymphoid cells to PPD or actively sensitized with antigen—antibody precipitates to HGG. Little significant difference was found until 12 hours after skin test.

[³H]thymidine was injected into tuberculin-sensitized donor guinea-pigs. 24 hours later spleen cells from these donors were injected into recipient guinea-pigs. These recipients had been previously actively sensitized so as to show delayed-type hypersensitivity to HGG. The arrival of the labelled cells in lesions produced in the recipient by tuberculin (passively transferred delayed-type hypersensitivity) and by HGG (actively induced delayed-type hypersensitivity) was compared at intervals up to 12 hours. No significant difference was found between the arrival of labelled cells in passively induced tuberculin lesions and actively induced HGG lesions.

     

Delayed-type hypersensitivity measured as an increase of ear thickness in guinea pigs.

Determination of swelling at an intracutaneous test site in the pinna of the ear of guinea pigs immunized with protein antigens in complete Freund's adjuvant was found to be a more sensitive assay of delayed-type hypersensitivity than the measurement of flank skin erythema and induration. In fact, 100 times less specific antigen was needed to detect 24-hour reactivity in the pinna of the ear. Reactivity early after sensitization (cutaneous basophil hypersensitivity), however, was best revealed as an erythematous lesion of the flank skin 24 h after testing.     

Cellular Immunity to Listeria monocytogenes Induced by Sensitization and Challenge with Bovine Gamma Globulin

Resistance to Listeria monocytogenes was demonstrated in guinea pigs undergoing systemic delayed-type hypersensitivity reactions to bovine gamma globulin.     

Cutaneous basophil hypersensitivity and inhibited macrophage migration in guinea-pigs with schistosomiasis

In guinea-pigs infected with schistosomes, delayed cutaneous reactions rich in basophils (CBH) were found to characterize skin test responses to schistosome egg antigens. In addition, strong contact hypersensitivity-like skin eruptions with large basophil infiltrates resulted from skin penetration challenge by live cercariae (larvae) in these animals. Oedema and diminished basophil granule staining were noted around schistosomula which had penetrated the skin of sensitized animals. CBH responses to egg antigens and to live cercarial challenges were also noted after immunization with a single injection of dead cercariae.

Using peritoneal exudates from guinea-pigs immunized with dead cercariae or infected with schistosomes, direct macrophage migration inhibition with schistosome antigens was found only in animals with infections. Thus, CBH correlated with intradermal exposure to schistosome cercarial antigens, while MMI correlated with live infections. It is suggested that cutaneous basophil responses may play a role in protection from re-infection with schistosomes, and that dead cercarial vaccines might stimulate this beneficial response, without immunizing for potentially harmful granulomatous hypersensitivity.

     

Dissociation of erythema and basophil accumulation in Jones-Mote type hypersensitivity in the guinea-pig.

Relationship between delayed-in-onset erythema and infiltrating leucocytes in the skin reaction site of Jones-Mote type hypersensitivity was observed in guinea-pigs. Guinea-pigs were immunized with formalin-fixed (F-SRBC) or native form (N-SRBC) of sheep red blood cells in incomplete Freund''s adjuvant (IFA). Elicitation was carried out with intradermal injection of F-SRBC or N-SRBC in saline 1 or 3 weeks after immunization. One week after immunization with F-SRBC or N-SRBC in IFA, erythema accompanied by basophil infiltration was detected, but antibody production was negative. Erythema reached a peak at 24 h, but basophil infiltration reached a peak at 48 h or later. In the skin reaction site elicited with N-SRBC, high levels of basophil infiltration were detected persistently even after erythema had disappeared. Three weeks after immunization, low titres of PCA (passive cutaneous anaphylaxis) were detected in guinea-pigs immunized with F-SRBC in IFA. At that time erythematous skin reaction was detected, but the level of basophil infiltration was lower than that of 1 week after immunization. In guinea-pigs immunized with N-SRBC in IFA, skin reaction of the Arthus type accompanied by haemorrhage was observed at the site elicited with N-SRBC.     

P-selectin directs T lymphocyte-mediated injury in delayed-type hypersensitivity responses: studies in glomerulonephritis and cutaneous delayed-type hypersensitivity

The role of P-selectin in T lymphocyte accumulation and injury was studied in delayed-type hypersensitivity (DTH) responses in the skin and glomeruli of rats. Sprague Dawley rats were sensitized to sheep globulin and challenged 5 days later in the skin by subcutaneous injection and simultaneously in glomeruli by intravenous injection of a subnephritogenic dose of sheep anti-rat glomerular basement membrane globulin. This resulted in cutaneous and glomerular T lymphocyte-dependent macrophage influx and injury characteristic of DTH. Up-regulation of P-selectin expression on endothelial cells was observed in both inflammatory lesions. Treatment of rats with anti-CD5 antibody immediately prior to antigen challenge prevented the development of injury as assessed by measurement of proteinuria and skin swelling, as well as local T cell and macrophage accumulation in the glomerulus and in the skin, but did not block up-regulation endothelial cell P-selectin. Treatment with anti-CD4 antibody produced similar results. Blocking P-selectin in vivo with a functionally inhibitory antibody prevented development of proteinuria and skin swelling following antigen challenge. Local accumulation of T cells and macrophages was markedly attenuated in glomeruli and the skin and up-regulation of endothelial cell P-selectin was prevented. These data demonstrate that P-selectin is locally up-regulated on endothelial cells in T cell-dependent glomerular and cutaneous inflammation and suggests a pivotal functional role for P-selectin in local T cell recruitment and subsequent injury in DTH.     

Delayed-type hypersensitivity responses in infected mice elicited by cytoplasmic fractions of Cryptococcus neoformans.

Four subcellular fractions of Cryptococcus neoformans prepared by differential centrifugation of disrupted whole yeast and a 3-day culture filtrate were examined for their ability to elicit delayed-type hypersensitivity in sensitized animals. The methods used to detect sensitization were (i) the footpad swelling test and inhibition of peritoneal macrophage migration in mice and (ii) skin testing in guinea pigs. Two entities, the post-mitochondrial supernatant and the culture filtrate, showed considerable activity in the footpad test, with 26- and 30-microliter 24-h swellings, respectively, at 6 weeks after infection. With the latter there was interference from a strong antibody-mediated 4-h skin reaction. The post-mitochondrial supernatant produced strong delayed-type hypersensitivity in guinea pigs at a dose of 69 microgram, and there was no demonstrable cross-reactivity in animals sensitized with heterologous fungi. The footpad swelling in mice correlated well with the macrophage migration inhibition test, with 71% inhibition in mice infected subcutaneously with C. neoformans at 6 weeks. However, mice infected intravenously developed poorer cell-mediated immunity than the subcutaneously infected mice. The post-mitochondrial supernatant was found to contain detectable amounts of cryptococcal capsular polysaccharide.     

Cell-mediated and humoral immune responses in mice. I. Necessary conditions for the detection of delayed-type hypersensitivity.

Footpad assay of delayed-type hypersensitivity to bovine serum albumin (BSA) was studied. Injection of saline solution of BSA (S-BSA) into footpads of sensitized mice resulted in the development of only the local Arthus-type reaction. Injection of alumprecipitated BSA (AP-BSA), however, was effective in generating both Arthus- and delayed-type reactions. The difference between S-BSA and AP-BSA in the ability of elicitation of delayed-type reaction may be attributable to the difference in the retention of these two forms of antigen in the footpad, since S-BSA was found to be eliminated much more rapidly from footpads than AP-BSA.     

Contact sensitivity to oxazolone in the chicken: evidence for Arthus type hypersensitivity of the cutaneous reaction.

Cutaneous hypersensitivity reaction can be induced in chickens by skin painting with oxazolone, 33 mg/Kg of body weight (KBW). The B cell contribution to the generation of the cutaneous reaction has been a matter of controversy. In an attempt to characterize this reaction we placed special interest on the possibility that the nature of this reaction could be Arthus type hypersensitivity. From the kinetics study on the cutaneous hypersensitivity after challenge with oxazolonated egg-albumin (EA-OX) it was excluded that the nature of this reaction could be delayed type hypersensitivity. Immune sera transfer experiments demonstrated that the cutaneous reaction was antibody dependent. Serum anti-oxazolone antibody titers in sensitized chickens were assayed by antiglobulin haemagglutination, using oxazolone coupled sheep erythrocytes (OX-SRBC). High titres of IgG were found in contact sensitized chickens. Furthermore this cutaneous reaction was characterized by neutrophils, inflammatory edema, rare thrombotic occlusion of small venules and on absence of monocytes. The utilization of complete Freunds' adjuvant (CFA) given at sensitization demonstrated that CFA enhanced oxazolone antibodies in the sera of immunized chickens without a correlated increase in the intensity of the cutaneous reaction to EA-OX. Animals sensitized to oxazolone (33 mg/KBW) without CFA and challenged intravenously seven days later with oxazolone coupled to autologous chicken red blood cells (OX-CRBC) died from anaphylactic shock; instead animals with the same treatment but with CFA given at sensitization did not die from anaphylactic shock. Taken collectively it was concluded that the cutaneous reaction to oxazolone in the chicken can be categorized as Arthus hypersensitivity. The relationship between cutaneous Arthus reaction and anaphylactic shock in chickens sensitized to oxazolone is discussed.     

Contact Sensitivity to Oxazolone in the Chicken: Evidence for Arthus Type Hypersensitivity of the Cutaneous Reaction

Cutaneous hypersensitivity reaction can be induced in chickens by skin painting with oxazolone, 33 mg/Kg of body weight (KBW). The B cell contribution to the generation of the cutaneous reaction has been a matter of controversy. In an attempt to characterize this reaction we placed special interest on the possibility that the nature of this reaction could be Arthus type hypersensitivity. From the kinetics study on the cutaneous hypersensitivity after challenge with oxazolonated egg-albumin (EA-OX) it was excluded that the nature of this reaction could be delayed type hypersensitivity. Immune sera transfer experiments demonstrated that the cutaneous reaction was antibody dependent. Serum anti-oxazolone antibody titers in sensitized chickens were assayed by antiglobulin haemagglutination, using oxazolone coupled sheep erythrocytes (DX-SRBC). High titres of IgG were found in contact sensitized chickens. Furthermore this cutaneous reaction was characterized by neutrophils, inflammatory edema, rare thrombotic occlusion of small venules and on absence of monocytes. The utilization of complete Freunds' adjuvant (CFA) given at sensitization demonstrated that CFA enhanced oxazolone antibodies in the sera of immunized chickens without a correlated increase in the intensity of the cutaneous reaction to EA-OX. Animals sensitized to oxazolone (33 mg/KBW) without CFA and challenged intravenously seven days later with oxazolone coupled to autologous chicken red blood cells (OX-CRBC) died from anaphylactic shock; instead animals with the same treatment but with CFA given at sensitization did not die from anaphylactic shock. Taken collectively it was concluded that the cutaneous reaction to oxazolone in the chicken can be categorized as Arthus hypersensitivity. The relationship between cutaneous Arthus reaction and anaphylactic shock in chickens sensitized to oxazolone is discussed.     

The Fate of [131I]Labelled Antigens in the Skin of Normal Guinea-Pigs and those with Delayed-Type Hypersensitivity

A comparison was made of the retention of three antigens PPD (protein purified derivative of tuberculin), HSA (human serum albumin) and HGG (human γ globulin) labelled with ¹³¹I in the skin of normal guinea-pigs. It was found that PPD was eliminated more rapidly than HSA or HGG in the first 24 hours after skin test. In contrast PPD was found to be more readily adsorbed to collagen and cellular tissues in vitro.

The retention of these antigens at 24 hours in normal and sensitized guinea-pigs was also compared. It was found that PPD was retained in the tuberculin lesion as compared with normal skin, whereas HSA was eliminated more rapidly from a delayed-type hypersensitivity lesion to HSA when compared with normal skin. It was concluded that retention of PPD was probably not specific to delayed-type hypersensitivity but was related to the difference in character and severity of the inflammatory process, which itself was related to the chemical nature of the antigen involved.

     

Cutaneous basophil anaphylaxis. Immediate vasopermeability increases and anaphylactic degranulation of basophils at delayed hypersensitivity reactions challenged with additional antigen.

Many delayed-type reactions contain large infiltrates of basophils whose function is unknown. We have studied these cutaneous basophil hypersensitivity (CBH) reactions in guinea-pigs to ascertain whether basophils that are recruited to delayed reaction sites could be triggered for immediate reactivity. We compared 24 h CBH reactions with nearby skin for immediate hypersensitivity by challenging each site with small amounts of antigen. CBH sites had augmented immediate increases in vascular permeability detected by extravasation of Evan's blue dye. The ability to elicit this augmented anaphylactic phenomenon correlated with the local presence of basophils, and light microscopy at CBH reactions 15 min after antigen challenge showed a 50% decline in basophil counts. Electron microscopy showed that progressive anaphylactic-type degranulation of local basophils occurred within minutes following reintroduction of antigen. There was fusion of vacuoles containing granules, exocytosis of granules, and dissolution of granules, without ultrastructural disruption of cellular integrity. These results establish that basophils in CBH reactions can be triggered with soluble antigen to undergo anaphylactic degranulation, with the immediate release of vasoactive mediators. We have termed this phenomenon 'cutaneous basophil anaphylaxis'. Thus, one function of basophils at sites of delayed hypersensitivity may be to provide the potential for augmented, local, immediate anaphylactic reactivity.     

Cutaneous basophil hypersensitivity in atopic dermatitis

The infiltration by basophils into delayed hypersensitivity skin lest sites was examined in patients with atopic dermatitis, contact dermatitis and in normal healthy persons. Atopic dermatitis patients, with large amounts of IgE, injected intradermally with staphylococcal antigens showed reactions that were more transient and erythemaious than those of normal persons. On hislological examination there were numerous, degranulating basophils among the perivascular mononuclear cells. Normal persons, with small amounts of IgE showed typical mononuclear cell infiltration and few basophils. Patients with contact dermatitis, one with much IgE, responded to patch tests to potassium dichromate or to nickel sulphate by delayed type reactions, but on histology, two of the four patients showed a significant infiltration by basophils. It is considered that the erythematous response to the antigen in atopic dermatitis patients is related to the basophil infiltration.     

Brucella ribosomal protein L7/L12 is a major component in the antigenicity of brucellin INRA for delayed-type hypersensitivity in brucella-sensitized guinea pigs.

A delayed-type hypersensitivity (DTH) reaction in the course of brucellosis in humans and animals can be revealed by the brucellin INRA (Brucellergen) skin test. Brucellergen is composed of more than 20 proteins of different molecular weights. A 12-kDa protein eliciting DTH in Brucella melitensis Rev1-sensitized guinea pigs was found to be a significant component for the allergenic properties of Brucellergen. Sequencing of the gene encoding this protein identified it as the L7/L12 ribosomal protein. The L7/L12 gene of B. melitensis was amplified by PCR and subcloned in the Escherichia coli pQE30 plasmid. The resulting recombinant protein did not produce a DTH reaction in sensitized animals. It was used to raise specific antibodies in a rabbit. Affinity chromatography with these antibodies was used to isolate a single protein from Brucellergen and from B. melitensis cytosol preparations which produced a DTH reaction in guinea pigs sensitized with B. melitensis Rev1. N-terminal amino acid sequencing of the protein confirmed that it was the L7/L12 ribosomal protein. This is the first complete report on the involvement of a defined bacterial ribosomal protein in the DTH response of animals infected with intracellularly multiplying bacteria.     

Contact Sensitivity to Oxazolone in the Chicken: Evidence for Arthus Type Hypersensitivity of the Cutaneous Reaction

Abstract

Cutaneous hypersensitivity reaction can be induced in chickens by skin painting with oxazolone, 33 mg/Kg of body weight (KBW). The B cell contribution to the generation of the cutaneous reaction has been a matter of controversy. In an attempt to characterize this reaction we placed special interest on the possibility that the nature of this reaction could be Arthus type hypersensitivity. From the kinetics study on the cutaneous hypersensitivity after challenge with oxazolonated egg-albumin (EA-OX) it was excluded that the nature of this reaction could be delayed type hypersensitivity. Immune sera transfer experiments demonstrated that the cutaneous reaction was antibody dependent. Serum anti-oxazolone antibody titers in sensitized chickens were assayed by antiglobulin haemagglutination, using oxazolone coupled sheep erythrocytes (DX-SRBC). High titres of IgG were found in contact sensitized chickens. Furthermore this cutaneous reaction was characterized by neutrophils, inflammatory edema, rare thrombotic occlusion of small venules and on absence of monocytes. The utilization of complete Freunds' adjuvant (CFA) given at sensitization demonstrated that CFA enhanced oxazolone antibodies in the sera of immunized chickens without a correlated increase in the intensity of the cutaneous reaction to EA-OX. Animals sensitized to oxazolone (33 mg/KBW) without CFA and challenged intravenously seven days later with oxazolone coupled to autologous chicken red blood cells (OX-CRBC) died from anaphylactic shock; instead animals with the same treatment but with CFA given at sensitization did not die from anaphylactic shock. Taken collectively it was concluded that the cutaneous reaction to oxazolone in the chicken can be categorized as Arthus hypersensitivity. The relationship between cutaneous Arthus reaction and anaphylactic shock in chickens sensitized to oxazolone is discussed.