Structure-activity profile of substituted purines and inflammation in the delayed hypersensitivity skin reaction

Substituted purines were tested for their effectiveness in inhibiting the delayed hypersensitivity skin reaction (DHSR) caused by tuberculin in the guinea-pig. Among the tested purines were naturally occurring derivatives of guanine and adenine, including cyclic AMP. Based on the structure-activity profile, a class of purines was identified, the members of which were very effective inhibitors of inflammatory aspects of the DHSR and are characterized by a benzyl group in position 9, an amino or alkylamino group in position 6, and various substituents in position 2. This class of 2-substituted-9-benzyladenines was more effective in the DHSR than some antimetabolites, particularly the structurally related mercaptopurines.     

Random monocyte migration: an in vitro correlation with the delayed hypersensitivity skin reaction.

The in vitro random migration and chemotactic activity of peripheral blood monocytes and neutrophils was compared with the delayed hypersensitivity skin test to streptokinase-streptodornase in fourteen normal subjects. A significant positive correlation (P less than 0.001) was observed between the random migration of monocytes and the size of the skin test reaction. No significant correlation was found with random neutrophil migration or monocyte and neutrophil chemotactic responses. These results indicate that the in vitro random mobility of monocytes is related to the in vivo expression of a delayed-type hypersensitivity skin reaction.     

Interaction among IgE-mediated hypersensitivity reaction, PCA reaction and delayed hypersensitivity reaction (at local skin sites of monkeys).

The effect of the IgE-mediated reaction on the passive cutaneous anaphylaxis (PCA) reaction was studied at local skin sites of monkeys, and we found that the IgE-mediated reaction appeared to enhance the PCA reaction. Interactions among IgE-mediated reaction, PCA reaction and delayed hypersensitivity reaction were also determined. Contact dermatitis induced with DNCB was utilized as the delayed hypersensitivity reaction. The IgE-mediated reactior or PCA reaction, as well as simple serum irritation, enhanced the delayed hypersensitivity reaction. It is thus assumed that the IgE-mediated reaction enhances the PCA reaction and that this in turn accelerates the delayed hypersensitivity reaction.     

Immunological studies on delayed hypersensitivity to phenytoin--II. The delayed skin reaction induced by BSA-phenytoin conjugate

Remarkable swelling of the foot pad was induced in guinea pigs sensitized with BSA-phenytoin by challenging with BSA-phenytoin or EA-phenytoin. Forty-eight hours after local injection of a mixture of exudate cells obtained from the abdominal cavity of sensitized guinea pigs. and BSA (EA)-phenytoin, both erythema and induration developed at the injection sites in normal guinea pigs. At the sites exhibiting these skin reactions, an exudation of mononuclear leukocytes was noted. In addition, macrophages obtained from the abdominal cavity of phenytoin-sensitized animals showed a low migration index in the presence of phenytoin. When phenytoin was administered to rats (p.o.), large amounts of the compound were detected in the gingiva and there was a good correlation between the tissue and serum phenytoin concentrations. These findings indicate that the etiology of gingival hyperplasia produced by chronic administration of phenytoin may be related in some way to a delayed-type hypersensitivity induced by the drug.     

Lymphocyte chemotaxis in inflammation. VIII. Demonstration of lymphocyte chemotactic lymphokines in PPD-induced delayed hypersensitivity skin reaction site in the guinea-pig.

The experiments were carried out to clarify whether lymphocyte chemotactic factors (LCFs) derived from activated lymphocytes, i.e. lymphocyte chemotactic lymphokines would exist in delayed-type hypersensitivity (DTH) reaction sites in guinea-pigs. To analyse the problem, we attempted to use an immunoadsorbent column conjugated with respective antibodies against LCFs (LCF-b, LCF-c and LCF-d) isolated from purified protein derivative (PPD)-induced DTH skin reaction sites in guinea-pigs. The chemotactic activity of culture supernatants from PPD- or concanavalin A (Con A)-stimulated lymph node (LN) cells was decreased to about 50% by the immunoadsorbent column with anti-LCF-c antibody or anti-LCF-d antibody, while its activity was little or not influenced by the columns with anti-LCF-b, anti-IgG or anti-IgM antibody. Further experiments using successive immunoadsorption with anti-LCF-c antibody followed by anti-LCF-d antibody showed almost the complete adsorption of the chemotactic activity in the above culture fluids. Additionally, the chemotactic lymphokine which was absorbed by anti-LCF-c antibody had a similar mol. wt. to that of LCF-c (mol. wt about 160,000). However, the chemotactic lymphokine which was absorbed by anti-LCF-d antibody had a mol. wt. of about 27,000; it was clearly distinct in mol. wt. from LCF-d (mol. wt. about 300,000). It is thus suggested that at least one of lymphocyte chemotactic lymphokines exists in the DTH reaction sites and functions as LCF-c.     

Role of thrombin and plasmin in development of delayed hypersensitivity reaction in guinea pig skin

Induration is a prominent feature of delayed hypersensitivity reaction (DHR), and fibrin deposition is the central mechanism. We studied the effects of two inhibitors of DHR on the activities of thrombin and plasmin in the extract of the skin site of DHR and compared the two activities in the site of DHR with those in the site of the Arthus reaction (AR) that lacks induration. Warfarin, an anticoagulant, inhibited thrombin activity and induration, but not plasmin activity. Ferritin, a blocker of a macrophage-dependent reaction, inhibited the two activities and induration. The lesion of DHR had three to four times the thrombin activity of the lesion of AR, and the activity paralleled the development of induration. In contrast, plasmin activity of the site of DHR was lower than that of the site of AR and was associated with the reduction of induration. The two protease activities in the site of AR did not correlate with the development of the AR lesion. These results suggest that thrombin and plasmin mediate the development of induration and that induration is produced by a synergistic effect of high thrombin activity and low plasmin activity in the site of DHR.This work was supported in part by grants from the Japanese Ministry of Education.     

Psychological modulation of the delayed type hypersensitivity skin test.

Considerable data have demonstrated that psychological states can influence the immune system in animals. Whether human immune function can be intentionally modulated by the central nervous system is unknown. This article presents data from two studies that sought to demonstrate intentional modulation of the immune system by psychological interventions. It also discusses the methodological complexities involved with this type of research in humans.     

Lymphocyte chemotaxis in inflammation. III. Demonstration of lymphocyte chemotactic activity in extract from PPD-induced delayed hypersensitivity skin reaction site in the guinea-pig.

Various skin extracts from inflammatory lesions in guinea-pigs were assayed for chemotactic activity for guinea-pig lymphocytes; distinct chemotactic activity was shown in extract from PPD-induced delayed hypersensitivity reaction sites, and the time-course of chemotactic activity almost paralleled the intensity of lymphocyte reaction in skin sites. However, extracts obtained from various control sites or passive Arthus reaction sites exhibited little chemotactic activity for the cells. It is therefore suggested that lymphocyte chemotactic factor(s) was produced in the soluble form in the delayed hypersensitivity reaction.     

Asymmetry of delayed type hypersensitivity reaction in mice

The intensity of delayed type hypersensitivity reactions in the left and right paws was studied in mice divided into left- and right-pawed by the motor asymmetry of the brain. The reaction was more pronounced in the left paw in all animals irrespective of motor asymmetry. Motor asymmetry of the brain hemispheres little contributed to the manifestation of differences in the delayed type hypersensitivity reactions in the left and right paws. The authors concluded that asymmetry of cellular immunity is determined by functional asymmetry of cells in regional lymph nodes.     

Lymphocyte chemotaxis in inflammation. VII. Isolation and purification of chemotactic factors for T lymphocytes from PPD-induced delayed hypersensitivity skin reaction site in the guinea-pig

Four types of lymphocyte chemotactic factor (LCF-a, -b, -c and -d) could be isolated from extract of 24-hr-old delayed-type hypersensitivity (DTH) skin reaction sites induced with purified protein derivative (PPD) in guinea-pigs by gel filtration on Sephadex G-100 followed by chromatography with DEAE-Sephadex. Partially purified LCF-b was thought to be a heat-stable protein with a molecular weight (mol. wt.) of about 14,000. LCF-c separated from LCF-d by chromatography with DEAE-Sephadex was highly purified by chromatography with CM-Sephadex, immunoadsorbent chromatography coupled with anti-IgG antibody, and chromatofocusing in that order. It was considered to be a heat-labile protein with a mol. wt. of about 160,000 and with pI of 8.1 +/- 0.2. LCF-d first separated from LCF-c was also highly purified by chromatography with CM-Sephadex followed by preparative isotachophoresis. The factor was considered to be a heat-labile protein with a mol. wt. of approximately 300,000 and with pI of 6.2 +/- 0.2. These factors were similarly active for non-adherent cells (mostly T cells) but not for cells (mostly B cells) adherent to anti-IgG antibody-coated petri-dishes. Since LCF-a was active for B cells as described earlier, it is thus suggested that LCF-b, LCF-c and LCF-d may be important for T cell migration in the DTH site to PPD.     

The role of protein component of T. pallidum in inducing skin reaction for delayed hypersensitivity

The protein component of T. pallidum was shown to be an important agent in inducing the skin reaction for delayed type of hypersensitivity (SDH) in syphilis. Treponema preparations which contained T. pallidum deprived of this component by treating the organisms with merthiolate, formalin or heat, were found unable to produce a distinct positive skin reaction in syphilitic rabbits. Using preparations containing treponemes with preserved protein component, it was possible to induce the skin reaction for DH in rabbits as early as 4 weeks after inoculation of T. pallidum and during the subsequent 64 weeks of the infection (and of the observation period). The SDH was fairly well correlated with the results of the MMI test in the whole course of the syphilitic infection; a disagreement of results of these two tests occurred only between the 16th and 26th week of the infection.     

Interference of simultaneous skin tests in delayed hypersensitivity.

The interference of two simultaneous skin test reactions of intermediate strength has been studied in the guinea-pig, using four different antigens, i.e. ovalbumin, horse cytochrome c, PPD and oxazolone. Skin test reactions were evaluated at 4, 24 and 48 h by measuring three parameters: increase in skin thickness, diameter of erythema and intensity of erythema. When an Arthus reaction was elicited simultaneously with a delayed hypersensitivity (DH) reaction, no effect on the DH reaction was observed. When two simultaneous DH reactions were elicited with different antigens, the risk of interference appeared to be rather small. When, however, the same antigen was used for both skin tests, suppression of at least one parameter of a DH-reaction was found in almost all experiments. Suppression of one skin test by another one could not be reduced by introducing a large distance between the two skin tests. As complete inhibition of either of the parameters never occurred, multiple skin testing may allow one to obtain a qualitative impression of the state of delayed hypersensitivity; when, however, reliable quantitative data are needed, the performance of more than one skin test at a time should be avoided.     

The chemical mediation of delayed hypersensitivity skin reaction. II. Characterization of a macrophage-chemotactic factor from bovine gamma-globulin-induced skin reaction in guinea pigs.

Macrophage-chemotactic factors were extracted from delayed hypersensitivity skin lesions induced by bovine gamma-globulin in guinea pigs. The most active factor, MCFS--1, was highly purified and found to be a heat-labile protein with a molecular weight of 150,000 and to possess in vivo as well as in vitro activity. This factor was homogeneous during polyacrylamide gel electrophoresis, and the chemotactic activity was associated exclusively with this band. Further characterization revealed that its isoelectric point was 6.7 to 6.9 and made a single arc in the beta-globulin region with rabbit antiserums against guinea pig serum on immunoelectrophoresis. This factor seemed to be antigenically different from immunoglobulin G (IgG) by immunodiffusion and immunoadsorption. On the other hand, the chemotactic activity of MCFS-2 was adsorbed by neither anti-IgG nor anti MCFS-1 and that of euglobulin fraction was partially adsorbed by anti-IgG. These indicate the presence of at least three types of antigenically different chemotactic factors for macrophages in the extracts of delayed hypersensitivity skin lesions.     

Enhancement of delayed skin hypersensitivity by neuraminidase in cancer patients

Delayed skin hypersensitivity to specific antigen was enhanced when the antigen was given intradermally with Vibrio cholerae neuraminidase in sensitive subjects. No enhancement of skin test response was seen when inactivated enzyme was used. Neuraminidase exhibited no effect on negative skin-test response in non-sensitive recipients and no reaction at the site of this enzyme was observed in any instance, suggesting that the enhancement effect is immunological in nature.     

The nature of retest reaction in delayed hypersensitivity. I. Light microscopic changes in the skin of the eyelid

The retest reaction is a variant of delayed hypersensitivity where in repeated application of an antigen at the same site leads to an intense eosinophil leucocytic infiltration. This reaction has been produced experimentally for the first time in the eyelids of rabbits and guinea-pigs and it is possible that such a mechanism is implicated in a variety of clinical ocular conditions in which IgE does not appear to feature. It was also found that tissue reaction was modified in the absence of suppressor cells, these having been temporarily inhibited by pretreatment of the animals with cyclophosphamide.     

Delayed hypersensitivity in mice induced by intravenous sensitization with sheep erythrocytes: evidence for tuberculin type delayed hypersensitivity of the reaction.

Delayed hypersensitivity (DH) reaction can be induced in mice by intravenous sensitization with sheep erythrocytes (SRBC). However, as the sensitizing procedure is quite different from a usual mode of sensitization for DH using complete Freund's adjuvant (FCA), the nature of this reaction has been a matter of controversy. In an attempt to characterize this reaction, we placed special interest on two possibilities regarding the nature of this reaction; Jones-Mote reaction or tuberculin type DH. From the kinetics study on the DH after challenge, the DH reaction to SRBC in mice by intravenous sensitization was clearly distinguished from the Arthus reaction. The dose-response pattern of this reaction also suggested that the contribution of Arthus reactivity to delayed reactivity was negligible. Cell reconstitution experiments revealed this DH to be quantitatively thymus cell dependent. Furthermore, this DH required macrophages at its manifestation stage, and appearance of basophil infiltration at the lesion was absent. In addition, strain difference and ageing of host mice influenced the DH reaction in exactly the same fashion in which these factors influence the tuberculin type-DH induced by subcutaneous sensitization with methylated human serum albumin (MHSA) in FCA. Taken collectively, it was concluded that this DH reaction can be categorized as the tuberculin type.