Development of a delayed-type hypersensitivity granuloma model in the mouse for the study of chronic immune-mediated inflammatory disease

A cutaneous model of delayed-type hypersensitivity granuloma (DTH GRA) was developed, using methylated bovine serum albumin (mBSA) which could be easily quantified and represented chronic cellular immune-mediated inflammation characteristic of rheumatoid arthritis (RA). Quantitative kinetics showed maximal DTH GRA wet tissue weights at d. 5 post-induction, diminishing between d. 15–22; DTH GRA dry tissue weights peaked between d. 8–22. Histological evaluation revealed early extensive fibrin deposition, edema, mixed PMN/mononuclear leucocyte infiltration and angiogenesis (d. 2–5), followed by increased macrophage recruitment, lymphocyte infiltration, angiogenesis and vasculitis (d. 10–15). Fibrin resorption and replacement by fibrous tissue resulted in resolution by d. 35. It is suggested that the DTH GRA reaction represents a relevant model for probing pathogenic mechanisms and potential therapeutics for RA.     

Quantitation of glycosaminoglycans of rabbit lung during delayed-type hypersensitivity reactions and granuloma formation

The specificity and kinetics of hyaluronic acid (HA) accumulation in relation to other glycosaminoglycans (GASs) were determined in rabbit lungs during an allergic granulomatous response to BCG, an allergic nongranulomatous response to tuberculoprotein, and during a foreign-body granulomatous response to carrageenan. Hyaluronic acid was the only GAG detected in the lung lavage fluids. Hyaluronic acid occurred in the airways on day two of the allergic granulomatous response, but its presence in the airway did not correlate with ensuing granuloma formation in the parenchyma. Generalized increases in GAG of the parenchyma also peaked on day two of the DTH responses. Generalized increases in GAG peaked on day five during the foreign-body granulomatous response to carrageenan. A persistently elevated level of HA in the lung tissue correlated with granuloma formation but not with the intensity of the response.     

A sensitive delayed-type hypersensitivity model in the rat for assessing in vivo cell-mediated immunity

Many drugs and other chemicals can alter cell-mediated immunity (CMI), a response that often correlates with delayed-type hypersensitivity (DTH). Several DTH assays were evaluated to determine a method best suited for assessing chemically induced modulation of CMI in rats. The effects of various antigens, adjuvants, doses, routes, and immunosuppressants were investigated. The DTH method which produced optimum results in rats uses a footpad swelling reaction elicited by specific preparations of bovine serum albumin (BSA) and Freund's complete adjuvant (FCA). The rats were sensitized with 100 μg BSA in FCA injected subcutaneously at the base of the tail, and were challenged 7 days later with 75 μl of 2% heat-aggregated BSA suspension injected into the left rear footpad. Footpad swelling was measured with pressure calipers 24 h later and compared to the contralateral footpad which was sham-injected with 75 μl of physiological saline. Antigen-injected footpads were nearly double the thickness (7–8 mm) of the control footpads (3–4 mm), and variation between animals was small (CV = 5%). Dexamethasone and cyclophosphamide significantly suppressed the DTH reaction. Histopathological examination of the DTH reaction sites revealed a mononuclear cell infiltrate which is characteristic of type IV hypersensitivity. In addition to being highly quantitative and sensitive, this method provides a simple and reproducible assessment of CMI responses in the rat.     

Regulation of delayed-type hypersensitivity IV. Antigen-specific suppressor cells for delayed-type hypersensitivity induced by lipopolysaccharide and sheep erythrocytes in mice.

Mice injected subcutaneously with 1 x 10(8) sheep red blood cells (SRBC) developed high levels of delayed-type hypersensitivity (DTH) to SRBC 4-8 days after injection. Such DTH was suppressed when 100 microgram lipopolysaccharide (LPS) was injected intravenously 1-2 days before or at the time of SRBC injection. This suppression of DTH was transferable by spleen, lymph node, thymus and bone marrow cells to sensitized or normal syngeneic recipients, but could not be transferred by serum. Suppressor cells were not induced by LPS alone or SRBC alone, and they were antigen-specific since DTH to chicken red blood cells was not affected. The suppressor cells appeared in the spleen in optimum number 3-4 days after induction. They were theta-negative and Ig-positive as judged by antiserum plus complement treatment and by Ig rosette separation. Attempts to obtain soluble suppressor factor from the suppressor cells by sonication or in vitro incubation were unsuccessful. Mitomycin C treatment of the suppressor cells completely abolished the suppressor activity. Thus, LPS, in conjunction with antigen, appears to induce a population of specific suppressor B cells which are capable of regulating T cell function.     

Lymphocyte subsets and their proliferation in a model for a delayed-type hypersensitivity reaction in the skin.

A delayed-type hypersensitivity (DTH) reaction was induced in the skin of young pigs, by local injection of phytohaemagglutinin, and evaluation was carried out on the resulting accumulation of lymphocyte subsets and lymphocyte production by incorporation of bromodeoxyuridine in the skin and the draining lymph node. There was a rapid increase in mononuclear cells, which were found in clusters around venules. These included very few B lymphocytes, and CD8+ lymphocytes far outnumbered CD4+ cells. Underlining the importance of determining absolute numbers, the relative and absolute numbers of lymphocyte subsets showed quite different patterns during the development of the skin reaction. Lymphocytes in the normal skin incorporated the DNA precursor bromodeoxyuridine at higher rates than have been found for peripheral lymphoid organs. After intradermal phytohaemagglutinin injections, all subsets showed high proliferation rates in the skin, with kinetics which differed from the reaction in the draining lymph node. The labelling indexes of cells labelled with bromodeoxyuridine in vitro and in vivo were comparable. The phytohaemagglutinin injections also caused a marked and rapid increase in the proliferation of the cells in the basal layer of the epidermis. This model DTH-like reaction in skin with major CD8+ T-cell accumulation and proliferation locally and in the lymph nodes provides a reliable model for study of such reactions and for investigation of the regulatory role of cytokines.     

Regulation of delayed-type hypersensitivity. III. Effect of cyclophosphamide on the suppressor cells for delayed-type hypersensitivity to sheep erythrocytes in mice.

A previous study (Eur. J. Immunol. 1977. 7: 714) has shown that mice injected intravenously (i.v.) with 4 x10(9) sheep red blood cells (SRBC) produce cells which suppress delayed-type hypersensitivity (DTH). These suppressor cells are theta-positive, antigen-specific and act via a soluble factor which does not bear immunoglobulin determinants (Eur. J. Immunol. 1978. 8: 168). The present paper demonstrates that these suppressor cells are inhibitable by cyclophosphamide (CY). Mice injected with graded amounts of CY two days prior to SRBC injection, showed maximum augmentation of DTH at 200 mg/kg body weight, a dose which completely suppressed the appearance of splenic plaque-forming cells (PFC) to SRBC. In contrast, lower doses of CY enhanced both DTH and PFC responses. Time course studies showed that CY inhibited the precursors of suppressor cells and had little or no effect on suppressor cells which have already encountered antigens. This was further confirmed by passive transfer studies which showed tha- suppressor cells were inhibited if CY was administered at the same time or 2 days before SRBC injection, but were not affected if CY was given after antigen stimulation. Direct evidence for the effect of CY on suppressor cells was obtained by cell fractination with a Ficoll density gradient. The denser suppressor cell population was absent from the spleens of mice treated with 200 mg/kg of CY 2 days before i.v. injection with 1 x 10(9) SRBC.     

An improved embryonated chicken egg model for the evaluation of antiviral drugs against influenza A virus

Influenza is a serious global public health problem and an economic burden. With the continual emergence of new influenza A virus strains, new antiviral drugs are needed urgently. In this study, an improved embryonated chicken egg model for evaluating antiviral activity against Influenza A virus was developed. In the model, the influenza A virus was injected into the allantoic cavity and ribavirin was injected into the albumen of the egg. The levels of influenza A virus in the allantoic fluid was titrated by the hemagglutination test after incubation for 72 h at 35.5 degrees C and 12 h at 4 degrees C. Ribavirin treatment at a dose of 25 mg/kg to 100 mg/kg decreased significantly the hemagglutination titers both of Influenza virus A/FM1, H1N1 (IVA1) (p < 0.01) and influenza virus A/Wuhan/359/95, H3N2 (IVA3) (p < 0.01). In a time-dependent drug addition assay, significant efficacy of ribavirin against both IVA1 and IVA3 was observed when the drug was administered before and shortly after viral inoculation (p < 0.01 or p < 0.05). In conclusion, ribavirin treatment showed significant antiviral activity against IVA1 and IVA3 in this model, suggesting that the improved model would be useful for evaluating the anti-influenza virus activity of potential inhibitors.     

The migration inhibition factor test for identification of hypersensitivity reactions to drugs

The migration inhibition factor (MIF) test detects the in vitro release of lymphokine from lymphocytes in in vitro contact with a drug that had sensitized them in vivo. The specificity and sensitivity of the MIF test in identifying a drug inducing an allergic reaction is presented. The MIF test detected the drugs responsible for 20 out of 21 allergic episodes (95.2%) while the basophil degranulation test detected only eight of them (P less than .001). The sensitivity of a positive MIF test was 95.2% and its specificity was 76.9%. The specificity of a negative MIF test was 94.7%. The positive MIF test assisted the physician in indicating the drugs responsible for an allergic reaction in half of the patients. The drugs for which the MIF test was negative could be considered innocent in 95% of the cases. It is concluded that although the results of the present studies are encouraging, the clinical utility of the MIF test is still limited and improvement of the test specificity is required.     

Interleukin 6 inhibits delayed-type hypersensitivity and the development of adjuvant arthritis

We studied the effect of interleukin 6 (IL 6) on the delayed-type hypersensitivity (DTH). In mice immunized with sheep red blood cells (SRBC), a DTH response was evoked by antigen challenge into the hind paw 5 days after immunization. The magnitude of the response was assessed by footpad swelling measured 24 h after antigen challenge. IL 6 significantly suppressed the DTH in its induction phase in a dose-dependent manner when administered s.c. into the back at a dose of greater than 2.5 micrograms twice a day (5 micrograms/day) for 5 consecutive days from the day of immunization (day 0) to 1 day before antigen challenge (day 4). Heat-inactivated IL 6 did not suppress the DTH response. Furthermore, the suppressive activity of IL 6 was completely abolished by affinity chromatography on an anti-IL 6 antibody. This suppression was also obtained when IL 6 was administered only on day 0 and day 1, but not on days 3 and 4. This indicates that IL 6 acts on the early part of the induction phase of DTH development. Furthermore, footpad swelling was suppressed even by the administration of IL 6 after antigen challenge. These results show that IL 6 suppresses both the induction and effector phases of DTH. To confirm further this inhibitory effect of IL 6, we examined its effect on the development of adjuvant arthritis in rats. Administration of IL 6 also significantly suppressed the development of adjuvant arthritis.     

Development and evaluation of an improved mouse model of meningococcal colonization

Studies of meningococcal pathogenesis have been severely restricted due to the absence of an adequate animal model. Given the significance of iron in meningococcal pathogenesis, we developed a model of Neisseria meningitidis colonization in outbred adult mice that included daily administration of iron dextran. While receiving iron, the animals were inoculated intranasally with the initial doses of bacterial suspension. Meningococci were recovered from the animals by nasopharyngeal washes. Approximately half of the animals inoculated with 10(7) CFU remained colonized 13 days after the initial bacterial inoculation. The model was further evaluated with genetically defined isogenic serogroup B mutant strains, and the colonization capabilities of the mutants were compared to that of the wild-type parent. A mutant that produces truncated lipooligosaccharide (KDO(2)-lipid A) and a mutant defective in capsule transport were dramatically impaired in colonization. A mutant defective in pilus transport (pilQ) showed moderately impaired colonization. The immunological aspect of the model was also evaluated by challenging mice after immunization with homologous whole-cell meningococci. The immunized mice were protected from colonization of the homologous strain. In this model, long-term meningococcal colonization was maintained, allowing us to study the effects of specific genetic mutation on colonization. In addition, this model allows investigation of the role of active immune response against meningococci.     

The effects of anticoagulants and other drugs on cellular and cutaneous reactions to antigen in guinea-pigs with delayed-type hypersensitivity

Delayed-type hypersensitivity to tuberculin was induced in guinea-pigs by vaccination with BCG. The effects of several drugs on the responses of peritoneal exudate cells to tuberculin (PPD) and on delayed skin reactions to PPD were investigated. In untreated animals intraperitoneal injections of PPD were followed by the virtually complete loss of macrophages from the exudates (the macrophage disappearance reaction), the partial loss of lymphocytes and a marked increase in the number of polymorphs in the exudates. The macrophage disappearance reaction was markedly or completely inhibited in animals treated with the anticoagulant drugs heparin or sodium warfarin, very slightly inhibited in animals treated with cortisone acetate or promethazine and not inhibited in animals treated with reserpine. The other peritoneal cellular responses were variably but only slightly affected by these drugs. Delayed skin reactions to PPD were partly inhibited in animals treated with heparin, sodium warfarin or cortisone acetate and more strongly inhibited in animals treated with a combination of sodium warfarin and cortisone acetate. Histological examination of the skin test sites of untreated animals and of animals treated with sodium warfarin and/or cortisone acetate showed that the accumulation of macrophages was more markedly inhibited in animals treated with sodium warfarin than in animals treated with cortisone alone. No correlation could be established between the effect of treatment with sodium warfarin on the macrophage disappearance reaction, on blood coagulation and on serum complement levels.     

Expression of systemic protection and delayed-type hypersensitivity to Listeria monocytogenes is mediated by different T-cell subsets.

The relationship between acquired cellular resistance and delayed-type hypersensitivity (DTH) during the immune response to Listeria monocytogenes was investigated. Treatment of concanavalin A-stimulated Listeria-immune spleen cells with anti-CD8 antibody plus complement abrogated the adoptive transfer of systemic antilisterial immunity but had no effect on the transfer of DTH. In contrast, in vitro depletion of the CD4+ T-cell subset eliminated the ability of culture-activated cells to transfer DTH reactivity but did not interfere with the adoptive transfer of protection. In vivo, the infusion of anti-CD8 antibody inhibited the expression of both actively and adoptively transferred protection but did not influence the development of DTH skin test reactivity to L. monocytogenes antigens. In vivo depletion of the CD4+ T-cell subset eradicated the DTH response, with only minor influence of the protective anti-Listeria response. The apparent functional dissociation of the CD4+ (DTH) and CD8+ (protection) T-cell populations was further emphasized by our findings that the adoptive transfer of protection was dependent on a cyclophosphamide-sensitive cell population, whereas DTH reactivity was mediated by a cyclophosphamide-resistant population.     

Glutathione depletion inhibits dendritic cell maturation and delayed-type hypersensitivity: implications for systemic disease and immunosenescence

BACKGROUND: Dendritic cells (DCs) play a key role as antigen-presenting cells in the immune system. There is growing evidence that the redox equilibrium of these cells influences their ability to induce T-cell activation and to regulate the polarity of the immune response. This could affect the outcome of the immune response during systemic diseases and aging. OBJECTIVE: Our aim was to elucidate the mechanism by which the redox equilibrium of antigen-presenting DCs affects the delayed-type hypersensitivity (DTH) response during experimental modification of glutathione levels, as well as during aging. METHODS: We looked at the effect of glutathione depletion by diethyl maleate in DCs as well as during systemic administration on the DTH response to the contact-sensitizing antigens, oxazolone, and 2,4-dinitro-1-fluorobenzene. We also determined whether glutathione repletion with N-acetyl cysteine could influence the decline of the DTH response in aged mice. RESULTS: Glutathione depletion in bone marrow-derived DCs interfered in their ability to mount a DTH response on adoptive transfer into recipient mice. Glutathione depletion interfered in IL-12 production and costimulatory receptor expression in DCs, leading to decreased IFN-gamma production in the skin of recipient mice. Systemic diethyl maleate treatment exerted similar effects on the DTH response and IFN-gamma production, whereas N-acetyl cysteine administration reversed the decline of the DTH response in aged animals. CONCLUSION: Glutathione depletion downregulates T(H)1 immunity through a perturbation of DC maturation and IL-12 production. CLINICAL IMPLICATIONS: These data show that the induction of oxidative stress in the immune system, under disease conditions and aging, interferes in T(H)1 immunity.     

Allergic skin disease: investigation of both immediate- and delayed-type hypersensitivity is essential

BACKGROUND: In our clinic we routinely patch test patients referred from occupational health for the investigation of latex contact urticaria. We also undertake both patch and prick testing (where indicated) in patients referred with persistent dermatitis/eczema. If investigation of allergic skin disease is undertaken by a non-dermatologist, it is unlikely that patch testing will be performed. OBJECTIVE: To carry out a retrospective analysis of patients who had been prick tested to establish whether an incomplete diagnosis would have been reached if patch testing had been omitted. METHODS: Details of patients who had attended for patch testing between July 2004 and December 2005 were analysed. Patients who had had prick tests and patch testing were identified. The outcomes of prick tests and patch testing were documented together with the clinical relevance. RESULTS: Three hundred and thirty out of 1060 patients referred to the clinic were prick tested. 54.2% patients were referred from dermatologists. 26.6% were referred from occupational health, 68 patients had positive reactions on prick testing of whom 36 had positive patch tests (52.9%), which were of current relevance in 27 patients (39.7%). Nine out of 106 health workers referred to exclude latex contact urticaria had positive prick tests to latex. Fifty of these patients demonstrated delayed-type hypersensitivity with nickel, cobalt, rubber and its additives being the most common allergens found. Of the 262 patients who had negative prick tests, 121 had positive patch tests (46.1%) of current relevance to patient history in 92 subjects (35.1%). While none of the six patients referred for investigation of reaction to local anaesthetics had a positive prick test, one was allergic to local anaesthetic on patch testing. CONCLUSION: Omission of patch testing from the investigation of allergic skin disease, even when contact urticaria may be the sole suspected diagnosis, would result in the frequent missed diagnosis of contact allergy. We recommend that patients with suspected allergic skin disease are investigated in an environment where investigation of both immediate- and delayed-type hypersensitivity can be undertaken. In particular, patients with atopic eczema, suspected latex rubber allergy, hand dermatitis (particularly occupational) and drug reactions should be targeted to receive both investigations.     

Relationship between delayed-type hypersensitivity and the progression of Mycobacterium lepraemurium infection.

The relationship between the level of delayed-type hypersensitivity (DTH) and the progression of Mycobacterium lepraemurium infection was examined after inoculation of mice with 10(8) M. lepraemurium in the left hind footpad. The expression of DTH developed over the first 4 weeks of infection, remained high up to week 8, and then dropped to a low level at which it remained for 12 more weeks. The development of DTH was concordant with an initial swelling of the inoculated foot, the appearance of a mononuclear infiltrate at this site, and a prevention of any increase in the number of mycobacteria in this foot and in other tissues studied. A decay of DTH reactivity was associated with a progressive increase in the number of M. lepraemurium initially at the original site of inoculation and subsequently in all other tissues. Although the expression of DTH was lost, adoptive immunization experiments showed that a population of sensitized lymphocytes persisted within host. Further experimentation offered evidence to suggest that the level of systemic antigen may be in part responsible for the loss of DTH reactivity.     

食管内脏高敏感性动物模型的建立和评价

目的建立食管内脏高敏感性动物模型。方法采用腹腔注射鸡卵清蛋白（OVA）基础致敏联合食管酸灌注的方法处理SD大鼠，并采用免疫组织化学方法和显微图像分析技术评价内脏高敏感性一食管化学刺激大鼠模型的可靠性。结果OVA基础致敏联合食管酸灌注组大鼠被激活了一个复杂而广泛的大脑网络，其在额顶皮质、岛叶、扣带皮质、中央杏仁核、Kfilliker-Fuse核、疑核、臂旁核、下丘脑室旁核、丘脑室旁核、三叉旁核、孤束核、最后区、延髓网状核等Fos样免疫活性（FLI）神经元的数目均显著高于其余各组（P〈0．05）。模型组大鼠在中央杏仁核、臂旁核、室旁核、三叉旁核、孤束核的FLI阳性产物的平均光密度（OD）值亦较其余各组明显增高（P〈0．05）。结论预先腹腔注射鸡卵清蛋白基础致敏联合食管酸灌注可成功建立食管内脏高敏感性动物模型。     

Tumour-induced suppression of delayed-type hypersensitivity in the mouse: independence of cyclophosphamide-sensitive suppressor cells

Delayed-type hypersensitivity (DTH) responses to sheep red blood cells and ovalbumin were markedly reduced in mice receiving viable Landschütz ascites tumour cells at the time of immunization. Similar inhibitory effects were observed in control immunized animals when cell-free ascitic fluid or tumour bearer serum was administered together with antigen at the challenge site. Normal mouse serum exhibited much less pronounced inhibitory activity over the range of concentrations tested. High-dose cyclophosphamide (Cy) prior to immunization enhanced DTH responses to both antigens in normal mice. However, in animals also given tumour or soluble tumour-associated material, the immunosuppressive effect which had been observed in non-Cy-treated controls was preserved. It is concluded that the observed suppression of DTH is not dependent on Cy-sensitive T suppressor cells or on the presence of immunosuppressive factors during induction of the immune response.     

A triple assay technique for the evaluation of metal-induced, delayed-type hypersensitivity responses in patients with or receiving total joint arthroplasty

The determination of biocompatibility has been dominated historically by the characterization of candidate materials based upon the observation of adverse host responses. However, some adverse responses are subtle in clinical settings and continue to foster debate and investigation. One of these responses is "metal allergy" or hypersensitivity to metallic biomaterials. Current methods used to diagnose hypersensitivity reactions, such as dermal patch testing and migration inhibition assays, are not well accepted in orthopedic practice as a means for the characterization of hypersensitivity to metallic joint-replacement components. An increasing need to resolve whether metal sensitivity may be a significant and/or predisposing factor for eliciting an over-aggressive immune response in patients with metallic implant components requires improved and standardized widespread study. Here we present three in vitro methodologies: (1) a proliferation assay, (2) cytokine analysis using ELISA, and (3) a migration inhibition assay. When in conjunction with one another, these assays may be used to more comprehensively quantify metal-induced hypersensitivity responses. Therefore, these methodologies are detailed with the intent of facilitating multi-center large-scale studies. In the following cases, a multi-assay approach for measuring the prevalence of delayed-type hypersensitivity in orthopedic patients shows the propensity to yield a more comprehensive and, therefore, more conclusive determination than currently employed patch testing or single assay techniques.