Drug actions on delayed-type hypersensitivity in rats with developing and established adjuvant arthritis

Rats were sensitized by i. d. injection in the base of the tail with Freund's complete adjuvant (FCA) and were challenged i. d. in the dorsal skin with mycobacterial antigen. The 24 hour dermal delayed-type hypersensitivity (DTH) response increased up to 10 days after FCA injection followed by a decrease by day 15 which coincided with the development of adjuvant arthritis (AA). Drug studies were performed, using a 4-day dosing schedule, on optimal DTH elicited on day 10, suboptimal DTH elicited on day 15, and AA (day 16). Cyclosporine, leflunomide and prednisolone significantly inhibited day 10 DTH and AA with no effect on day 15 DTH. Indomethacin and tiaprofenic acid significantly inhibited AA with no effect on either DTH response. Chloroquine, levamisole,d-penicillamine, diazepam and RU38468 had no significant effect on DTH or AA. These findings suggest a complex temporal relationship between AA, DTH and drug actions.     

Anti-neutrophil monoclonal antibody therapy inhibits the development of adjuvant arthritis

The aim of this study was to determine the contribution of neutrophils to adjuvant arthritis (AA) by in vivo depletion of peripheral blood neutrophils. Specific anti-neutrophil MoAb, RP3 (10 mg), or a control antibody was given twice daily on days 8–11 after injection of Mycobacterium tuberculosis in inbred male Sprague-Dawley rats. RP3 treatment inhibited the neutrophil leukocytosis associated with AA (3.3 ± 0.6 × 10³/mm³versus 21.2 ± 6.9 × 10³/mm³; P < 0.001). On day 12, control animals exhibited severe arthritis as assessed by articular index (AI) (9.2 ± 1.3), increase in paw volume (149.3 ± 10.6%), and synovial fluid (SF) cell count (5.3 ± 0.5 × 10⁵). RP3 treatment significantly reduced AI (1 ± 0.1; P < 0.001), paw volume (103.6 ± 5.8%; P < 0.001) and SF cells (0.6 ± 0.1 × 10⁵; P < 0.001) without affecting cutaneous DTH (treated 0.6 ± 0.1 mm change in thickness, control 0.8 ± 0.2 mm; NS). Additional experiments demonstrated that CD4⁺ cell depletion but not decomplementation inhibited AA development and synovial neutrophil accumulation. Depletion of circulating neutrophils prevented joint inflammation and synovial leucocyte influx in AA, suggesting a pivotal role for neutrophils in the effector phase of AA. Inhibition of neutrophil accumulation by CD4⁺ cell depletion and not by decomplementation suggests that neutrophil accumulation in AA is T cell-dependent.     

Thalidomide analogue CC1069 inhibits development of rat adjuvant arthritis.

The cytokine tumour necrosis factor-alpha (TNF-alpha) has been implicated in the aetiology of rheumatoid arthritis in humans as well as of experimental arthritis in rodents. Thalidomide, and to a greater extent the new thalidomide analogue CC1069, inhibit monocyte TNF-alpha production both in vitro and in vivo. The aim of the present study is to establish whether these drugs block production of TNF-alpha as well as IL-2 by rat leucocytes and whether this inhibition affects the development of rat adjuvant arthritis (AA). Cultured splenocytes were stimulated with either lipopolysaccharide (LPS) or concanavalin A (Con A) in the presence of thalidomide, CC1069, or solvent, and the production of TNF-alpha and IL-2 were compared. Next, adjuvant was injected into the base of the tail of rats without or with daily intraperitoneal injections with 100-200 mg/kg per day thalidomide or 50-200 mg/kg per day CC1069. Disease activity, including ankle swelling, hind limb radiographic and histological changes, weight gain, and ankle joint cytokine mRNA levels, were monitored. CC1069, but not the parent drug thalidomide, inhibited in vitro production of TNF-alpha and IL-2 by stimulated splenocytes in a dose-dependent manner. In vivo, a dose-dependent suppression of AA disease activity occurred in the CC1069-treated animals. In contrast, thalidomide-treated rats experienced comparable arthritis severity to placebo-treated animals. There was also a reduction in TNF-alpha and IL-2 mRNA levels in the ankle joints of CC1069-treated rats compared with thalidomide- and placebo-treated arthritic rats. Early initiation of CC1069 treatment suppressed AA inflammation more efficiently than delayed treatment. We conclude that thalidomide, which did not suppress TNF-alpha or IL-2 production in vitro by Lewis rat cells, did not suppress development of rat AA. However, the development of rat AA can be blocked by the thalidomide analogue CC1069, which is an efficient inhibitor of TNF-alpha production and IL-2 in vitro.     

Specific delayed-type hypersensitivity responses to ESAT-6 identify tuberculosis-infected cattle

Human and bovine tuberculosis have long been detected by skin testing with purified protein derivative (PPD), a complex mix of partly denatured mycobacterial antigens with suboptimal specificity. In the present study, skin tests based on ESAT-6, a recombinantly produced antigen highly specific for tuberculosis infection, were investigated. Although ESAT-6 was strongly recognized in vitro and induced high levels of gamma interferon, initial investigations demonstrated that higher doses of ESAT-6 than of PPD were needed to induce substantial delayed-type hypersensitivity reactions. Also, the kinetics of the skin test response differed for the two reagents; PPD showed maximal response at 72 h, but the response to ESAT-6 often peaked later at 96 h. Tests based on an optimized strategy (400 micro g of ESAT-6 measured between 72 and 96 h), in cattle infected with Mycobacterium bovis (n = 22) and animals sensitized by exposure to environmental mycobacteria showed ESAT-6 to have a promising diagnostic potential (sensitivity, 82%; specificity, 100%; optimal cutoff, 3 mm), compared with PPD (sensitivity, 86%; specificity, 90%; optimal cutoff, 4 mm). Larger investigations are required to refine cutoff points for any new diagnostic test, but the present results indicate great potential for skin tests based on specific antigens for accurate in vivo diagnosis of tuberculosis.     

Interleukin 17 inhibits progenitor cells in rheumatoid arthritis cartilage

Mesenchymal stem cells are known to exert immunomodulatory effects in inflammatory diseases. Immuneregulatory cells lead to progressive joint destruction in rheumatoid arthritis (RA). Proinflammatory cytokines, such as tumour necrosis factor α (TNF‐α) and interleukins (ILs) are the main players. Here, we studied progenitor cells from RA cartilage (RA‐CPCs) that are positive for IL‐17 receptors to determinate the effects of inflammation on their chondrogenic potenial. IL‐17A/F reduced the chondrogenic potential of these cells via the upregulation of RUNX2 protein and enhanced IL‐6 protein and MMP3 mRNA levels. Blocking antibodies against IL‐17 positively influenced their repair potential. Furthermore, treating the RA‐CPCs with the anti‐human IL‐17 antibody secukinumab or the anti‐TNF‐α antibody adalimumab reduced the proinflammatory IL‐6 protein level and positively influenced the secretion of anti‐inflammatory IL‐10 protein. Additionally, adalimumab and secukinumab in particular reduced RUNX2 protein to promote chondrogenesis. The amelioration of inflammation, particularly via IL‐17 antagonism, might be a new therapeutic approach for enhancing intrinsic cartilage repair mechanisms in RA patients.     

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.     

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.     

Interleukin 12 as an adjuvant to WI-1 adhesin immunization augments delayed-type hypersensitivity, shifts the subclass distribution of immunoglobulin G antibodies, and enhances protective immunity to Blastomyces dermatitidis infection

Cell-mediated immunity is pivotal in host resistance to Blastomyces dermatitidis infection. Immunization of mice with the WI-1 adhesin enhances resistance against experimental pulmonary infection but elicits features of a mixed T-helper-cell immune response. Immune mice acquire delayed-type hypersensitivity (DTH) but also high titers of WI-1-specific immunoglobulin G1 (IgG1) and IgG2b, a result indicative of T-helper-2 cellular immunity. We report that interleukin-12, used as an adjuvant for WI-1 immunization, augments DTH, shifts the balance of the T-helper phenotype toward Th1, and enhances resistance to B. dermatitidis infection.     

Lysolecithin analogs as adjuvants in delayed-type hypersensitivity in mice. I. Characterization of the adjuvant effect.

The adjuvant activity of 5 different lysolecithin analogs (LLA) has been studied in delayed-type hypersensitivity (DTH) using fowl gamma globulin, bovine serum albumin and the terpolymer L-glutamic acid 60-L-alanine30-L-tyrosine10(GAT), as antigens. Increased DTH responses, by factors of 1.8--2.0 in CBA and BALB/c mice, showed that LLA are immunopotentiators if given intraperitoneally (i.p.) or subcutaneously (s.c.) together with the antigen. The concentration range, within which LLA are active, is limited to 10--20 micrograms/mouse s.c. and 100--300 micrograms/mouse i.p. Adjuvanticity was tested as a function of the LLA structure. The most pronounced immunopotentiation was obtained with racemic 1-octadecyl-2-methylglycero-3-phosphorylcholine (ET18-O-CH3). The LLA became less active with decreasing numbers of C atoms in the alkyl chain.     

Regulation of delayed-type hypersensitivity to pathogens and alloantigens

Recent studies reinforce the notion that delayed-type hypersensitivity plays a key role in the host defence against microbial and intracellular parasitic infection, and in the rejection of skin allografts. F. Y. Liew reviews these studies and discusses the observation that this T-cell mediated immunity is profoundly regulated by antigen-specific suppressor T cells, some of which are restricted by products of the I-J subregion of the MHC.     

Adoptive immunotherapy with IL-2 results in the loss of delayed-type hypersensitivity responses and the development of immediate hypersensitivity to recall antigens

Skin testing represents a direct method of assessing immune responses in vivo. Twenty-six patients with metastatic cancer of the lung, kidney, or melanoma were treated with adoptive transfers of autologous tumor-infiltrating or blood lymphocytes and continuous infusions of interleukin-2 (IL-2). Prior to therapy, cutaneous anergy to recall antigens was observed in 19 patients (73%), whereas 6 (27%) displayed normal delayed-type hypersensitivity (DTH) responses. When tested again at the end of therapy, DTH responses could not be elicited in any of the patients. Proliferative responses to skin test antigens, lectins, and IL-2 diminished progressively during therapy but returned to baseline values at 1 month. Unexpectedly, 14 of these patients (53%) developed immediate skin test responses to candida antigens and 5 (19%) to mumps antigens. These immediate responses were characterized by local erythema and induration that developed within minutes of injecting antigen. Biopsies displayed marked dermal edema and infiltration by eosinophils. Although serum IgE levels were not increased, immediate reactivity could be transferred by a heat-sensitive serum factor. The implications of this novel response are uncertain, and its development did not correlate directly with the anti-tumor effects of therapy. We conclude that adoptive immunotherapy with IL-2 produces a reduction in cutaneous DTH and diminished responses to mitogens while simultaneously promoting cutaneous allergy. We hypothesize that this may reflect diminished IL-2 production by antigen-specific helper T cells and that other lymphokines may promote these immediate hypersensitivity responses.     

Cyclosporin A inhibits the delayed-type hypersensitivity reaction: impaired production of early pro-inflammatory mediator(s)

The effect of cyclosporin A (CsA) on the delayed-type hypersensitivity (DTH) elicited in mice by sheep red blood cells was investigated. Evidence is presented that a single injection of CsA adversely affects the inflammatory reaction. Sensitized T lymphocytes initiate the DTH reaction by their recruiting activity on phagocytic cells which infiltrate the cutaneous site of antigen deposition. CsA administration has no adverse effect on the recruitment of phagocytic cells at the site of the inflammatory reaction. The present studies show that CsA acts on specific T cells: (a) in adoptive transfer, T-DTH-mediating cells cannot elicit a response in mice treated with CsA 8 h before; (b) when collected 8 h after a single injection of CsA, T-DTH-mediating lymphocytes are no longer able to adoptively transfer the reaction. This conclusion is strengthened by in vitro studies: (a) the frequency of T-DTH-mediating lymphocytes is 50-fold decreased after a short in vitro incubation with CsA; (b) in vitro production by concanavalin A-activated lymphocytes of chemotactic pro-inflammatory mediator(s) is abolished in presence of CsA.     

Establishment and comparison of delayed-type hypersensitivity models in the B6C3F1 mouse

The objective of these studies was to establish and compare delayed-type hypersensitivity (DTH) models, using keyhole limpet hemocyanin (KLH), sheep red blood cells (SRBC), and Candida albicans as sensitizing antigens, for their capability to assess a DTH response (utilizing footpad swelling as the endpoint) with minimal confounding factors resulting from antigen-specific antibody (Ab) production. The key elements of the DTH are the sensitization dose, time interval between sensitization and challenge [i.e. the challenge interval (CI)], and the challenge dose. Models were established by first determining the challenge dose, or the amount of antigen that produced no greater footpad swelling 24-h post-injection than the trauma induced by injection of physiological saline. Time-course studies determined the CI that produced a peak response for each antigen. Dose-response sensitization studies were conducted to determine the optimum sensitization concentration (i.e. maximum footpad swelling with minimal impact by antigen-specific Ab production). Footpad swelling decreased dose-responsively with increasing KLH sensitization concentration and corresponded to a dose-responsive increase in KLH-specific Ab levels. In the SRBC model, footpad swelling decreased at the high dose (1?×?10⁹ SRBC/mouse), and a corresponding increase in SRBC-specific Ab was observed at this dose level. A dose-responsive increase in footpad swelling was observed in the C. albicans model up to 3?×?10⁷ organisms/mouse, while antigen-specific antibody levels were not different from background (unsensitized) levels following sensitization with any concentration of C. albicans (up to 1.2?×?10⁸ organisms/mouse, the highest concentration tested). Finally, each model was evaluated for its ability to detect immunosuppression following exposure to benzo[a]pyrene (B[a]P), with the C. albicans model demonstrating greater sensitivity than the other models. These results indicate that, of the three models examined here, the C. albicans DTH model may be the most appropriate model for evaluating effects on cell-mediated immunity when conducting immunotoxicological investigations.     

Regulation of delayed-type hypersensitivity V. Suppressor cell memory in antigen-specific suppression of delayed-type hypersensitivity to sheep erythroeytes

Mice primed i. v. with 10⁹ sheep red blood cells (SRBC) produce antigen-specific T suppressor (Ts) cells which inhibit both the induction and the expression of delayed-type hypersensitivity (DTH). These Ts cells are detectable in the spleen and lymph nodes 3–5 days after priming but are largely absent by 6 days. The transient detect-ability of the Ts cells contrasts sharply with the profound antigen-specific suppression which persists in primed donor mice for at least a year. Evidence is presented that this long-term impairment of DTH is maintained, at least in part, by memory Ts cells which are Thy-1⁺, cyclophosphamide-resistant and antigen-specific. Although they appear to be co-induced with the short-lived primary Ts cells and localize initially in the lymphoid organs, they are present in the long-lived circulating pool of T cells and can be adoptively transferred by celomic parabiosis. Memory Ts cells are readily reactivated by lower doses of SRBC which would induce T effector cells rather than Ts cells in naive animals. Reactivated memory Ts cells seem to generate a population of antigen-specific secondary Ts cells which again localizes in the lymphoid organs and can adoptively suppress the induction and expression of DTH to SRBC.     

In vitro induction of delayed-type hypersensitivity.

Murine spleen cells, cultured in vitro for 6 days in the presence of high concentrations of burro erythrocytes (BRBC), are sensitized to exhibit delayed-type hypersensitivity (DTH) specific for this antigen. Such cells, on being injected with antigen into the footpads of normal mice, cause a 24-h swelling reaction. This activity of the cultured cells requires the presence of BRBC both during the in vitro incubation and in the footpad. The activity of the sensitized cells in causing swelling is sensitive to anti-Thy-1 antibody and complement, and the kinetics of the swelling reaction are characteristic of a DTH response. In vivo low-dose priming of the spleen cell donors considerably enhances the ability of the cultured cells to cause swelling. This system provides a means of studying the regulation of the induction of DTH in vitro.     

Development of delayed-type hypersensitivity to hog cholera

Leukocyte migration inhibition test showed that pigs with classical swine fever develop delayed type hypersensitivity. The highest migration inhibition (65-85%) was observed in animals with the acute form, on day 3 after infection, or directly before death, or in animals immunized with reactogenic strain with clinical signs of disease. In some pigs with acute form the migrating capacity of leukocytes was restored on days 7-8 postinfection. Leukocyte migration inhibition factor is detected in the sera of pigs starting from day 3 after infection with the virulent strain. The degree of delayed type hypersensitivity correlated with the outcome of classical swine fever.