Cyclic AMP and the mechanism of leucocyte lysosomal enzyme release during an immediate hypersensitivity reaction in vivo.

The pleural cavity of rats was used to study the effect of altering leucocyte cyclic AMP content on the release of B-glucuronidase activity during an immediate hypersensitivity reaction. The effect on intravenous colchicine was also studied. Despite an increase of 135 to 235% in leucocyte cyclic AMP content no decrease in B-glucuronidase release was observed. Similarly, colchicine had no effect on enzyme release. It was concluded that the cyclic nucleotides and leucocyte microtubules may have no significant role to play in the release of lysosomal enzymes during acute inflammation in vivo.     

Monitoring leukocyte traffic in vivo into human delayed-type hypersensitivity reaction

Leukocyte traffic from blood to sites of inflammation has been elaborately studied in animal models and in vitro. However, to date, little understanding has accumulated on the process in humans in vivo. A noninvasive light confocal microscopy technique has enabled us to image leukocyte rolling, arrest and transmigrated cells in vivo in human tissues. In the current study, a delayed-type hypersensitivity (DTH) reaction was elicited in the lower lip of healthy, Bacille Calmette-Guerin (BCG)-vaccinated volunteers by injection of respective purified protein derivate (PPD), mimicking the classic cutaneous Mantoux reaction. Subjects were imaged with real-time confocal microscopy at baseline and 2, 4, 24 and 48 h after the injection. The number of rolling leukocytes did not increase significantly until at 48 h. Even then, rolling cells were seen in only a minority (23%) of blood vessels. The frequency of engaged blood vessels was, nevertheless, significantly greater than at baseline. As the inflammation generated with this challenge was mild, transmigrated leukocytes could be detected in the confocal microscopy only occasionally. Histology of biopsies taken immediately after imaging at 48 h showed a T cell-dominated leukocyte population at the site of the DTH reaction. We have thus developed a method to monitor noninvasively leukocyte traffic in vivo in human subjects during the classic inflammatory model of delayed-type hypersensitivity reaction.     

A modification of the in vivo mixed lymphocyte reaction and rapamycin's effect in this model.

Rapamycin, a novel macrocyclic immunosuppressive agent, suppresses murine T cell activation in vitro by mechanisms distinct from cyclosporin A (CsA). This study was designed to examine rapamycin and CsA in the host vs graft popliteal lymph node (PLN) model, an in vivo system of T cell-dependent lymphocyte activation. The PLN procedure was modified by using irradiated CTLL-2 cells of C57BL/6 origin, instead of primary mouse splenocytes, as the allogeneic stimulus in C3H/HeN recipient mice. PLN cell proliferation was determined by [3H]-thymidine uptake. We found that the host lymphocyte proliferative response to CTLL-2 cells (H-2b) is greater than the response to mouse Balb/c splenocytes (H-2d). Rapamycin (ip or po) produced a dose-related inhibition of the in vivo mixed lymphocyte reaction. By contrast, the effects of CsA and FK-506 were not dose related within the same dose range (0.006-12 mg/kg). These data indicate that rapamycin is an effective immunosuppressive agent and confirm its ability to affect the allogeneic T cell response in vivo. Furthermore, the pharmacological data suggest that this PLN model utilizing irradiated CTLL-2 cells as an allogeneic stimulus provides a reproducible system to examine mixed lymphocyte reactions in vivo.     

The relation of cyclic AMP levels to phagocytosis and enzyme release in acute inflammation in vivo.

The effect of altering endogenous leucocyte cyclic AMP levels on phagocytosis and lysosomal enzyme release was studied in vivo. Acute pleural exudates were produced in rats using either calcium pyrophosphate dihydrate crystals or rat serum. Despite marked increases in leucocyte cyclic AMP concentration produced by injection of dibutyryl cyclic AMP and theophylline, there was no reduction in crystal phagocytosis or in enzyme discharge.     

The macrophage disappearance reaction (mdr) as an in vivo test of delayed hypersensitivity in mice.

The macrophage disappearance reaction (MDR), as an invivo analogue of in vitro tests for delayed hypersensitivity, was utilized in mice with M. tuberculosis or mouse thyroid extract (MTE). The optimal schedule for the induction of macrophages in peritoneal exudates and the optimal concentration of antigen for the MDR were determined. Macrophage disappearance occurred 4 h after immunized mice received an intraperitoneal injection of 200 microng of soluble antigen. The MDR was found to be antigen specific. Intraperitoneal injection of an unrelated antigen or tissue culture medium alone did not cause macrophage disappearance; however, the re-injection of the antigen used for immunization caused a 70-80% reduction of macrophages. Macrophage disappearance was greater in mice immunized with M. tuberculosis than in mice immunized with MTE. Comparison of the MDR with the footpad test in these 2 groups showed that mice immunized with M. tuberculosis developed a higher degree of delayed hypersensitivity than the group immunized with MTE. These results demonstrate that the MDR represents a specific and quantitative method for detecting the delayed type of cellular immune response in mice.     

The in vivo effects of concanavalin A on the cellular immune response in BALB-c mice. Inhibition of the delayed hypersensitivity reaction to tuberculin

The suppressive effect of concanavalin A (Con A) on the cell-mediated immune response was demonstrated in BALB/c mice made immune to challenge with tuberculin. 400 μg of Con A administered intraperitoneally simultaneously with injection of antigen into the footpad of primed animals resulted in suppression of the delayed hypersensitivity reaction as detected by a sensitive radioisotopic footpad assay. If Con A was given in a dose of 100 μg three times weekly to normal mice being sensitized to tuberculoprotein, the maximum immune response following complete immunization was significantly suppressed but not abolished. The suppressive effect of Con A appeared to be due to its binding to cell membranes, since splenic lymphocytes obtained from Con A-treated immune mice failed to respond in the local adoptive transfer reaction to tuberculin. The lymphocytes regained their immune reactivity to tuberculin if they were first incubated in α-methyl-D -mannoside. These findings represent the first demonstration of in vivo immunosuppression by Con A of the tuberculin immune response in the mouse. Both the inductive and established responses of the delayed hypersensitivity reaction appear to be affected. The reversible inhibition of the local adoptive transfer of tuberculin immunity suggests the depression of the response is due to binding to or alteration of antigen receptor sites by Con A.     

In vivo macrophage suppression of delayed hypersensitivity in the guinea-pig.

The nature of the suppressive activity in the peritoneal exudate cells (PEC) of guinea-pigs immunized with dinitrophenyl bovine gamma globulin (DNP50-BGG) was investigated. A method was developed to isolate from the peritoneal exudate large numbers of macrophages. Using density gradient centrifugation on Percoll it was possible to obtain a population of cells which contained over 90% macrophages. This macrophage preparation was found to respond to lymphokine but to be incapable of passively transferring delayed hypersensitivity reactions. When these immune macrophages were transferred into antigen immunized animals, which had been pretreated with cyclophosphamide (CY), the skin reactions were suppressed to the same extent as when the total PEC was transferred. PEC from guinea-pigs immunized with ovalbumin in Freund's incomplete adjuvant did not suppress the skin reactions in CY-pretreated DNP50BGG immunized animals. However, in contrast, macrophages from these animals did suppress the skin reactions in the recipient guinea-pigs indicating that the macrophage suppression was not antigen specific.     

Delayed-type hypersensitivity reaction to the meta-cresol component of insulin.

BACKGROUND: Reactions to recombinant human insulin preparations occur at a rate of 2.4%. Although most have been traced to immunological reactions to the insulin, recent reports suggest that some adverse reactions can occur to the nonmedicinal excipients or preservatives of commercially available insulin preparations. OBJECTIVES: To investigate a localized delayed cutaneous reaction to human insulin. METHODS: Intradermal and patch testing were performed on a patient to evaluate sensitivity to commercial human insulins, meta-cresol, and other sensitizers. RESULTS: Patch test results were positive to the meta-cresol preservative. CONCLUSIONS: This is the first report, to our knowledge, of a reaction to meta-cresol in commercial preparations of insulin. This reaction should be considered in the differential diagnosis of adverse reactions to insulin injections.     

IgE-induced changes in human basophil cyclic AMP levels.

We studied a unique patient with 77% basophils, not different from normal by a number of criteria, in order to measure the changes in cyclic AMP level associated with IgE-mediated histamine release. In accordance with previous hypothesis and circumstantial evidence, anti-IgE challenge led to a significant fall in the cyclic AMP level which preceded histamine release.     

Cyclic AMP potentiation of interferon antiviral activity and effect of interferon on cellular cyclic AMP levels.

Treatment of L cells with 3 to 10 mM 3':5'-cyclic adenosine monophosphate (cAMP) in the presence of interferon was found to potentiate the development of antiviral activity. The dose response of interferon activity at various time periods in the presence and absence of cAMP indicated that potentiation of interferon activity by cAMP occurred at an early stage in the development of antiviral activity. Among the analogues of cAMP tested for interferon-potentiating activity, only the acylated derivatives were found to be active. Combined L-epinephrine and theophylline treatment of cells elevated cellular cAMP levels and also potentiated interferon-mediated antiviral activity. Interferon was also found to elevate cAMP levels in L cells. This activity was limited to biologically active interferon and antagonized the depression of cAMP associated with vesicular stomatitis virus (VSV) infection of L cells. These observations suggest that some aspects of interferon's biological activity is associated with an alteration in cellular levels of cAMP.     

Trans vivo analysis of human delayed-type hypersensitivity reactivity.

There are clinical situations in which it may be advantageous to monitor delayed type hypersensitivity (DTH) responses, an index of cell-mediated immunity, without exposing patients directly to the challenge antigens. For example, transplant patients may be at risk for becoming sensitized to donor antigens if injected with donor antigen during traditional skin tests. We describe an alternative method for human DTH testing, which involves the transfer of human peripheral blood mononuclear cells plus antigen into the pinnae or footpads of naive mice. This induces a measurable DTH-like swelling response, which we refer to as the "trans vivo DTH response." As proof of principle, we provide data obtained during trans vivo DTH studies with tetanus toxoid, cytomegalovirus (CMV) and alloantigens. In general, human T cells must be co-localized with antigen and human macrophages to produce swelling responses, and such responses are antigen-specific and require prior antigen sensitization. Not only does this assay offer a simple, reliable clinical monitoring device, but it also provides a model with which to study the in vivo mechanisms of human DTH responses.     

Effects of noise on monoamine levels in the rat brain using in vivo microdialysis

The concentrations of monoamines in the striatum, dorsal raphe and cortex of the brain of male Sprague-Dawley rats were monitored using in vivo microdialysis before, during and after noise exposure. Some 120 h after implantation of the microdialysis probe in the desired area, the rats were exposed to white noise (110 dB) for 20 min. The concentrations of monoamines in the dialysate were determined by HPLC. Exposure to white noise increased epinephrine in the striatum (42%) and dorsal raphe (39%), values then declined to baseline. In contrast, white noise decreased 3,4-dihydroxyphenylacetic acid (DOPAC) in the striatum (99%) and cortex (53%); these levels remained depressed following noise cessation. Noise also decreased homovanillic acid (HVA) in the striatum and in 5-hydroxy-indoleacetic acid (5-HIAA) in the striatum and dorsal raphe nucleus. Other neurotransmitters (norepinephrine etc.) did not change significantly in any region. These results suggest that noise influences monoamine levels differently in different brain regions.     

Effects of relaxation on the delayed-type hypersensitivity (DTH) reaction to diphenylcyclopropenone (DCP)

Delayed-type hypersensitivity (DTH) reactions to the experimental allergen diphenylcyclopropenone (DCP) were measured in four groups, which either trained (+) or did not train in relaxation (−) during the sensitization and/or the challenge phase. All groups consisted of high and low hypnotic susceptible subjects. While there were no differences in erythema, the mean induration of the group which trained in relaxation in both the sensitization and the challenge phase (+/+) was significantly greater than that of the group which trained in relaxation in the challenge phase only (−/+). Significant correlations were found between induration and hypnotic susceptibility scores, and between induration and degree of perceived relaxation during challenge. High hypnotic susceptible subjects experienced a higher degree of perceived relaxation and exhibited greater indurative and erythematous DTH reactions to DCP than low hypnotic susceptible subjects in all four experimental conditions. Though the mediating mechanisms remain unclear, our results suggest that relaxation may affect the DTH reaction, and support previous findings of higher psychophysiologic reactivity of high hypnotic susceptible subjects.     

Effect of histamine on cyclic AMP levels in the submandibular gland

Histamine and 4-methylhistamine increased the cyclic AMP level in chopped guinea-pig submandibular glands. 4-Methylhistamine was more active than histamine. 2-(2-Pyridyl)ethylamine was less active than histamine or almost inactive. Metiamide markedly antagonized the histamine-induced cyclic AMP increase but mepyramine had no inhibitory effect on this increase.These results suggest that there are H₂-receptors which mediate the cyclic AMP response to histamine in the guinea-pig submandibular gland.