Cytokine measurement techniques for assessing hypersensitivity.

One of the most potentially useful tools in immunotoxicology is the assessment of cytokines, the molecules responsible for regulating a variety of processes including immunity, inflammation, apoptosis, and hematopoiesis. The particular profile of cytokine production may provide important information regarding the nature of an immunotoxic response such as hypersensitivity (i.e. TH(1) vs. TH(2) response). Proper evaluation of the role of cytokines in understanding hypersensitivity reactions requires attention to a multitude of technical details. Some of the details discussed in this review include the source of the sample to be tested (circulating, local, or ex vivo isolated cells), the potential effects of collection, processing, and storage on the results of the assays, potential variables associated with the source material (matrix effects, relevance, inhibitory substances), and factors influencing the choice of assay used (bioassay, immunoassay, molecular biology technique, flow cytometry). Other often-overlooked issues are discussed, including species considerations and quality control issues such as the use of reference standards and the expression of results.

Modulation of natural killer cell function after exposure to 60 Hz magnetic fields: confirmation of the effect in mature B6C3F1 mice.

In a previous study, we demonstrated that subchronic exposure to pure, linearly polarized 60 Hz magnetic fields (MFs) at flux densities ranging from 0.002 to 1.0 mT induced a modest but statistically significant and reproducible suppression of NK cell activity in young adult B6C3F(1) mice. NK cell activity in mice declines with age and is known to be suboptimal in older animals. The present study was designed to determine if the same MF exposure regimens will suppress NK cell activity in mature (i.e. more than 1 year old) animals. Extending our previous findings, a modest suppressive effect of MFs on NK cell activity in B6C3F(1) mice was found when subchronic exposure was initiated in animals held in quarantine for 1 year prior to exposure. These data demonstrate that MF exposure suppresses NK cell activity in both young and mature adult B6C3F(1) mice. However, because chronic exposure to the same MF parameters used in the NK function studies does not increase the incidence of neoplasia in B6C3F(1) mice, this statistically significant inhibition of NK cell function appears to be of limited biological significance.

Theory and practice of cytokine assessment in immunotoxicology.

The tools and concepts of immunotoxicology are increasingly being used in novel ways, such as using toxic reagents to understand immune system function. One of the most potentially useful of these new tools is the assessment of cytokines, the molecules responsible for regulating a variety of processes including immunity, inflammation, apoptosis, and hematopoiesis. Cytokine production or bioactivity may be affected by a variety of toxic mechanisms including direct toxicity to cytokine-producing cells, inhibition of cytokine production, inhibition of cytokine release, induction of immunosuppressive factors, alterations in cellular homeostasis, alterations in cellular activational or transcriptional mechanisms, and miscellaneous or undefined mechanisms. Moreover, alterations in the profile of cytokine production may provide important information regarding the nature of an immunotoxic insult (i.e., TH1 vs TH2 response). Proper evaluation of the role of cytokine modulation in immunotoxicology requires attention to myriad details. Some of the details discussed in this review include the source of the sample to be tested (circulating, local, or ex vivo isolated cells); the potential effects of collection, processing, and storage on the results of the assays; potential variables associated with the source material (matrix effects, relevance, inhibitory substances); and factors influencing the choice of assay used (bioassay, immunoassay, molecular biology technique, flow cytometry, hybrid assays). Other often-overlooked issues are discussed, including species considerations and quality control issues such as the use of reference standards and the expression of results.

Cytokine bioassays: an overview.

Although eclipsed in recent years by immunoassays and molecular biology techniques, bioassays remain a vital research tool for cytokine biology. Like any type of biological system, cytokine bioassays require meticulous technique to obtain accurate and reproducible results. However, these minor difficulties are more than compensated for by their exclusive detection of biologically active molecules, a feature as yet unmatched by other assay methods.

Formation of interleukin-6 in the brain of the febrile cat: relationship to interleukin-1.

Previous investigations have shown that interleukin-6 (IL-6), unlike other cytokines, is produced in larger amounts in the brain of the febrile animal regardless of the route, peripheral vs. central, of pyrogen administration. In addition, depending on the experimental condition IL-6 production may or may not require the prior induction of interleukin-1 (IL-1). The present study was carried out in the conscious cat to assess the importance of brain-derived IL-6 in the pathogenesis of fever and the interaction at that site between this cytokine and IL-1. IL-6 was detected in cerebrospinal fluid (CSF) collected at rest and its levels increased during the fever to intravenous (i.v.) endotoxin. The IL-6 elevation, but not the fever, was reversed by pretreatment with intracerebroventricular (i.c.v.) IL-1 receptor antagonist (hIL-1ra). Conversely, when pyrogens (endotoxin, IL-1) were given i.c.v., i.c.v. hIL-1ra reduced the fever without altering significantly the associated rise in CSF IL-6. We conclude that IL-6 is formed in brain in response to both i.v. and i.c.v. pyrogens; however, its formation, whether requiring the prior induction of IL-1 or not, does not appear to be critical for the development of the fever. Blood-borne IL-6, unlike brain-derived IL-6, may still play a role in fever as a trigger of signal-transducing mechanisms operating across the blood-brain barrier.

Assessment of the skin sensitization potential of topical medicaments using the local lymph node assay: an interlaboratory evaluation.

The murine local lymph node assay (LLNA) is a method for the predictive identification of chemicals that have a potential to cause skin sensitization. Activity is measured as a function of lymph node cell (LNC) proliferative responses stimulated by topical application of test chemicals. Those chemicals that induce a threefold or greater increase in LNC proliferation compared with concurrent vehicle controls are classified as skin sensitizers. In the present investigations we have evaluated further the reliability and accuracy of the LLNA. In the context of an international interlaboratory trial the sensitization potentials of six materials with a history of use in topical medicaments have been evaluated: benzoyl peroxide, hydroquinone, penicillin G, streptomycin sulfate, ethylenediamine dihydrochloride, and methyl salicylate. Each chemical was analyzed in the LLNA by all five laboratories. Either the standard LLNA protocol or minor modifications of it were used. Benzoyl peroxide and hydroquinone, both human contact allergens, elicited strong LLNA responses in each laboratory. Penicillin G, another material shown previously to cause allergic contact dermatitis in humans, was also positive in all laboratories. Streptomycin sulfate induced equivocal responses, in that this material provoked a positive LLNA response in only one of the five laboratories, and then only at the highest concentration tested. Ethylenediamine dihydrochloride dissolved in a 3:1 mixture of acetone with water, or in 4:1 acetone:olive oil (one laboratory), was uniformly negative. However, limited further testing with the free base of ethylene diamine yielded a positive LLNA response when applied in acetone:olive oil (AOO). Finally, methyl salicylate, a nonsensitizing skin irritant, was negative at all test concentrations in each laboratory. Collectively these data serve to confirm that the local lymph node assay is sufficiently robust to yield equivalent results when performed independently in separate laboratories and indicate also that the LLNA is of value in assessing the skin sensitization potential of topical medicaments.

Successful treatment of herpetic infections by autohemotherapy.

Herpes zoster (shingles) affects a significant number of individuals over age 50. To date, no satisfactory treatment has been available. The clinician author (JHO) witnessed a dramatic response of a shingles patient to autohemotherapy: the pain was completely relieved and lesions gone within 5 days with no recurrence of either. Treatment of other herpetic patients then began with autohemotherapy. Twenty-five patients with herpes were given an autologous blood transfer of 10 mL of blood from the antecubital vein into the gluteal bundle and followed for clinical signs. A 100% favorable response occurred in 20 patients who received autohemotherapy within 7 weeks of the onset of clinical signs and 1 other who received autohemotherapy at a 9-week interval. No untoward signs or symptoms of the treatment occurred. Autohemotherapy has been demonstrated to be effective in elimination of clinical sequelae in these cases of herpes infections and these results justify further rigorous clinical investigation.

In vitro exposure to peptidic delta opioid receptor antagonists results in limited immunosuppression.

Previous studies by our group have demonstrated that in vitro exposure to delta-opioid receptor agonists results in a significant immunostimulation, whereas in vitro exposure to non-peptidic delta-opioid receptor antagonists results in significant suppression of various immune functions. The present study assessed potential immunomodulation by the peptidic delta-opioid receptor antagonists TIPP, D-TIPP, and ICI 174864 using a panel of in vitro immune function assays. Splenocytes from female B6C3F1 mice were cultured with the peptides at concentrations of 0.00001-10 microM. B cell proliferation was quantified following cellular activation, T cell function was assessed by cytokine production following stimulation with anti-CD3 monoclonal antibody, natural immunity was assessed by quantitating natural killer (NK) cell activity following a 24-h exposure, and macrophage function was assessed by quantification of interleukin-6 (IL-6) production. None of the peptides examined significantly affected B cell proliferation. Production of IL-2 by T cells was not consistently affected by exposure to either TIPP or D-TIPP, but was significantly suppressed at 10 microM ICI 174864. Production of IL-4, however, was significantly suppressed by low concentrations of either TIPP or D-TIPP, and by 10 microM ICI 174864. IL-6 production by macrophages was unaffected except for sporadic incidents of enhanced production in cells exposed to ICI 174864. NK cell function exhibited a differential pattern of suppression, with the greatest degree of suppression observed following exposure to TIPP and only slight suppression in cells exposed to either D-TIPP or ICI 174864. These data suggest that peptidic delta-opioid receptor antagonists do not exhibit the same pattern or degree of immunosuppressive activity as the non-peptidic antagonists at equivalent in vitro concentrations.

Immune function and host defense in rodents exposed to 60-Hz magnetic fields.

This study was conducted to evaluate the influence of subchronic exposure to pure, linearly polarized 60-Hz magnetic fields (MF) on the host immune response in mice. The experimental design was as follows: three groups were exposed continuously (18.5 hr/day) to MF at field strengths of 0.02, 2, or 10 gauss (G), one group was exposed intermittently (1 hr on/1 hr off) to MF at a field strength of 10 G, and one group served as a sham control. Experimental endpoints included spleen and thymus weights and cellularity, antibody-forming cell (AFC) response, delayed-type hypersensitivity (DTH) response, splenic lymphocyte subset analysis, susceptibility to infection with Listeria monocytogenes, and natural killer (NK) cell activity. No differences in body weight, lymphoid organ weight, or lymphoid organ cellularity were observed in any MF-exposed group in comparison to sham controls. Likewise, no statistically significant differences were found in comparisons of AFC responses. Isolated statistically significant differences from control were observed in MF-exposed mice in the DTH assay, although no clear dose-related pattern of altered activity was seen. Splenic lymphocyte subset parameters examined were within normal limits in all groups, and no differences between control and MF-exposed mice were found. Host resistance to bacterial infection was not altered at any MF exposure examined in this study. Finally, although apparently dose-related, statistically significant alterations were observed in an initial study of NK cell function, repeat studies failed to demonstrate a consistent pattern of alteration.

Effects of chronic administration of 7-benzylidene-7-dehydronaltrexone and naltriben on the antinociceptive actions of delta 1- and delta 2-opioid receptor agonists.

The effects of chronic administration of 7-benzylidene-7-dehydronaltrexone, a delta 1-opioid receptor antagonist and naltriben, a delta 2-opioid receptor antagonist, on the antinociceptive responses to [D-Pen2, D-Pen5] enkephalin and [D-Ala2, Glu4]deltorphin II, delta 1- and delta 2-opioid receptor agonists, respectively, were determined in the mouse. Female B6C3F1 mice were given 7-benzylidene-7-dehydronaltrexone (3 mg/kg/day), naltriben (1 mg/kg/day) or the vehicle by subcutaneously implanted Alzet osmotic minipumps for 7 days. Both [D-Pen2, D-Pen5]enkephalin and [D-Ala2, Glu4]deltorphin II administered intracerebroventricularly (i.c.v.) produced antinociceptive as measured by the tail-flick test with ED50 values of 6.76 and 6.68 micrograms/mouse, respectively. Chronic administration of 7-benzylidene-7-dehydronaltrexone lowered the ED50 of [D-Pen2, D-Pen5]enkephalin but not of [D-Ala2, Glu4]deltorphin II. Chronic administration of naltriben lowered the ED50 of [D-Ala2, Glu4]deltorphin II but had no effect on the ED50 of [D-Pen2, D-Pen5]enkephalin. The binding of [3H][D-Pen2, D-Pen5]enkephalin to whole brain membranes of chronic 7-benzylidene-7-dehydronaltrexone-treated mice did not differ from chronic vehicle-treated mice. On the other hand, chronic administration of naltriben resulted in slight but reproducible elevation in the Bmax value of [3H][D-Pen2, D-Pen5]enkephalin to bind to whole brain membranes in comparison to vehicle-injected controls. The results suggest that chronic treatment with delta 1- and delta 2-opioid receptor antagonist cause behavioral supersensitivity to their agonists, respectively, and provides further evidence for the existence of delta-opioid receptor subtypes.

Cellular immune function in mice tolerant to or abstinent from l-trans-delta 9-tetrahydrocannabinol.

The effects of tolerance to l-trans-delta 9-tetrahydrocannabinol (THC) following repeated administration and of subsequent abstinence following drug withdrawal on the cellular immune function were determined in female B6C3F1 mice. Mice were injected with THC (10 mg/kg s.c.) twice daily for 4 days. On day 5, analgesic response to various doses of THC was determined using the tail flick test. Similarly, hypothermic response to THC was also determined. Multiple injections of THC resulted in the development of tolerance to both the analgesic and hypothermic effects of THC. Immune function studies were performed on mice rendered tolerant to or abstinent from THC by these procedures. Neither tolerance nor abstinence subsequent to a 4-day THC administration had any effect on either body weight or thymus weight and cellularity, although both spleen weight and cellularity were decreased in THC-abstinent animals. Likewise, no significant effects on B cell proliferation were observed in either tolerant or abstinent mice. The production of the cytokine interleukin-2 by T helper cells was markedly suppressed in both tolerant and abstinent mice, whereas the production of interleukin-4 was significantly suppressed only in THC-abstinent mice. Significant suppression of tumor cell cytolysis mediated by cytotoxic T cells and natural killer cells was only observed in THC-abstinent mice. These results suggest that THC-mediated modulation of the immune response may result from a differential effect on cellular populations.

Further evaluation of the local lymph node assay in the final phase of an international collaborative trial.

The local lymph node assay (LLNA) is a method used for the prospective identification in mice of chemicals that have the potential to cause skin sensitization. We report here the results of the second and final phase of an international trial in which the performance of the assay has been evaluated using seven test materials in five independent laboratories. The additional chemicals examined here included compounds which are considered less potent allergens than some of those tested in the first phase of the investigation, and includes hexylcinnamic aldehyde (HCA), a chemical recommended by the Organization for Economic Cooperation and Development (OECD) as a positive control for skin sensitization studies. In each laboratory all skin sensitizing chemicals examined (2,4-dinitrochlorobenzene {DNCB}, HCA, oxazolone, isoeugenal and eugenol) elicited positive responses of comparable magnitude as judged by the derived lowest concentration of test chemical required to elicit a 3-fold or greater increase in the proliferative activity of draining lymph node cells compared with vehicle-treated controls. We observed that sodium lauryl sulphate, considered to be a non-sensitizing skin irritant, also induced a positive response in the assay. Para-aminobenzoic acid (pABA), a nonsensitizing chemical, was negative at all test concentrations in each laboratory. Some laboratories incorporated minor modifications into the standard assay procedure, including the evaluation of lymph nodes pooled from individual mice rather than treatment groups and the use of statistical analyses. The use of statistics did not markedly change the determination of the lowest concentration yielding a positive response. These data confirm that the local lymph node assay is robust and yields equivalent results when performed independently.

A comparative study of immunomodulation produced by in vitro exposure to delta opioid receptor agonist peptides.

The present study assessed the direct immunomodulatory effect of a panel of synthetic peptides exhibiting delta-opioid receptor agonist activity. Murine splenic lymphocytes and peritoneal macrophages were cultured in vitro with peptides at concentrations of 0.00001-10 microM. Assessment was made of B-cell function by quantitating cellular proliferation, T-cell function by measuring cytokine production, natural immunity by quantitating basal and cytokine-augmented natural killer (NK) cell activity, and macrophage function by production of IL-6. These peptides had minimal effects on B-cell proliferation at any concentration examined. In comparison, enhancement of cytokine production by T-helper cells occurred following exposure to several of the compounds, to a significant extent with DPDPE, DPDPE-trifluoroacetate, or deltorphin-1 and most pronounced at concentrations between 0.00001 and 0.1 microM. Likewise, IL-6 production by macrophages was significantly augmented by exposure to these three peptides. NK cell function was significantly enhanced by in vitro exposure to several of the peptides, with enhancement generally noted at concentrations between 0.00001 and 0.01 microM. However, some of the peptides (most notably DADLE) greatly suppressed NK cell activity. These data suggest that delta opioid agonists are broadly immunomostimulatory.

Response of newborn and adult sheep to pyrogens: relation between fever and brain eicosanoid changes.

We investigated whether the weak febrile response to pyrogens in newborns is due to a diminished activation of the putative pyrogen mediator, prostaglandin (PG)E2. Indwelling cannulas in the third ventricle of lambs (age, 5-31 days) and adult ewes were used to collect cerebrospinal fluid (CSF) for radioimmunoassay of PGE2. Intravenous (i.v.) endotoxin caused a smaller increase in body temperature but a larger increase in CSF PGE2 in lambs compared to adults. PGE2 by intracarotid infusion raised body temperature in 5 of 7 trials in 3 lambs and in 4 of 4 trials in 1 adult. Endotoxin given intracerebroventricularly (i.c.v.) induced a rise in temperature and CSF PGE2 in the lamb but, in the adult, these responses were delayed and smaller. Interleukin-1 i.c.v. and PGE2 i.c.v. were weak pyretic agents at both ages. We conclude that the lamb's diminished febrile response to endotoxin i.v. is not caused by a lesser rise in CSF PGE2, rather it may be due, at least in part, to reduced responsiveness to this putative mediator. Regardless of age, the sheep differs from other species in that pyrogen/PGE2 coupling occurs primarily at a site in brain that is better accessible from blood than CSF.

An international evaluation of the murine local lymph node assay and comparison of modified procedures.

The murine local lymph node assay is a predictive test for the identification of skin-sensitizing chemicals. The method has been the subject both of national inter-laboratory studies and of extensive comparisons with guinea pig tests. In the investigations reported here, the local lymph node assay has been evaluated further in the context of an international study comprising five independent laboratories. In addition, the influence of minor modifications to the standard assay procedure on the performance of the test has been examined. The modified procedures investigated were exposure of mice for 4 rather than 3 consecutive days, excision of lymph nodes 4 rather than 5 days after the initiation of exposure and the use of an alternative isotope. All five laboratories, irrespective of whether the standard or a modified protocol was used, were able to identify accurately, and with comparable sensitivity, potassium dichromate and 2,4-dinitrochlorobenzene as skin sensitizers. Using standard criteria, none of the laboratories recorded positive responses with methyl salicylate, a non-sensitizer. In the standard protocol, lymph nodes are pooled for each experimental group and the vigor of responses measured as a stimulation index relative to vehicle controls. A stimulation index of 3 or greater is considered to indicate skin-sensitizing potential. One further modification adopted by three of the laboratories was to analyze nodes from individual animals and, thereby, permit statistical evaluation. This allowed a direct comparison of statistical significance with the conventional stimulation index as criteria for a positive response. The data indicate that, while statistical evaluation may provide, in some instances, for small increases in sensitivity, this may be at the expense of some loss of selectivity. There are, however, insufficient data presently to draw firm conclusions regarding the relative value of statistical analysis. These studies demonstrate that the local lymph node assay is sufficiently robust to accommodate minor procedural and technical modifications without material changes in test performance.

Suppression of immune function by non-peptidic delta opioid receptor antagonists.

Previous studies in this laboratory and elsewhere have provided evidence that compounds acting as delta opioid receptor agonists exhibit marked immunostimulatory potential. Conversely, the delta opioid receptor antagonists have previously been shown to demonstrate immunosuppressive effects as assessed by proliferation of T-cells following allogeneic or xenogeneic stimulation. The present study was performed to further characterize this immunosuppressive activity using the compounds benzylidene naltrexone (BNTX), naltrindole (NTI), and naltriben (NTB). In vitro exposure to BNTX resulted in an apparent dose-related suppression of B-cell proliferation, cytokine production by T-helper cells, and natural killer (NK) cell activity, with statistically significant suppression observed at concentrations between 1 and 10 microM. NTI was also immunosuppressive for all immune function parameters examined, although this compound was less active than BNTX. In vitro exposure to the structurally related compound NTB had no significant effect on any immune function examined in this study. In all cases, immunosuppression occurred in the absence of any detectable alteration in cellular viability, suggesting a specific immunosuppressive effect rather than overt toxicity.

Local versus systemic immunotoxicity of isobutyl nitrite following subchronic inhalation exposure of female B6C3F1 mice.

Female B6C3F1 mice were exposed to isobutyl nitrite (IBN) by inhalation at 0, 37.5, 75, or 150 ppm for 6 hr per day, 5 days per week for 15 weeks. The potential of this compound to induce immunotoxicity was assessed during the 3rd, 13th, 14th, and 15th week of exposure and after 2 weeks of recovery following the 15 weeks of exposure. Both systemic and lung immune functions were examined, including body and lymphoid organs weights, pulmonary macrophage function and host defense, expression of splenic lymphocyte cell-surface markers, natural killer cell function, mixed lymphocyte reaction, and induction of specific antibody to a T-cell-dependent antigen. There was a dose-related suppression of T-cell-dependent antibody-forming cell responses in the spleen following IBN exposure; however, other measures of T-cell and nonspecific immunity were not significantly affected. A dose-related increase of H202 production by alveolar macrophages was present after 12 but not after 68 exposures to IBN. In contrast, pulmonary host defense mechanisms against Klebsiella pneumoniae were unaffected. These results suggest that in the absence of changes in host resistance, IBN may have selective and partially reversible effects on the immune system.