Aleksandra Popov Aleksandrov

Acute cadmium administration to rats exerts both immunosuppressive and proinflammatory effects in spleen.

Conflicting data (both suppression and augmentation as well as lack of the effect) exist in respect to cadmium (Cd) and splenic T cell-based immune cell activity. Spleen is also the site of innate immune responses but impact of Cd on this type of immunity has been less explored. In the present study the effects of acute Cd administration on basic aspects of both T cell-based and innate immune spleen cell activity were examined in rats. Intraperitoneal injection of 1mg of Cd/kg resulted in decrease in concanavalin A (ConA) induced proliferation which seems to be more related to altered spleen cells responsiveness to IL-2 than to apoptosis. Differential effects on proinflammatory T cell derived cytokines were observed (decreases of IFN-γ gene expression and ConA-stimulated production, but increases in IL-17 mRNA levels with no effect on concentrations of protein product). Reduction of IFN-γ production seemed not to rely on IL-4 and IL-10, but at least partly on nitric oxide (NO). Increased activity relevant for innate immunity (granulocyte and CD11b(+) cell accumulation in the spleen, inducible nitric oxide synthase/iNOS expression and NO production by spleen cells) was observed, but there was a decrease in respiratory burst (dihydrorhodamine/DHR oxidation and nitroblue tetrazolium/NBT reduction). Increases of TNF-α and IL-1β gene expression and IL-1β protein product were noted as well. Administration of 0.5mg Cd/kg resulted in less pronounced (ConA-induced proliferation) or lack of the effect (IFN-γ production) on spleen T cell activities and on innate activities (granulocyte accumulation, NO production) as well. However, increases of spleen cell respiratory burst activity and IL-1β production were observed. Effects of lower cadmium doses (5ppm and 50ppm) on several aspects of spleen cell immune activity were observed in intermediate period of exposure (30 days, oral intake) as well. Differential effects of Cd on immune activities of spleen cells might contribute to our understanding of the complexity of immunomodulatory effects of this metal.

Toxicity of oral cadmium intake: Impact on gut immunity

Gastrointestinal tract is one of the main targets of cadmium (Cd), an important food and drinking water contaminant. In the present study, the effect of subchronic (30 days) oral (in water) intake of 5ppm and 50ppm of cadmium on immune responses in the gut was examined in rats. Cadmium consumption resulted in reduction of bacteria corresponding to Lactobacillus strain, tissue damage and intestinal inflammation [increases in high mobility group box 1 (HMGB1 molecules), superoxide dismutase (SOD) and catalase (CAT) activity and proinflammatory cytokine (TNF, IL-1β, IFN-γ, IL-17) content]. Draining (mesenteric) lymph node (MLN) stress response was observed [elevation of MLN glutathione (GSH) and metallothionein (MT) mRNA levels] and stimulation of both adaptive [cellularity, proliferation, proinflammatory (IFN-γ and IL-17) MLN cell cytokine responses] as well as innate immune activity (increases in numbers of NK and CD68(+) cells, oxidative activities, IL-1β). In contrast to proinflammatory milieu in MLN, decreased or unchanged antiinflammatory IL-10 response was observed. Stimulation of immune activities of MLN cells have, most probably, resulted from sensing of cadmium-induced tissue injury, but also from bacterial antigens that breached compromised intestinal barrier. These effects of cadmium should be taken into account when assessing dietary cadmium as health risk factor.

Proinflammatory cytokine responses in skin and epidermal cells following epicutaneous administration of anticoagulant rodenticide warfarin in rats.

Abstract Context: Dermal toxicity of coumarin anticoagulant rodenticides, such as warfarin, represents potential risk for workers handling these agents and for individuals applying easily available rodenticides in their households as well. Objective: In this study, proinflammatory effects of repeated epicutaneous administration of warfarin in rats were explored by examining inflammatory cytokine skin responses. Materials and methods: Ex vivo production of IL-1β, IL-6, TNF-α and IL-17 by skin explants and by epidermal cells isolated by enzyme (dispase/trypsin) digestion from skin repeatedly (once a day, three consecutive days) exposed to 10 µg of warfarin was measured 24 h and 72 h following the last warfarin application by ELISAs for respective rat cytokines. Results: Warfarin treatment resulted in histological changes, but skin or epidermal cell viability were not compromised, judging by MTT reduction assay. Both skin and epidermal cells responded to administration of this agent by production of all examined inflammatory cytokines (skin explants by TNF-α and IL-17; epidermal cells by IL-1β and TNF-α) except IL-6. Discussion: Along with histomorphological changes, cytokines indicate functional consequences in treated skin. IL-1β production, that precede production of TNF-α, might be responsible for production of the latter cytokine. Sustained production of IL-1β suggests persistence of epidermal cell stimulation or existence of some amplification mechanisms. Requirements for T cells seem to exist concerning epidermal cell IL-17 production. Conclusion: Presented data provide additional new information concerning proinflammatory effects of warfarin.

Regional cytokine responses to pulmonary aspergillosis in immunocompetent rats.

Rat models of pulmonary aspergillosis are used widely in diagnostic studies and in exploring antifungal therapeutic modalities, but there is lack of data concerning antifungal immunity in rats. In this study, cytokine response to pulmonary infection to Aspergillus fumigatus in non-immunosuppressed rats is explored. Temporal display (from the start of infection up to its eradication) of proinflammatory cytokines (IFN-γ and IL-17) as well as Th2/anti-inflammatory ones (IL-4 and IL-10) was explored by measuring their presence in the environment in which elimination of infection occur (lung homogenates), by production of these mediators by lung cells (recovered by enzyme digestion or by bronchoalveolar lavage) as well as by cells of draining lymph nodes (as sites of generation of cytokine-producing cells). Reduction of infection (1 × 10⁷conidia) was associated with an increase of IFN-γ and IL-17 content in lung homogenates, but with unchanged IL-4 and IL-10 content. Lung cells produced proinflammatory cytokines with differential dynamics (IFN-γ earlier than IL-17). Differential pattern of Th2/anti-inflammatory cytokine production by lung cells was observed (unchanged IL-4 and increased IL-10), with the levels of the latter higher than proinflammatory cytokines. Upregulation of IFN-γ, IL-17 and IL-10 production and gene expression, but downregulation of IL-4, by draining lymph node cells (dLN cells) accounted essentially for the observed ex vivo cytokine response in lungs. Similar pattern of cytokine production by dLN cells following restimulation with A. fumigatus conidia confirmed the specificity of cytokine response to the fungus. Draining lymph node CD4⁺ cells seems to be the main source of proinflammatory cytokines, significant contributors to IL-10 production and the target for down regulation of IL-4. The knowledge of immune-based mechanisms of defense against A. fumigatus in rats might be helpful in the future use of rat models of pulmonary aspergillosis particularly those that develop immune-based therapeutic interventions as an adjunct treatment of fungal diseases.

Strain differences in the immune mechanisms of resistance of immunocompetent rats to pulmonary aspergillosis

Although the relevance of genetically-based variations in susceptibility to pulmonary aspergillosis was shown in immunocompromised mice and is indicated in humans, there is virtually no information concerning variations in antifungal immune responses in resistant individuals. We have shown recently the relevance of proinflammatory cytokine (interferon-γ/IFN-γ and interleukin-17/IL-17) responses in resistance to sublethal Aspergillus fumigatus infection of non-suppressed Dark Agouti (DA) rats (strain known of a substantial immune reactivity to noxious insults). In this study, anti-fungal immune activities of leukocytes recovered from lungs by enzyme digestion (phagocytosis, oxidative activity, hyphal killing, CD11b expression, as well as production of IFN-γ, IL-17 and Th2/anti-inflammatory cytokines interleukin-4/IL-4 and interleukin-10/IL-10) were investigated in less reactive Albino Oxford (AO) and compared to DA rats. Elimination of fungus from lungs of AO rats was associated with lower degree of leukocyte infiltration and of the majority of their basic effector activities in comparison to DA rats. Lower production of IFN-γ by pulmonary leukocytes was observed early (day 1) post infection (p.i.) in AO compared to DA rats, but without changes in IL-4. Both strains responded to infection by an increase of IL-17 and IL-10, but production of cytokines was higher (from days 7 p.i. and 3 p.i. for IL-17 and IL-10, respectively) in AO compared to DA rats. The levels and pattern of IFN-γ and IL-4 responses by draining lymph node (dLN) cells were similar in both strains and basically corresponded to those of lung leukocytes. In contrast, similar levels of draining lymph node cell production of IL-17 and IL-10 were observed in both strains with lack of changes in mRNA, what suggests additional stimulation of these cytokines in lungs of AO rats. The knowledge of strain differences in the immune-based strategies in response of immunocompetent hosts to A. fumigatus might contribute to our understanding of variations in underlying mechanisms that enable of resistance to this fungus.

Strain differences in contact hypersensitivity reaction to dinitrochlorobenzene (DNCB) in rats.

Genetic factors are among the most important determinants of susceptibility to induction of allergic contact dermatitis. A limited number of studies of experimental contact hypersensitivity (CHS) in animals has shown differences in the severity of CHS; however, the underlying mechanisms are unknown. In this study comparative analysis of CHS to low and high dinitrochlorobenzene (DNCB) doses regimen of sensitization/challenge in inbred Dark Agouti (DA) and Albino Oxford (AO) rats was examined. Basic aspects of draining lymph node (dLN) activity (cellularity, proliferation), proinflammatory (IFN-γ, IL-17) and anti-inflammatory (IL-10) cytokine gene expression and production, as well as IL-12 and IL-23 subunits mRNA expression, were examined in challenge and sensitization phase of CHS reaction. Lower (compared to DA) intensity of CHS in AO rats was associated with lack of (or negligible) dLN responses in challenge phase (ex vivo, hapten- or IL-2-stimulated cell proliferation and proinflammatory cytokine mRNA and production levels) but with lack of changes in IL-10 response. Less pronounced dLN activity of sensitized animals of this strain was observed as well. Higher proliferative activity and more pronounced proinflammatory cytokine response during challenge and sensitization phase suggest these activities as underlying mechanisms of higher susceptibility of DA rats to CHS response to DNCB.

Strain differences of cadmium-induced toxicity in rats: Insight from spleen and lung immune responses.

The impact of genetic background on effects of acute i.p. cadmium administration (0.5mg/kg and 1mg/kg) on basic immune activity of spleen and lungs was examined in two rat strains, Albino Oxford (AO) and Dark Agouti (DA), known to react differently to chemicals. More pronounced inhibition of Concanavalin A (ConA)-induced and Interleukin (IL)-2 stimulated spleen cell proliferation as well as higher levels of nitric oxide (known to decrease cells proliferative ability) in DA rats at 1mg/kg, along with greater inhibition of ConA-induced Interferon (IFN-γ)-production by total and mononuclear (MNC) spleen cells and IL-17 production by spleen MNC in DA vs. AO rats at this dose show greater susceptibility of this strain to Cd effects on spleen cells response. More pronounced infiltration of neutrophils/CD11b(+) cells to lungs of DA rats treated with 1mg/kg of Cd and decreased IL-17 lung cell responses noted solely in DA rats speaks in favor of their higher susceptibility to this metal. However, lack of strain disparity in lung cells IFN-γ responses show that there are regional differences as well. Novel data from this study depict complexity of the influence of genetic background on the effects of cadmium on host immune reactivity.

Cadmium administration affects circulatory mononuclear cells in rats

Abstract Although numerous investigations have demonstrated a direct effect of cadmium (Cd) on peripheral blood mononuclear cell (PBMC) activity in humans, there is virtually no data concerning the in vivo impact of this metal on circulatory mononuclear cells. In this study, the effects of a sub-lethal Cd (1 mg/kg) dose were examined in rats 48 h following a single intraperitoneal injection. Cd treatment resulted in increased total peripheral blood leukocyte levels; however, decreases in PBMC numbers were seen. These changes coincided with an accumulation of mononuclear cells in the lungs and an increase in mononuclear cells expressing CD11b. A lack of effect of Cd on spontaneous nitric oxide (NO) production and on iNOS mRNA levels in the PBMC was also noted. Differential effects of Cd on PBMC inflammatory cytokine (IL-1β, TNFα, IL-6, IFNγ, and IL-17) gene expression and production were also seen. Specifically, except for IL-1β (levels increased), there were decreases (relative to controls) in mRNA levels for all the other cytokines examined. While there were no Cd treatment-related changes in spontaneous production of the cytokines assessed, there seemed to be a trend (p = 0.06) toward a decrease in spontaneous IL-6 release. When these harvested cells were stimulated ex vivo, there was no effect from Cd exposure on LPS-stimulated IL-1β and TNFα or on ConA-stimulated IFNγ or IL-17 production, but a decrease in IL-6 production in response to LPS was, again, noted. A preliminary study with a lower Cd dose (0.5 mg/kg) revealed some of the same outcomes noted here (mononuclear cell infiltration into lungs, increases in PBMC IL-1β mRNA levels), but differential (increased IL-17 mRNA levels) or newly detected outcomes (increased levels of IL-1α mRNA) as well. The described effects of the single in vivo exposure to Cd on PBMC might contribute to a better overall understanding of the immunomodulatory potential of this environmental contaminant.

Pulmonary Immune Responses to Aspergillus Fumigatus in Rats

OBJECTIVE To evaluate immunologic mechanisms underlying Aspergillus fumigatus pulmonary infections in immunocompetent Dark Agouti (DA) and Albino Oxford (AO) rats recognized as being susceptible to some inflammatory diseases in different manners. METHODS Lung fungal burden (quantitative colony forming units, CFU, assay), leukocyte infiltration (histology, cell composition) and their function (phagocytosis, oxidative activity, CD11b adhesion molecule expression) and cytokine interferon-γ (IFN-γ) and interleukin-17 and -4 (IL-17 and IL-4) lung content were evaluated following infection (intratracheally, 1x10(7) conidia). RESULTS Slower reduction of fungal burden was observed in AO rats in comparison with that in DA rats, which was coincided with less intense histologically evident lung cell infiltration and leukocyte recovery as well as lower level of most of the their activities including intracellular myeloperoxidase activity, the capacity of nitroblue tetrazolium salt reduction and CD11b adhesion molecule expression (except for phagocytosis of conidia) in these rats. Differential patterns of changes in proinflammatory cytokine levels (unchanged levels of IFN-γ and transient increase of IL-17 in AO rats vs continuous increase of both cytokines in DA rats) and unchanged levels of IL-4 were observed. CONCLUSION Genetically-based differences in the pattern of antifungal lung leukocyte activities and cytokine milieu, associated with differential efficiency of fungal elimination might be useful in the future use of rat models in studies of pulmonary aspergillosis.

Transdermal toxicity of topically applied anticoagulant rodenticide warfarin in rats.

Occupational/accidental exposure data have showed hemorrhage as a result of transdermal exposure to warfarin, however, other effects are not known. In the present study, the impact of epicutaneous application of 10 μg or 100 μg of warfarin (three times, once a day) on peripheral blood polymorphonuclear (PMN) and mononuclear cells (PBMC) was examined in rats. Both doses resulted in prolongation of prothrombin time and changes in hematologic parameters. Increases in PMN intracellular myeloperoxidase (MPO) activity were seen at higher warfarin dose and both doses resulted in higher percentages of granular CD11b(+) cells. In contrast, a decrease in PMN TNF and IL-6 production (ELISA) and gene expression (RT-PCR) was observed. Epicutaneous application of warfarin resulted in decreased numbers of PBMC, higher numbers of mononuclear CD11b(+) cells, but without effect on PMBC cytokine production. The data obtained showed differential effects of transdermal exposure to warfarin depending on leukocyte type and activity.