Popliteal lymph node responses to acetone and ethanol differ from those induced by streptozotocin

The popliteal lymph node (PLN) assay was proposed to detect the potential of immunotoxicants for inducing systemic autoimmune-like reactions, but also xenobiotics that are sensitizing or exert immunostimulatory properties. Results on over 100 chemicals, mostly pharmaceuticals, are available with the PLN assay and show many correlations between rodent data and the clinical experience. A major issue is that the mechanisms involved have not been fully elucidated. In order to provide mechanistic clues to improve the predictability of the PLN assay, the effects of streptozotocin (STZ) were compared to those of ethanol and acetone in normal C57Bl/6 mice as well as mice depleted in CD4⁺ or CD8⁺ T-cells by treatment with specific monoclonal antibodies. STZ, ethanol and acetone gave similar positive responses in normal mice. Neither CD4⁺ nor CD8⁺ T-cell depletion influenced the PLN responses to ethanol or acetone, whereas CD8⁺ in contrast to CD4⁺ T-cell depletion abolished the response to STZ. There was an increase in the production of IL-6 and IFN- mRNAs measured by RT-PCR in STZ-, but not in ethanol- or acetone-treated normal mice. The production of TNF, IL-1, IL-1, IL-2R and IL-12 mRNAs was increased whatever the treatment, but increases were 2- to 3-fold greater after STZ than ethanol or acetone. These results suggest that PLN responses to primary irritants such as ethanol and acetone essentially reflect non-specific inflammation, whereas PLN responses to an autoimmunogenic compound such as STZ involve CD8⁺ T lymphocytes and the production of IFN- and IL-6. These findings may prove useful to improve the predictability of the PLN assay.     

Increased production of interferon-gamma, but not IL-4 mRNA, by streptozotocin in the popliteal lymph node assay

The popliteal lymph node (PLN) assay has been proposed as a tool to predict systemic autoimmune reactions induced by medicinal products and chemicals, the mechanisms of which are poorly understood. To determine whether PLN responses involved Th1 or Th2 cell control, or both, the effects of streptozotocin (STZ), a prototypic immunotoxic compound, were analysed on the production of interferon-gamma (IFN-gamma) and interleukin-4 (IL-4) mRNA by lymph node cells after injection into the hind footpad of C57 BL/6 mice. Streptozotocin induced a dramatic increase in IFN-gamma mRNA production, which correlated with PLN responses as evidenced by augmented weight and cellularity indices. No effect on IL-4 mRNA synthesis was noted. These results suggest that a Th1 response is involved in the PLN response to STZ.     

Diclofenac activates T cells in the direct popliteal lymph node assay and selectively induces IgG(1) and IgE against co-injected TNP-OVA

Non-steroidal anti-inflammatory drugs (NSAIDs) are frequently associated with immune-mediated hypersensitivity reactions. The NSAID diclofenac is associated with several distinct allergic and autoimmune-like reactions including anaphylaxis, idiosyncratic hepatotoxicity and autoimmune hemolytic anemia. The aim of this study was to examine the immunostimulating potential of diclofenac in the direct popliteal lymph node assay (PLNA) and reporter antigen PLNA. In BALB/c mice, diclofenac caused dose-dependent increases in PLN weight and PLN cellularity in the direct PLNA; 0.25 mg was non-immunostimulating whereas 0.50-1.00 mg caused a significant PLN reaction. In the direct PLNA, diclofenac also increased the percent of T cells in the PLN with activated phenotypes (CD44(high)CD62L(low) and CD44(high)CD62L(high)). Finally, the magnitude of the diclofenac-induced direct PLN reaction was significantly reduced when the assay was conducted in T-cell-deficient mice. When co-injected with the reporter antigen TNP-Ficoll (trinitrophenyl Ficoll), 0.50 mg diclofenac caused significant increases in PLN weight, PLN cellularity, and induced IgM and IgG(1) anti-TNP antibody forming cells (AFCs) in the PLN. In a final set of studies, a TNP-OVA PLNA was conducted using diclofenac, phenobarbital (negative control) and streptozotocin (positive control). As expected, phenobarbital (1.00 mg) failed to cause an increase in PLN cellularity or induce AFCs in the PLN. Streptozotocin (1.00 mg) caused significant increases in PLN cellularity, IgM AFCs, and selectively induced IgG(2a) and IgG(2b) AFCs against TNP-OVA. Likewise, diclofenac caused dose-dependent increases (0.25-1.00 mg) in PLN cellularity and IgM AFCs. However, in contrast to streptozotocin, diclofenac caused a selective dose-dependent increase in both IgG(1) and IgE AFCs. Finally, an increase in the intracellular level of IL-4, but not INFgamma, was detected in CD4(+) PLN cells following the injection of diclofenac mixed with TNP-OVA. Collectively, these data suggest that diclofenac: (i) induces a T-cell-dependent direct PLN reaction that; (ii) provides non-cognate help for IgG AFC production when co-injected with TNP-Ficoll, possibly through the formation of neo-antigens; and (iii) possesses intrinsic adjuvant activity that selectively induces IL-4 mediated production of IgG(1) and IgE against co-injected TNP-OVA.     

应用腘窝淋巴结实验评价干扰素的免疫毒性

目的用小鼠腘窝淋巴结实验(Popliteallymphnodassay,PLNA)以及检测淋巴细胞表面分子来预测干扰素的免疫毒性,探讨该方法的预测价值。方法采用小鼠腘窝淋巴结实验方法测定腘窝淋巴结重量及细胞指数,淋巴细胞亚群测定采用流式细胞技术法。结果干扰素可引起小鼠腘窝淋巴结肿大,并造成腘窝淋巴结CD4+/CD8+T细胞比例改变。结论本实验中干扰素的测试结果呈阳性,提示用腘窝淋巴结实验对干扰素(Interferon,IFN)的免疫毒性进行初步评估。     

Differentiation of responses to allergenic and irritant compounds in mouse popliteal lymph node assay

The mouse popliteal lymph node assay (PLNA) has been proposed as an immunotoxicological test to predict allergenic chemicals. However, PLN response is also observed in association with non-specific activation induced by some irritants. We, therefore, examined the kinetics of the PLN cellularity indices of primary and secondary responses. Flow cytometric analysis was used to measure the proportions of T and B cells in PLN. Male ICR mice were subcutaneously injected with TNBS (an allergenic compound) or SDS (an irritant compound) in the left hind footpad, and with vehicle in the right one. On day 28 after first injection, mice were injected with 1/10 or 1/100 dose of the initial injection. On day 7 after first injection of TNBS, primary response reached maximal PLN cellularity index (16.5). On day 2 after second injection, secondary response reached maximal PLN cellularity index (13.1). A marked increase in proportion of B cells was observed in the PLN. On the other hand, after first injection of SDS, primary response reached maximal PLN cellularity index (2.8) on day 10, but neither secondary response nor increase in the B cell proportion were observed. These results suggest that the detection of secondary response and the flow cytometric analysis are useful in differentiating responses to allergenic and irritant compounds in the PLNA.     

Non-antibiotic properties of tetracyclines as anti-allergy and asthma drugs

All available therapies for human allergic disease target IgE mediated pathologic responses after IgE has been produced. We are developing tetracyclines as anti-allergy drugs to prevent IgE production, based on our findings that minocycline or doxycycline treatment of allergic asthmatic humans significantly improves their asthma symptoms, reduces their oral steroid requirements, and strongly suppresses their ongoing IgE responses (ELISA, mast cell mediated cutaneous late phase responses); the tetracyclines also strongly suppress peak IgE responses of BPO-KLH sensitized mice (ELISPOT assay, ELISA, skin tests). The antibiotic activity of the tetracyclines is not required for suppression of IgE responses; inclusion of minocycline or doxycycline in sterile culture prevents anti-CD40/IL-4 mediated induction of memory IgE responses by PBMC of allergic asthmatic patients (ELISA), and induction of specific memory IgE responses by spleen cells of BPO-KLH sensitized mice (ELISPOT assay, ELISA). The tetracyclines affect an epsilon specific pathway because IgM, IgG and IgA responses did not decrease. Further, in humans, DTH responses to recall antigens did not decrease. In related studies, we found that two distinct T cell subsets: CD4+CD60 negative and CD8+CD60+ (CD60 is a ganglioside) (humans) and CD4+ Asialo GM1 ganglioside negative and CD8+Asialo GM1 ganglioside+ (mice), both are required for induction of memory IgE responses. Phosphorylated (phos) p38 MAP kinase, but not phos ERK or phos JNK expression by CD4+ and CD8+, including CD8+CD60+, T cells is increased in allergic asthmatic humans, as is IL-4 and IL-10 production. The tetracyclines appear to target T cell pathways to induce suppression of IgE responses because they suppress phos p38 MAP kinase expression by both CD4+ and CD8+, including CD8+CD60+, T cell subsets, and IL-4 and IL-10, while upregulating IL-2 and IFN gamma, and suppressing IgE responses. Our finding that tetracyclines do not require antibiotic activity to suppress IgE responses opens the door to development of new tetracycline-based and other therapeutics for human allergic disease.     

Oxazolone and diclofenac-induced popliteal lymph node assay reactions are attenuated in mice orally pretreated with the respective compound: potential role for the induction of regulatory mechanisms following enteric administration

The murine popliteal lymph node assay (PLNA) was examined as a preclinical assay with the potential to identify low-molecular-weight compounds (LMWCs) that are likely to be associated with immune-mediated drug hypersensitivity reactions (IDHRs) in humans. We hypothesized that the contact sensitizer oxazolone (OX) would cause a strong PLN reaction in naive mice and that the PLN reaction would be attenuated in mice orally pretreated with OX due to the induction of oral tolerance. In naive mice, OX induced a strong PLN reaction and caused dose-dependent increases in PLN size, weight, cellularity, percentage of CD4(+) PLN T cells, and percentage of PLN B cells, with a concomitant decrease in the percentage of CD8(+) PLN T cells. Next, the PLNA was conducted in mice gavaged three times with either OX or vehicle alone (olive oil). Mice pretreated with OX had suppressed PLN reactions following the footpad injection of OX (decrease in PLN size, weight, and cellularity), which was associated with an increase in the percentage of PLN CD8(+)T cells. In contrast, oral pretreatment with OX had no observable effect on the PLN reaction induced following footpad injection of the irrelevant hapten dinitrochlorobenzene (DNCB). Adoptive transfer studies were conducted to examine the mechanism of PLN hyporesponsiveness. It was found that either (1) unfractionated splenocytes or (2) purified CD8(+) splenocytes, but not (3) purified CD4(+) splenocytes isolated from mice gavaged with OX adoptively transferred PLN suppression to naive BALB/c mice. Because OX is not a pharmaceutical, we also examined the NSAID diclofenac (DF) (Voltaren). Like OX, DF caused dose-dependent increases in PLN size, weight, and cellularity in naive mice. Furthermore, like OX, the diclofenac-induced PLN reaction was attenuated in mice that had been orally pretreated three times with DF. However, splenocytes from mice orally treated with DF were not able to adoptively transfer PLN hyporesponsiveness. Collectively, these observations demonstrate that both OX and DF are potent immunostimulators in the PLNA. As importantly, these results demonstrate that the immunostimulating potential of OX and DF in the PLNA is significantly decreased in mice orally exposed to the respective drug, possibly due to the presence of a cellular mechanism of oral tolerance. For OX, the mechanism appears to involve, in part, CD8(+) T cells, whereas the mechanism(s) associated with PLN hyporesponsiveness using DF remain to be defined.     

Tritiated thymidine incorporation does not enhance sensitivity of the popliteal lymph node assay

The popliteal lymph node (PLN) assay has been proposed as a tool to predict drugs and chemicals with the potential to induce systemic autoimmune reactions in man. In this assay, weight and cellularity indices typically are the measured endpoints. The present study was conducted to test whether incorporation of tritiated thymidine could improve sensitivity of the PLN assay. Male and female Balb/c mice were injected with 20 microCi of [3H]-methyl-thymidine intravenously 7 days after receiving 0.5, 1 or 2 mg of diphenylhydantoin, streptozotocin, sulfamethoxazole, ofloxacin, phenobarbital, or metformin intradermally. Results obtained with incorporation of tritiated thymidine were compared to weight indices. No consistent or marked differences in these endpoints were noted whatever the compound used. This study shows that incorporation of tritiated thymidine does not improve sensitivity of the PLN assay.     

Curcumin inhibits Th1 cytokine profile in CD4+ T cells by suppressing interleukin-12 production in macrophages.

1 Interleukin-12 (IL-12) plays a central role in the immune system by driving the immune response towards T helper 1 (Th1) type responses which are characterized by high IFN-gamma and low IL-4 production. In this study we investigated the effects of curcumin, a natural product of plants obtained from Curcuma longa (turmeric), on IL-12 production by mouse splenic macrophages and the subsequent ability of these cells to regulate cytokine production by CD4+ T cells. 2 Pretreatment with curcumin significantly inhibited IL-12 production by macrophages stimulated with either lipopolysaccharide (LPS) or head-killed Listeria monocytogenes (HKL). 3 Curcumin-pretreated macrophages reduced their ability to induce IFN-gamma and increased the ability to induce IL-4 in Ag-primed CD4+ T cells. Addition of recombinant IL-12 to cultures of curcumin-pretreated macrophages and CD4+ T cells restored IFN-gamma production in CD4+ T cells. 4 The in vivo administration of curcumin resulted in the inhibition of IL-12 production by macrophages stimulated in vitro with either LPS or HKL, leading to the inhibition of Th1 cytokine profile (decreased IFN-gamma and increased IL-4 production) in CD4+ T cells. 5 These findings suggest that curcumin may inhibit Th1 cytokine profile in CD4+ T cells by suppressing IL-12 production in macrophages, and points to a possible therapeutic use of curcumin in the Th1-mediated immune diseases.     

Controlled-release particulate cytokine adjuvants for cancer therapy

Cytokine therapy can induce tumor regression in cancer patients but systemic administration of cytokines is accompanied with severe toxicity. Loco-regional delivery represents an effective and less toxic alternative to systemic injection. However; the requirement for frequent repeated injections of recombinant cytokine or the logistical difficulties associated with gene-modification have limited wide-spread use of loco-regional therapy. A simpler alternative local delivery strategy involves the use of controlled-release cytokine depot formulations. These formulations provide the advantage that physiological doses of cytokines are directly released to the tumor microenvironment in a sustained manner. Anti-tumor efficacy of IL-2; IL-12; GM-CSF or TNFalpha-encapsulated polymer microspheres has been evaluated in syngeneic murine and human tumor /SCID mouse xenograft models. A single intra-tumoral injection of these formulations; particularly that of IL-12 in combination with GM-CSF or TNFalpha; promoted the regression of established primary tumors; induced systemic anti-tumor T- and NK-cell responses and achieved complete eradication of disseminated disease. Cellular and molecular analysis of post-therapy tumor microenvironment demonstrated that treatment promoted the activation of tumor-associated T-effector/memory cells; the elimination of CD4+ CD25+ Foxp3+ T-suppressors and the de novo priming of tumor-specific CD8+ T-effector cells. Long-term monitoring of post-therapy tumors revealed that reversal of intra-tumoral immune suppression was transient and that T-suppressor cells rapidly re-infiltrated tumors. Repeated treatment resurrected anti-tumor activity; however, therapeutic efficacy declined with each treatment cycle. The observed loss of therapeutic efficacy was associated with a progressive intensification of the post-treatment T-suppressor cell rebound. In contrast; depletion of T-suppressor cells with low dose chemotherapy prior to each cycle of treatment resulted in a dramatic enhancement of long-term therapeutic efficacy leading to complete remissions. Clinical implications of these findings are discussed herein.     

Cytokine release does not improve the sensitivity and specificity of the direct popliteal lymph node assay

The popliteal lymph node assay (PLNA) is being considered as a tool to predict the potential of drugs for inducing systemic autoimmune and hypersensitivity reactions. Despite the use of different technical approaches and the evaluation of over 130 compounds, the sensitivity and specificity of the PLNA are still debatable due to many false positive and negative responses. In this study, cytokine production was assessed as a possible endpoint to improve the direct (primary) PLNA. Diclofenac, imipramine, hydralazine, glafenin and minocycline were tested using the classical procedure. TH₁ cytokines (IL-2 and IFN-γ), TH₂ cytokines (IL-4 and IL-5) and pro-inflammatory cytokines (IL-6, TNF-, monocyte chemoattractant protein-1 (MCP-1), IL-12p70 and IL-10) were measured in the serum and in suspensions of popliteal lymph node cells of female Balb/c mice by flow cytometry 7 days after drug administration. Only diclofenac and imipramine induced a cellularity index above 5 (considered as a positive response). Of the five tested drugs, only diclofenac induced a slight increase in TH₁ cytokines, but there were no effects on TH₂ cytokine production whatever the drug tested. Diclofenac increased the production of pro-inflammatory cytokines, whereas the production of MCP-1 was increased by minocycline and decreased by imipramine. No changes in serum cytokine levels were evident. These results suggest that measuring cytokine release is unlikely to improve the sensitivity and specificity of the direct PLNA.     

The IgE adjuvant effect of particles: characterisation of the primary cellular response in the draining lymph node

Diesel exhaust particles, and polystyrene particles (PSP) as a model for the insoluble particle core, have an adjuvant effect on allergen-specific IgE production in mice. We therefore examined the primary immune response in the draining popliteal lymph node (PLN) to the allergen ovalbumin (OVA) injected together with polystyrene particles into the footpad of BALB/cA mice. Similar numbers of particle-containing cells were observed in the draining lymph node on day 1 after injection of PSP alone or OVA + PSP, the numbers increasing continuously until day 21. The total lymph node cell numbers increased three to four times in the OVA + PSP group compared to both OVA and PSP groups, peaking on day 5. The increase in B cell numbers was twice the increase in T cell numbers. On day 5, OVA + PSP increased the expression of most surface markers measured (MHC class II, CD86, CD23, CD69) compared to OVA and PSP. Further, the ex vivo production of IL-4 and IL-10 by PLN cells from OVA + PSP-injected animals was increased. In conclusion, whereas PSP alone did not influence any of the immunologic markers studied, the adjuvant effect of PSP on the IgE antibody response to OVA was associated with an early increased primary cellular response in the draining lymph node.     

Direct evidence for immunomodulatory properties of ribavirin on T-cell reactivity to hepatitis C virus

BACKGROUND/AIM: An understanding of ribavirin's beneficial effects on treatment outcome in chronic hepatitis C (CH-C) may help to develop new treatment approaches. Here we investigated whether ribavirin directly affects HCV-specific reactivity of CD4+T-lymphocytes from patients with CH-C. METHODS: Peripheral blood mononuclear cells from forty HCV RNA positive patients were cultured ex vivo with HCV core, NS3, NS4 alone, and with different concentrations of ribavirin. Virus-specific CD4+ T-cell reactivity was analysed by a proliferation assay; quantitation of cytokine (interferon-gamma, IL-10, IL-5, IL-12p35, IL-12p40) mRNA levels; measurement of interferon-gamma and IL-10 production (by ELISA) and enumeration of interferon-gamma and IL-10 producing T-cells by Elispot assays. RESULTS: At 2-5 microM ribavirin induced de novo or enhanced T-cell proliferation to HCV antigens in a proportion of patients. Increased T-cell proliferation was associated with decreased IL-10 production in response to HCV core and reduced frequency of IL-10 producing CD4+ T-cells, while interferon-gamma levels remained unchanged. At 20 microM ribavirin markedly suppressed T-cell proliferation, and interferon-gamma mRNA expression to HCV antigens. CONCLUSIONS: Ribavirin, at clinically achievable plasma levels, modulates directly the T-cell responses to HCV antigens in some CH-C patients. Suppression of IL-10 production may represent a useful strategy to induce/augment T-cell reactivity to HCV.     

Evaluation of the use of reporter antigens in an auricular lymph node assay to assess the immunosensitizing potential of drugs.

Immune-mediated idiosyncratic drug reactions are a major problem for susceptible patients, physicians, and the pharmaceutical industry. Validated screening tools to assess the immunosensitizing capacity of orally or intravenously administered pharmaceuticals are currently not available. To date, the popliteal lymph node assay (PLNA) seems the most promising tool for this purpose. The PLNA has recently been extended with the use of reporter antigens (RA) that are coinjected together with the drug of interest. The measurement of isotypes of RA-specific antibody-secreting cells (ASC) enables the distinction of sensitizing chemicals and (nonsensitizing) irritants without radio-isotopic end points. However, the use of footpad injections raises ethical concerns. Therefore, we examined the use of RA after intradermal injection into the ear of BALB/c mice and measured RA-specific ASC in the draining auricular lymph node (ALN). We show that RA-specific IgG isotype ASC numbers are very useful and sensitive parameters to identify drug-induced hypersensitivity in both PLN and ALN. However, the type 1-associated parameters (CD8(+) cells, macrophages, IFN-gamma, TNF-alpha, and IL-1 beta) that are induced in the PLN by streptozotocin were less pronounced in the ALN. Thus, the PLNA may provide more immunologically relevant information on the mechanisms of certain chemical-induced hypersensitivity reactions. The RA-ALN assay may provide an alternative for the RA-PLNA; both assays can be used to distinguish sensitizing compounds from nonsensitizing ones.     

Diesel exhaust, carbon black, and silica particles display distinct Th1/Th2 modulating activity

Certain particulate air pollutants may play an important role in the increasing prevalence of respiratory allergy by stimulating T helper 2 cell (Th2)-mediated immune responses to common antigens. The study described here examined different particles, diesel exhaust particles (DEP), carbon black particles (CBP), and silica particles (SIP) for their immunomodulating capacity in both primary and secondary immune responses in female BALB/C mice. The primary response was studied after subcutaneous injection of 1 mg of particle together with 10 microgram of reporter antigen TNP-OVA (2,4,6-trinitrophenyl coupled to ovalbumin) into the hind paw. Interferon-gamma (IFN-gamma) and interleukin 4 (IL-4) production was assessed in the popliteal lymph node (PLN) at Day 2 and Day 5 after injection by flow cytometry and ELISA. The number of IL-4-containing CD4(+) T cells increased between Day 2 and Day 5 in DEP- and CBP-exposed mice, in contrast to SIP-treated animals. IL-4 production by cultured PLN cells was also significantly increased for DEP- and CBP-treated animals. The secondary response was studied in different organs after an intranasal challenge with TNP-OVA (50 microgram), which was given 4 weeks after the initial subcutaneous injection. Five days after challenge the number of antibody-forming cells (AFCs) was assessed in peribronchial lymph nodes (PBLN), spleen, bone marrow, and PLN, and antibody levels were determined in weekly obtained blood samples. It appeared that all particles acted as adjuvant, but the different particles stimulated distinct types of immune responses to TNP-OVA. DEP-treated animals show high IgG1 and IgE levels in serum and high IgG1 and IgE-forming AFC numbers in PBLN, bone marrow, and spleen. CBP-treated animals show even higher IgG1 and IgE levels and AFC numbers, and in addition display IgG2a production. SIP-injected animals display predominantly IgG2a responses. It is concluded that DEP are able to skew the immune response toward the T helper 2 (Th2) side, whereas SIP stimulate a Th1 response and CBP have a mixed activity, stimulating both Th1 and Th2 responses in this model.     

Extracorporeal human whole blood in motion,as a tool to predict first-infusion reactions and mechanism-of-action of immunotherapeutics

First infusion reactions along with severe anaphylactic responses can occur as a result of systemic administration of therapeutic antibodies. The underlying mechanisms by which monoclonal antibodies induce cytokine release syndrome (CRS) can involve direct agonistic effects via the drug target, or a combination of target-engagement along with innate receptor interactions. Despite the wide variety of pathways and cells that can play a role in CRS, many currently used assays are devoid of one or more components that must be present for these responses to occur. One assay that has not been assessed for its capacity to predict CRS is the modified Chandler loop model. Herein we evaluate a plethora of commercially available monoclonal antibodies to evaluate the modified Chandler loop model's potential in CRS prediction. We demonstrate that in a 4-hour loop assay, both the superagonistic antibodies, anti-CD3 (OKT3) and anti-CD28 (ANC28.1), display a clear cytokine response with a mixed adaptive/innate cytokine source. OKT3 induce TNFα and IFN-γ release in 20 out of 23 donors tested, whereas ANC28.1 induce TNF-α, IL-2 and IFN-γ release in all donors tested (n = 18–22). On the other hand, non-agonistic antibodies associated with no or low infusion reactions in the clinic, namely cetuximab and natalizumab, neither induce cytokine release nor cause false positive responses. A TGN1412-like antibody also display a clear cytokine release with an adaptive cytokine profile (IFN-γ and IL-2) and all donors (n = 9) induce a distinct IL-2 response. Additionally, the value of an intact complement system in the assay is highlighted by the possibility to dissect out the mechanism-of-action of alemtuzumab and rituximab. The loop assay can either complement lymph node-like assays or stand-alone to investigate drug/blood interactions during preclinical development, or for individual safety screening prior to first-in-man clinical trial.     

Pertussis toxin-induced mitogenesis in human T lymphocytes

Pertussis toxin (PT) has previously been shown to affect a wide variety of immune responses and to cause lymphocyte proliferation. We have investigated the biochemical basis for the mitogenic activity of PT by using human peripheral blood lymphocytes. PT was found to induce a rapid rise in cytosolic free calcium concentration and an alkalinization of the cytosol through the Na+/H+ antiporter. The toxin was also found to induce expression of IL-2-receptor on CD3+ cells and to stimulate IL-2 production. PT induced proliferation of both CD4+ and CD8+ T cells in the presence (but not in the absence) of accessory cells. PT also stimulated IL-1 production by monocytes but neither IL-1, IL-6 alone nor a combination of the two lymphokines could replace accessory cells suggesting that cell:cell contact is required. Low doses of PT induced ADP-ribosylation of G proteins but this treatment did not affect significantly PHA-induced [Ca2+]i increase and IL-2-induced DNA synthesis suggesting that the substrates of the ADP-ribosyltransferase activity of PT are not involved in the signalling pathways leading to DNA replication.     

Cytokines as adjuvants for the induction of mucosal immunity

Safe nasal vaccines capable of promoting both mucosal and systemic immunity are needed for effective protection against bacterial and viral pathogens. While parenteral cytokine treatment could lead to unwanted toxicity, the nasal delivery route results in low but biologically active serum cytokine levels. Interleukin (IL)-6, IL-1 and IL-12, which promote either Th2- or Th1-type responses, respectively, also enhance systemic immunity to co-administered antigens. The chemoattractants lymphotactin (Lptn), RANTES and defensins also exerted adjuvant activity for systemic immunity when nasally administered with antigens. However, each cytokine or innate factor promoted a distinct pattern of T helper cell responses and corresponding IgG subclass response. Interleukin-12, IL-1, and the chemokines Lptn and RANTES promote mucosal immunity. In contrast, nasal IL-6 and defensins failed to induce mucosal S-IgA Ab responses, suggesting that mechanisms more complex than T cell activation and chemotaxis are required for the development of mucosal immunity after nasal delivery of cytokines.     

Fluoxetine treatment to rats modifies serotonin transporter and cAMP in lymphocytes,CD4+ and CD8+ subpopulations and interleukins 2 and 4

Several lines of evidences indicate that antidepressants produce various immunomodulatory effects. Fluoxetine, an antidepressant and selective serotonin reuptake inhibitor, modulates immune cells in vitro. To explore the in vivo influence of fluoxetine on lymphocytes, male Sprague–Dawley rats were treated daily, 10 mg/kg, or with saline solution for 1, 2 and 3 weeks. The presence of serotonin transporter in CD3+, CD4+ and CD8+ subpopulations of T lymphocytes was determined by immunofluorescence. Serotonin transporter was also labeled with [³H]paroxetine, specific binding defined with imipramine. Plasma levels of pro-inflammatory interleukin 2 (IL-2), and anti-inflammatory interleukin 4 (IL-4), were measured by ELISA; and cAMP concentration by radioimmunoassay. Fluoxetine significantly increased the number of lymphocytes expressing serotonin transporter and elevated the binding of [³H]paroxetine. The percentage of CD4+ cells decreased, that of CD8+ increased, and CD3+ did not change. The ratio CD4+/CD8+ was significantly lowered. Fluoxetine administration elevated the levels of IL-4 at 1, 2 and 3 weeks; and of IL-2, at 2 and 3 weeks. IL-4/IL-2 ratio was significantly increased in fluoxetine group respecting the controls and was similar during the 3 weeks of treatment. Fluoxetine produced a significant decrease in cAMP concentrations in lymphocytes, probably by secondary activation of serotonin receptors. Treatment with fluoxetine modified immune parameters in plasma and lymphocytes of rats, which might be relevant for its systemic therapeutic action as an antidepressant.