Allergen-driven limiting dilution analysis of human IL-4 and IFN-{gamma} production in allergic rhinitis and clinically tolerant individualsz

Difficulties in detecting human IL-4 synthesis in antigen-drivenprimary culture have led to widespread reliance on less physlologicappraoches to T cell activation. Although there is general agreementof a T 2-like blas,l the precise defects in ctokine responsivenessremain controverslal. Analysis of cytokine protein productionby fresh, unselected cell populations in response to gognate,antigen-driven stimulation remains an important goal. Here,limitting dilution analysis (LDA) was sued to evaluate antigen-stimulatedcytokine gene expression by fresh peripheral blood mononuclearcells(PBMC)(.PBMC from 19 grass pollen sensitive, allergicrhinitis subjects and normal, non-atopic contrls were evaluated1 mont after natural relmmunization (the peak of te local grasspollen season). Surprisingly, highly atopic subjects and cllnicallynon-allergic individuals exhiboited virtually equlvalent antigen-specific,CD4-dependent cytokine production in response to short-termculture with these common environmental antigens. As anticipated,pronounced increases in T_h2-like activity were evidnet in tecirculating immune repertolre of grass of 117:1 among normalsubjects versus 4:1 among those with allergic rhinitis (Mann-WhitneyU-test, P=0.0067). This T_h2-like blas reflected both a lowerfrequencey of IFN-prducing cells and a markedly increased frequencyof IL-4-producing cells in the circulatin grass-pollen specificrepertoire of atopic donors. The observation that every atopicand normal subject produced IFN- (±IL-4) following antigenre-stimulation arguses that the decision as to whether allergyor (clinical) tolerance results, hinges not on a geneticallydetermined capacity of whether allergen-reactive T cells canbe stimulated in any given individual by cronic exposure toubiqultous enveronmental antigens, but on the nature of thecokine response that comes to dominate that individual's response     

Induction of human hsp60 expression in monocytic cell lines.

Both bacterial and mammalian heat shock proteins (HSP) are recognized by some T cells, and hsp60 recognition has been implicated in rheumatoid arthritis. We have developed a model to study the induction of hsp60 in human monocytic cell lines. An anti-mycobacterial hsp65 mAb (ML30), cross-reacting with human hsp60 was used to screen 21 human tumor cell lines in Western blot analysis. All T cell and B cell lymphomas constitutively expressed hsp60 protein at moderate to high levels, while little or no hsp60 protein was detected in two monocytic leukemia lines. Moderate to high levels of hsp60 mRNA and protein could be induced in the THP-I monocytic leukemia cell line by heat shock, retinoic acid, interferon (IFN)-gamma or tumor necrosis factor (TNF)-alpha treatment, the highest levels obtained with a combination of IFN-gamma/TNF-alpha. This was also seen using two rabbit anti-hsp60 antisera directed against the N-terminal or C-terminal part of the human hsp60 protein. The determinants detected by the ML30 mAb or the two rabbit anti-hsp60 antisera were not cell surface expressed, as measured with immunofluorescence (FACS) analysis on control cultured or cytokine treated cell lines. This could be a useful model for studies related to the induction of hsp60 in human cells.     

In vitro and in vivo recovery of IFN-{gamma}, but not IL-2, production by IL-12 in mice with plasma ceIL tumors

We have previously found that T ceILs from mice bearing plasmaceIL tumors (PCT mice) demonstrate decreased proliferation asweIL as decreased production of the T^h 1-associated cytokinesIL-2 and IFN- in response to polyclonal stimulation. In thepresent study, we have examined soluble factors as possibleelements required to rescue this decreased proliferation andcytokine production by splenocytes from PCT mice. We find thatthe addition of supernatants from stimulated normal splenocyteshas no effect on proliferation or IL-2 production by splenocytesfrom PCT mice. In contrast, these supernatants completely restoreIFN- production by splenocytes from PCT mice. We have foundthat IL-12 is responsible for the observed increase in IFN-production because: (i) addition of anti-IL-12 antibody blocksthis recovery of IFN- production by these supernatants, (ii)the addition of recombinant IL-12 to cultures of splenocytesfrom PCT mice results in increased IFN- production and (iii)In vivo treatment of PCT mice in IL-12 also results in increasedIFN- production by the subsequently activated splenocytes, buthas little effect on proliferation or IL-2 production. Theseresults demonstrate that both in vitro and in vivo, IL-12 selectivelyrestores the decreased production of IFN- by splenocytes fromPCT mice.     

Maternal serum levels of interferon-gamma and interleukin-2 soluble receptor-alpha predict the outcome of early IVF pregnancies

BACKGROUND: Elevated maternal serum levels of interleukin-2 soluble receptor-alpha (IL-2 sRalpha), tumour necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) have been associated with pregnancy loss. The aim of our study was to evaluate the predictive value of these cytokines in the outcome of early IVF pregnancies. METHODS: One hundred and fifty-nine consecutive IVF patients who were subsequently diagnosed to have a biochemical pregnancy (n = 23), a first-trimester miscarriage (n = 19) or a normal term delivery (n = 117) were included in this study. Serum was collected from the initial pregnancy test, 11 days after a day 3 embryo transfer, and all samples were analysed for IL-2 sRalpha, TNF-alpha and IFN-gamma by commercially available enzyme-linked immunosorbent assay (ELISA) kits. RESULTS: IL-2 sRalpha levels were significantly higher in patients with an early pregnancy loss compared with patients with a normal term delivery (849.5 +/- 69.6 versus 693.5 +/- 31.2 pg/ml, P = 0.02), and a cut-off point of IL-2 sRalpha >1000 pg/ml predicted a poor pregnancy outcome (44.4 versus 22.7% pregnancy loss, IL-2 sRalpha >or=1000 versus IL-2 sRalpha <1000 pg/ml; P = 0.02). IFN-gamma-positive patients had twice the risk for poor IVF pregnancy outcome compared with IFN-gamma-negative subjects (40.8 versus 20.0%, respectively; P < 0.02), including a significantly lower implantation rate (37.6 +/- 0.05 versus 50.0 +/- 0.03%, respectively; P = 0.02). There was no difference in pregnancy outcome based upon serum levels, or the ability to detect the presence of TNF-alpha. No differences in levels of these cytokines were found based on the aetiology of the patients' infertility. CONCLUSIONS: Elevated maternal serum levels of IL-2 sRalpha and IFN-gamma as early as 11 days after embryo transfer are associated with poor IVF pregnancy outcome.     

Activation of Vgamma9Vdelta2 T cells by non-peptidic antigens induces the inhibition of subgenomic HCV replication

Hepatitis C virus (HCV) has evolved complex strategies to evade host immune responses and establish chronic infection. Since human Vgamma9Vdelta2 T lymphocytes play a critical role in the immune response against viruses, we analyzed their antiviral functions on Huh7 hepatoma cells carrying the subgenomic HCV replicon (Rep60 cells). In a transwell culture system, Rep60 cells were co-cultured with either PBMCs or highly purified gammadelta T cells stimulated by non-peptidic antigens. Vgamma9Vdelta2 T cell activation was associated with a dramatic reduction of HCV RNA levels. Neutralizing antibodies targeting IFN-gamma revealed a critical role for this cytokine in the inhibition of HCV replication. Interestingly, drugs already in clinical use, such as Phosphostim and Zoledronate, known to activate gammadelta T cells, were shown to induce the inhibition of HCV replication mediated by Vgamma9Vdelta2 T cells of HCV patients. Our data suggest that the therapeutic activation of Vgamma9Vdelta2 T lymphocytes may represent an additional strategy to inhibit HCV replication and to restore a Th1-oriented immune response in HCV-infected patients.     

Type 1 cytokine/chemokine production by mouse NK cells following activation of their TLR/MyD88-mediated pathways

It is well established that IL-18R- and toll-like receptor (TLR)-mediated signalings share a common signal pathway mediated by signal adaptor, MyD88, and that IL-18 synergizes with IL-12 for IFN-gamma production by NK cells. Here, we investigated whether TLR agonists can replace IL-18 for production of IFN-gamma by NK cells. Freshly isolated NK cells possessed functional LPS receptor composed of TLR4/MD2 complex and of CD14, and also expressed other various tlrs. Hepatic CD3(-)DX5(+) NK cells produced IFN-gamma in response to TLR2 or TLR7 agonists only when co-stimulated with IL-12, indicating that TLR agonists synergize with IL-12 for IFN-gamma. The tlr2(-/-) or tlr7(-/-) NK cells could not produce IFN-gamma in response to IL-12 plus TLR2 or TLR7 ligands, respectively, indicating requirement of the corresponding TLRs. Furthermore, upon stimulation with these combinations, wild-type NK cells produced type 1 chemokines, such as CCL3, CCL4 and CCL5 as well. NK cells from bacterium (e.g. Propionibacterium acnes)-inoculated rag2(-/-) mice, when compared with those from naive mice, exhibited significantly enhanced capacity to produce these CC chemokines and IFN-gamma, suggesting that microbial infection enhances responsiveness of NK cells to TLR agonists. These results indicate that upon microbial infection, macrophages produce IL-12 that renders NK cells highly responsive to TLR agonists to produce IFN-gamma and chemokines, which might in turn recruit and fully activate macrophages, leading to the development of inflammatory foci presumably necessary for efficient microbial eradication. Thus, NK cells, like T cells, induce orchestrated immune responses in collaboration with macrophages to show potent host defense effects during early infectious phase.     

Fel d 1 peptides: effect on skin tests and cytokine synthesis in cat-allergic human subjects

We tested peptide immunotherapy in cat-allergic humans, usinga formulation of two synthetic peptides, IPC-1 and IPC-2, eachof which is 27 amino acids long and contains T cell-reactiveregions of Fel d 1, the major cat allergen. In this exploratory,randomized, double-blind, parallel-group study, 42 subjectsreceived s.c. injections of treatment peptides 250 µgor placebo weekly for four consecutive weeks. Changes in immediate-and late-phase skin test reactivity, and in antigen-driven cytokinesynthesis were assessed. Epicutaneous (end-point titration)and intradermal tests were performed with cat extract (ALK SQCat Hair) containing Fel d 1, before the first injection, then2, 6 and 24 weeks after the fourth and last injection of peptidesor placebo. IL-4, IL-10 and IFN- expression by circulating peripheral bloodmononuclear cells (PBMC) in response to cat extract was measuredusing short-term bulk culture of PBMC and short-term limitingdilution analysis. Subjects who received peptide immunotherapydid not tolerate significantly more cat extract containing Feld 1 in the skin tests 2, 6 or 24 weeks after the last injectionthan they did at baseline, and their late-phase responses didnot decrease significantly compared to baseline. SubstantialIL-4, IL-10 and IFN- responses were observed following primaryculture of cat antigen-stimulated PBMC; however, the intensityof cytokine synthesis and the IFN-: IL-4 ratio were unchangedin peptide- and placebo-treated groups 6 and 24 weeks afterthe last injection. A few hours after the injections, subjectsreceiving peptides reported more allergic rhinitis and asthmasymptoms and more pruritus than those receiving placebo. Weconclude that under the conditions tested, peptide immunotherapydid not reduce immediate- or late-phase skin reactivity to catextract containing Fel d 1 or modify cat antigen-specific cytokineproduction significantly.     

Systemic administration of rIL-12 induces complete tumor regression and protective immunity: response is correlated with a striking reversal of suppressed IFN-{gamma} production by anti-tumor T cells

Unfractionated spleen cells taken from tumor-bearing mice 2weeks after tumor implantation contained tumor-primed T cellswhich produced cytokines including IL-2 and IFN- when culturedin vitro. With progressive tumor growth this initial lymphokine-producingcapacity decreased. Here, we investigated the ability of IL-12to (I) restore suppressed IFN- production, (II) cause tumorregression and (II) induce anti-tumor protective immunity. Additionof rIL-12 to spleen cell cultures from 4- to 10-week-old tumor-bearingmice resulted in a striking enhancement in the production ofIFN- compared with cultures of these cells in the absence ofrIL-12 or of normal spleen cells in the presence of rIL-12.Five I.p. injections of rIL-12 into mice bearing s.c. tumorsinduced complete tumor regression. This was found when rIL-12was given at early (1–2 weeks), intermediate (4–5weeks) or even late (7 weeks) stages of tumor growth. Furthermore,IL-12-treated mice which rejected the primary tumor exhibitedcomplete resistance to a rechallenge with the same tumor butdid not reject a second syngenetic tumor. Immunohistochemicalanalyses following IL-12 treatment revealed that CD4⁺ and CD8⁺T cells infiltrate the tumor. More importantly, IFN- mRNA expressionwas observed in fresh tumor masses from tumor-bearing mice receivingIL-12 treatment The importance of IFN- was further demonstratedby the observation that the systemic administration of anti-IFN-mAb prior to IL-12 treatment completely abrogated the anti-tumoreffect of IL-12. Thus, these results indicate that administrationof modest levels of rIL-12 to tumor-bearing mice results intumor regression through mechanisms involving reversal of suppressedIFN- production by anti-tumor T cells and the establishmentof a tumor-specific protective immune response.     

Differential effects of IL-4 and IL-10 on IL-2-induced IFN-gamma synthesis and lymphokine-activated killer activity.

Culture of human peripheral blood mononuclear cells (PBMC) with IL-2 stimulates synthesis of cytokines and generation of lymphokine-activated killer (LAK) activity. Both IL-4 and IL-10 [cytokine synthesis inhibitory factor (CSIF)] inhibit IL-2-induced synthesis of IFN-gamma and tumor necrosis factor (TNF)-alpha by human PBMC. However, unlike IL-4, IL-10 inhibits neither IL-2-induced proliferation of PBMC and fresh natural killer (NK) cells, nor IL-2-induced LAK activity. Moreover, IL-4 inhibits IL-2-induced IFN-gamma synthesis by purified fresh NK cells, while in contrast the inhibitory effect of IL-10 is mediated by CD14+ cells (monocytes/macrophages). IL-10 inhibits TNF-alpha synthesis by monocytes or monocytes plus NK cells, but not by NK cells alone. These results suggest that IL-4 and IL-10 act on NK cells via distinct pathways, and that IL-2-induced cytokine synthesis and LAK activity are regulated via different mechanisms.     

Differential production of IL-12 in BALB/c and DBA/2 mice controls IL-4 versus IFN-{gamma} synthesis in primed CD4 lymphocytes

The profile of cytokines produced by CD4 T cells is profoundlyinfluenced by the presence of IL-12. Here we demonstrate thatduring re-stimulation of antigen-specific immune responses invitro, antigen-primed lymph node cells from DBA/2 mice produced3- to 30-fold more IL-12 than did cells from BALB/c mice, whichare identical at the major histocompatibility locus. The straindifferences in IL-12 production were observed only in antigen-drivenresponses (and not in responses induced by bacterial products),and were dependent upon an interaction between CD4 T cells andlymph node adherent cells. In addition, differences in the quantityof IL-12 produced by DBA/2 and BALB/c antigen-presenting cells(APC) was not dependent on differential production of IFN- byT cells, since APC from DBA/2 mice still produced much greaterquantities of IL-12 than did BALB/c APC when each was culturedwith the same H-2^d-restricted T_h2 clones, in the complete absenceof IFN, or when each was cultured with primed (BALB/c x DBA/2)F₁T cells. The level of IL-12 produced in the cultures criticallyaffected cytokine production in CD4 T cells, since neutralizationof endogenous IL-12 in DBA/2 cultures, which are predisposedtowards developing T_h1 responses, reduced IFN- production andenhanced IL-4 synthesis to levels normally seen in BALB/c cultures,which are predisposed toward developing Th2 responses. We proposetherefore that differential production of antigen-driven IL-12is a mechanism by which the genetic background in DBA/2 andBALB/c mice can affect the pattern of cytokine synthesis byT cells during the development of adaptive immune responses.     

Osteopontin affects the persistence of beta-glucan-induced hepatic granuloma formation and tissue injury through two distinct mechanisms

Osteopontin (OPN) plays a pivotal role in various immune responses and inflammatory diseases. OPN is expressed in various granulomatous diseases; however, the cellular and molecular role of OPN in these diseases is not well known. We analyzed the role of OPN in a beta-glucan-induced hepatic granuloma model. First, we found that neither OPN deficiency nor overexpression of OPN affected the number and the size of hepatic granulomas at day 7, indicating that OPN is not involved in the formation of hepatic granulomas at the early stages. Importantly, OPN did not influence the liver tissue damage as defined by alanine aminotransferase and aspartate aminotransferase levels at early stages. Second, OPN deficiency resulted in the reduction of IL-12 and IFN-gamma production at early stages. Third, at late stages, OPN deficiency resulted in a decrease in the number and size of hepatic granulomas, and a reduction of liver tissue injury. This was due to the reduction of the cellular recruitment including macrophages, CD4 T cells and dendritic cells into the liver, and the reduction of tumor necrosis factor (TNF)-alpha production in the liver. In contrast, overexpression of OPN resulted in the persistence of granuloma formation. These data suggest that OPN affects the persistence of hepatic granuloma formation. Our results indicate that OPN up-regulates the production of IL-12 and IFN-gamma within the granulomas at early stages, and OPN has an additional role in the regulation of cellular recruitment and TNF-alpha production at late stages that determine the severity of liver tissue injury.     

Up-regulation of Borrelia-specific IL-4- and IFN-gamma-secreting cells in cerebrospinal fluid from children with Lyme neuroborreliosis

The clinical course and outcome of several infectious diseases are dependent on the type of immune response elicited against the pathogen. In adults with neuroborreliosis (NB), a type 1 response with high production of Borrelia-specific IFN-gamma, but no IL-4, has been reported. Since children have a more benign course of NB than adults, we wanted to investigate type 1 and type 2 responses in children with NB. Cerebrospinal fluid (CSF) and blood were collected from children during the acute stage of 'confirmed NB' (n = 34), 'possible NB' (n = 30) and 'non-NB' (n = 10). The number of Borrelia-specific IL-4- and IFN-gamma-secreting cells was measured by enzyme-linked immunospot assay. Borrelia-specific secretion of both IL-4 and IFN-gamma was increased in CSF in confirmed (P < 0.05) and possible (P < 0.01) NB, when compared with non-NB controls. Furthermore, children with NB had significantly higher Borrelia-specific IL-4 secretion in CSF than an adult reference material with NB (P < 0.05). There were no differences in cytokine secretion in relation to onset or recovery of neurological symptoms. Since IL-4 is known to down-regulate the pro-inflammatory and possibly harmful effects of prolonged IFN-gamma responses, the prominent IL-4 response observed in the central nervous system compartment might contribute to the more benign disease course seen in children with Lyme NB.     

CD4(+) T cells induced by virus-like particles expressing a major T cell epitope down-regulate IL-5 production in an ongoing immune response to Der p 1 independently of IFN-gamma production

It has been previously demonstrated that hybrid Ty virus-like particles (VLP) prime effective CD8(+) and CD4(+) T cell responses. In this study, we investigated the effect of treating mice with Ty VLP carrying the immunodominant epitope of Der p 1 after sensitizing them to the group 1 allergen of the house dust mite Dermatophagoides pteronyssinus (Der p 1), under conditions that induce T(h)2 immunity. We show that i.p. treatment with the hybrid VLP abrogated allergen-specific IL-5 production and reduced allergen-specific cell proliferation. This suppression of the response was mediated by CD4(+) T cells and was not accompanied by an increase in IFN-gamma production.     

Induction of Ly-6A/E expression by murine lymphocytes after in vivo immunization is strictly dependent upon the action of IFN-alpha/beta and/or IFN-gamma

Ly-6A/E is a phosphatidylinositol-linked membrane protein which mediates murine T and B cell signalling. IFN-gamma, IFB-alpha/beta, LPS, and IL-4 have all been reported to induce or upregulate Ly-6A/E by normal lymphocytes. Since no systematic study has addressed the stimulant selectivity of Ly-6A/E expression by murine lymphocytes nor investigated its induction and regulation during primary in vivo immune responses we analyzed in vitro Ly-6A/E expression after murine stimuli and during a number of distinct in vivo immunizations. We show that LPS induces B cell Ly-6A/E in vitro by stimulating the release of IFN-alpha/beta by 'contaminating' adherent cells. In the presence of anti-IFN-gamma + anti-IFN-alpha/beta antibodies, no Ly-6A/E was induced upon addition of multiple cytokines, including IL-4, or mitogenic doses of anti-Ig antibody. Furthermore, IFN-gamma-containing, CD4+ T cell (Th1) supernatants potently induced Ly-6A/E by murine B cells whereas IL-4-containing (Th2) supernatants were either weak or ineffective; anti-IFN-gamma + anti-IFN-alpha/beta inhibited Ly-6A/E induction by both Th1 and Th2 supernatants. Immunization of mice with Brucella abortus or poly (I).poly (C) resulted in induction of Ly-6A/E expression by virtually all B and T cells, whereas injection of G alpha M delta led to peak induction of Ly-6A/E by approximately 50% of both B and T cells. Lymphocytes from mice infected with the nematode parasites Nippostrongylus brasiliensis or Heligmosomoides polygyrus expressed no Ly-6A/E.(ABSTRACT TRUNCATED AT 250 WORDS)     

Monoclonal anti-tumor necrosis factor antibody renders non-obese diabetic mice hypersensitive to irradiation and enhances insulitis development.

In attempt to evaluate biological roles of tumor necrosis factor (TNF), we studied the effects of anti-TNF mAb in non-obese diabetic (NOD) mice. Anti-murine TNF mAb rendered NOD mice hypersensitive to the lethal effects of radiation and prevented the reconstitution of lethally irradiated mice with adoptively transferred lymphocytes. While TNF-alpha reduced the incidence of diabetes development in the adoptive transfer system even when given 6 days post-transfer, mAb to TNF could not reduce or increase the incidence of diabetes compared to control mice. Administration of TNF-alpha for 4 or 8 weeks significantly reduced the incidence of spontaneous insulitis in NOD mice, while anti-TNF mAb given for 8 weeks increased the incidence of insulitis significantly.     

Defensins act as potent adjuvants that promote cellular and humoral immune responses in mice to a lymphoma idiotype and carrier antigens

Defensins released by neutrophils are able to kill a broad spectrum of microbes. They also induce leukocyte migration in vitro and elicit inflammatory leukocyte responses at s.c. injection sites in mice. In vitro experiments showed that human defensins enhanced concanavalin A-stimulated murine spleen cell proliferation and IFN-gamma production. This led us to examine the effects of human defensins on specific immune responses in vivo. BALB/c mice were immunized with 50 microg of keyhole limpet hemocyanin (KLH) adsorbed to aluminum hydroxide and administered with defensins in aqueous solution. Intraperitoneal administration of defensins significantly increased the production of KLH-specific IgG1, IgG2a and IgG2b antibodies 14 days after immunization. In vitro splenic KLH-specific proliferative responses were higher in mice treated with KLH and defensins than in those treated with KLH alone. Increased IFN-gamma and, to a lesser extent, IL-4 production were also detected in the supernatants of ex vivoKLH-activated spleen cells from mice treated with defensins. Finally, defensins significantly enhanced the antibody response to a syngeneic tumor antigen, lymphoma Ig idiotype and also augmented resistance to tumor challenge. These results indicate that defensins act as potent immune adjuvants by inducing the production of lymphokines, which promote T cell-dependent cellular immunity and antigen-specific Ig production. Thus, defensins appear to function as neutrophil-derived signals that promote adaptive immune responses.