Interleukin-6 Bioactivity Research Articles

Parathyroid hormone induces hepatic production of bioactive interleukin-6 and its soluble receptor.

Interleukin-6 (IL-6) is an important mediator of parathyroid hormone (PTH)-induced bone resorption. Serum levels of IL-6 and its soluble receptor (IL-6sR) are regulated in part by PTH. The PTH/PTH-related protein type 1 receptor is highly expressed in the liver, and in the current study we investigated whether the liver produces IL-6 or IL-6sR in response to PTH. Perfusion of the isolated rat liver with PTH-(1-84) stimulated rapid, dose-dependent production of bioactive IL-6 and the IL-6sR. These effects were observed at near physiological concentrations of the hormone such that 1 pM PTH induced hepatic IL-6 production at a rate of approximately 0.6 ng/min. In vitro, hepatocytes, hepatic endothelial cells, and Kupffer cells, but not hepatic stellate cells, were each found to produce both IL-6 and IL-6sR in response to higher (10 nM) concentrations of PTH. Our data suggest that hepatic-derived IL-6 and IL-6sR contribute to the increase in circulating levels of these cytokines induced by PTH in vivo and raise the possibility that PTH-induced, liver-derived IL-6 may exert endocrine effects on tissues such as bone.

Inhibitory Effects of Anti-Inflammatory Drugs on Interleukin-6 Bioactivity.

Interleukin-6 (IL-6) is known as a proinflammatory cytokine involved in immune response, inflammation, and hematopoiesis. Inhibitory effects of anti-inflammatory drugs on IL-6 bioactivity using IL-6-dependent hybridoma have been evaluated. Three out of 16 nonsteroidal anti-inflammatory drugs (NSAIDs) showed IC50 values of less than 100 microM, which were in the order of oxyphenylbutazone hydrate (IC50=7.5 microM)>meclofenamic acid sodium salt (31.9 microM)>sulindac (74.9 microM). Steroidal anti-inflammatory drugs (SAIDs) exhibited significant inhibitory effects at 100 microM on the IL-6 bioactivity, and their inhibitory potencies were in the order of budesonide (IC50=2.2 microM)>hydrocortisone 21-hemisuccinate (6.7 microM), prednisolone (7.5 microM), betamethasone (10.9 microM)>dexamethasone (18.9 microM) and triamcinolone acetonide (24.1 microM). The results would provide an additional mechanism by which anti-inflammatory drugs display their anti-inflammatory and immunosuppressive effects at higher concentrations.

Interferon-?-inducing activity of interleukin-18 in the joint with rheumatoid arthritis

To examine the levels of interleukin-18 (IL-18) bioactivity within the rheumatoid arthritis (RA) joint, and the differential effects of IL-12 and IL-18 on interferon-gamma (IFNgamma) production by T cell infiltrates. Expression of IL-18 protein and messenger RNA (mRNA) was determined by enzyme-linked immunosorbent assay and reverse transcriptase-polymerase chain reaction, respectively. The biologic activity of IL-18 was detected on the basis of IFNgamma secretion from IL-18-responding human myelomonocytic KG-1 cells. To determine the extent of inhibitory activity on binding of IL-18 to its receptor, a [125I]-IL-18 binding inhibition assay was performed, using a Chinese hamster ovary cell line transfected with a murine IL-18 receptor. The amount of IL-18 protein detected in both the serum and synovial fluid of RA patients was markedly larger than that detected in the serum and synovial fluid ofosteoarthritis (OA) patients, and serum IL-18 levels correlated with the levels of serum C-reactive protein. IFNgamma production by KG-1 cells was more strongly stimulated in synovial fluid samples from RA patients than in samples from OA patients, and this activity was largely diminished in the presence of anti-IL-18 antibody. In contrast, the activity of IL-18 binding inhibition in the serum and synovial fluid of RA patients was not significantly elevated compared with that in OA patients. RA synovial tissues showed increased expression of IL-18 mRNA and increased IL-18 protein synthesis compared with that in OA tissues. Purified CD14+ macrophages, but not activated fibroblast cell lines, from RA synovium were able to release mature IL-18, although both cell types expressed its transcripts. IL-18 alone showed a negligible effect on IFNgamma production by RA synovial tissue cells, in contrast to IL-12, which was directly stimulatory. However, IL-12-induced IFNgamma production was synergistically enhanced by IL-18, and yet was >50% reduced by neutralization of endogenous IL-18 with anti-IL-18 antibody. These results indicate that IL-18, produced predominantly by tissue macrophages, primarily potentiates IL-12-induced IFNgamma production by T cell infiltrates in RA synovium. Detection of significant IL-18 bioactivity in the joints, despite the presence of IL-18 binding inhibitors, supports an integral role of this cytokine in perpetuating the IFNgamma-dominant T cell cytokine response in RA.

Interleukin-18 expression and modulation in human corneal epithelial cells

Purpose. The interferon-?-inducing factor Interleukin-18 (IL-18) is a recently described cytokine that appears to have multiple important pro-inflammatory effects including the induction of interferon-? (IFN-?) by activated T-cells. The expression of IL-18 by human cornea has not been previously reported. In the present study, we examine the possibility that human corneal epithelial cells are capable of producing this leukocyte-activating factor which may play an important role in IFN-?-dependent inflammation responses in the cornea. Methods. Northern blot analysis and ELISA were used to investigate the in vitro expression of IL-18 mRNA and protein respectively in primary (HCEC) and transformed human corneal epithelial cells (HCET). To determine if IL-18 expression was modulated by pro-inflammatory mediators, cells were treated with lipopolysaccharide (LPS), phorbol 12-myristate 13-acetate (PMA) or synthetic double stranded RNA (poly dI : dC). IL-18 bioactivity was determined in a leukocyte interferon-? induction assay and IL-18 was immunolocalized in whole human cornea by immunohistochemistry using a specific anti-IL-18 antibody. Results. IL-18 mRNA and bioactive protein was constitutively expressed by human corneal epithelial cells and upregulated by PMA, LPS and poly dI : dC. The constitutive expression of IL-18 protein immunoreactivity was also demonstrated in the epithelial cells of whole human cornea tissue. Conclusions. This is the first study demonstrating that corneal epithelial cells are capable of producing the IFN-? inducing factor IL-18. Increased bioactive corneal IL-18 production can be induced by a number of pro-inflammatory agents and may play an important role in initiating ?-interferon-mediated inflammatory responses in the cornea.

Levels of interleukin-18 and its binding inhibitors in the blood circulation of patients with adult-onset Still's disease.

Interleukin-18 (IL-18) is a proinflammatory cytokine that is involved in immunologically mediated tissue damage, but its bioactivity is regulated in vivo by its soluble decoy receptor, IL-18 binding protein (IL-18BP). This study was undertaken to determine levels of IL-18 and IL-18 binding inhibition in the blood of patients with adult-onset Still's disease (ASD). Serum concentrations of IL-18 in ASD patients were compared by enzyme-linked immunosorbent assay (ELISA) with those in patients with other systemic rheumatic diseases and healthy controls. The biologically active mature protein of IL-18 was detected by Western blot analysis with anti-IL-18 antibody and its induction of interferon-gamma (IFNgamma) secretion from IL-18-responding human myelomonocytic KG-1 cells. The inhibitory activity on IL-18 binding to its receptor was determined by 125I-IL-18 binding inhibition assay using the Chinese hamster ovary cell line transfected with a murine IL-18 receptor (CHO-K1/mIL-18R). Concentrations of serum IL-18 were extremely elevated in patients with active ASD compared with those in patients with rheumatoid arthritis, systemic lupus erythematosus, systemic sclerosis, polymyositis/dermatomyositis, Sjogren's syndrome, or healthy individuals. Levels of IL-18 were found to correlate with serum ferritin values and disease severity in ASD. Western blot analysis revealed that serum samples from patients with active ASD contained an 18-kd polypeptide of IL-18, corresponding in size to the mature form. Accordingly, the samples were able to induce IFNgamma secretion from KG-1 cells, which was largely abolished by neutralizing anti-IL-18 antibody. However, the level of IL-18 bioactivity was more than 10-fold weaker than the concentration of IL-18 protein measured by ELISA. Serum samples from patients with active ASD showed an inhibitory effect on the binding of 125I-IL-18 to CHO-K1/mIL-18R cells, and this activity was associated with elevation of IL-18. These data indicate that systemic overproduction of IL-18 may be closely related to the pathogenesis of ASD, despite the restriction on its inflammatory activity by IL-18 binding inhibitors such as IL-18BP. The disease activity appears to be determined on the basis of the relative levels of IL-18 and its specific inhibitors.

Controlled local delivery of interleukin-2 by biodegradable polymers protects animals from experimental brain tumors and liver tumors.

The purpose of our study was to develop an injectable polymeric system for the long-term localized delivery of bioactive interleukin-2 for antitumor immunotherapy. IL-2 was encapsulated into gelatin and chondroitin-6-sulfate using an aqueous-based complex coacervation. CTLL-2 cells were used to measure the bioactivity of released IL-2 and radiolabeled IL-2 was used for release studies in the rat brain and mouse liver. Antitumor efficacy studies were carried out in primary (9L gliosarcoma) and metastatic (B16-F10 melanoma) brain tumor models in rats and mice, respectively, as well as a murine liver tumor model (CT26 carcinoma). Survivors of the metastatic brain tumor challenge were rechallenged with tumor in the opposite lobe of the brain to confirm that antitumor immunologic memory had developed. Bioactive IL-2 was released for over 2 weeks in vitro and in vivo IL-2 release showed significant IL-2 levels for up to 21 days. Polymeric IL-2 microspheres injected intratumorally were statistically more effective in protecting animals challenged with fatal tumor doses in the brain and the liver than placebo or autologous tumor cells genetically engineered to secrete IL-2. Immunologic memory was induced following IL-2 microsphere therapy in the B16-F10 brain tumor model that was capable of protecting 42% of animals from a subsequent intracranial tumor challenge, suggesting that tumor destruction was mediated by the immune system. Local IL-2 therapy using novel polymeric carriers. aimed at stimulating long-lasting antitumor immunity, may provide an improved method of treating a variety of cancers.

Interleukin-6 decreases estrogen production and messenger ribonucleic acid expression encoding aromatase during in vitro cytodifferentiation of rat granulosa cell

To elucidate the influence of interleukin-6 (IL-6) on the maturation of rat granulosa cell (GC), we have established a differentiation model of rat GC in vitro and examined the expression of IL-6 and its receptors, and its possible actions during GC maturation. Bioactive IL-6 was detectable in the conditioned media of GC at ∼2.5 ng/ml/24 h per 5×10 5 cells. Reverse transcription-polymerase chain reaction (RT-PCR) analysis revealed that messenger RNAs encoding components of functional IL-6 receptor, namely both ligand-binding subunit (IL-6R) and gp130, were expressed in GC. Treatment of GC with IL-6 for 72 h during the process of in vitro GC maturation dose-dependently inhibited the accumulation of estradiol-17β (E 2) and the expression of cytochrome P450 aromatase (P450arom). IL-6 did not change nitric oxide (NO) production and inducible NO synthase expression, implying that IL-6-induced suppression on E 2 levels is dissociated with NO expression. Further, GC which had been incubated with neutralizing anti-IL-6 antibody showed a distinct increase in the levels of P450arom mRNA. These results suggest that IL-6 may attenuate E 2 production partially by inhibiting the expression of aromatase mRNA as an intraovarian regulator for suppressing GC maturation.

Collagens Serve as an Extracellular Store of Bioactive Interleukin 2

The binding of certain growth factors and cytokines to components of the extracellular matrix can regulate their local availability and modulate their biological activities. We show that interleukin 2 (IL-2), an important stimulator of T cell growth, preferentially binds to collagen types I, III, and VI and to a lesser degree to collagen types II, IV, and V, immobilized on polystyrene or nitrocellulose. These interactions are inhibited by denatured, single collagen chains or a subset of their cyanogen bromide peptides in a dose-dependent manner. Cross-inhibition experiments and ligand blotting of collagen-derived peptides point to a limited set of collagenous consensus sequences mediating the binding of IL-2. This interaction is saturable, with dissociation constants of approximately 10(-)(8) m, and estimated molar ratios of 4-6 molecules of IL-2 bound to one molecule of triple helical collagen. Furthermore, collagen-bound IL-2 stimulates proliferation of mouse lymphocytes. We conclude that its specific binding to the abundant interstitial collagens leads to a spatial pattern of bioavailable IL-2 which is dictated by the local organization of the collagenous extracellular matrix. This interaction may contribute to the particular phenotype of stromal lymphocytes and could be exploited for devising collagenous peptide analogues that modulate IL-2 bioactivity.

A potential role for tumour necrosis factor-alpha in synergy between porcine respiratory coronavirus and bacterial lipopolysaccharide in the induction of respiratory disease in pigs.

This study examined whether exposure of pigs to both porcine respiratory coronavirus (PRCV) and bacterial lipopolysaccharide (LPS) can potentiate respiratory disease and lung secretion of tumour necrosis factor-a (TNF-alpha) and interleukin-1 (IL-1). Caesarian-derived colostrum-deprived pigs were inoculated intratracheally with PRCV, with LPS from Escherichia coli O111:B4 (20 microg/kg), or with a combination of the two, and killed at set times after inoculation. Clinical signs, virus replication and (histo)pathological changes in the lungs, percentage of neutrophils and bioactive TNF-alpha and IL-1 in broncho-alveolar lavage (BAL) fluids were examined. The effects of separate virus or LPS inoculations were subclinical and failed to induce high and sustained cytokine levels. In a preliminary study, pigs were inoculated with PRCV and then with LPS 24 h later and killed sequentially. Severe respiratory disease and significantly enhanced TNF-alpha titres (208-3601 U/ml versus 40-89 U/ml after LPS only) were seen during the first 12 h after LPS inoculation. IL-1 levels (106-1631 U/ml versus 28-654 U/ml after LPS only) were also increased, but persisted for longer after clinical recovery than TNF-alpha. In a second study, pigs were inoculated with PRCV and subsequently with LPS at various time intervals ranging from 0 to 24 h, and killed 5 h after inoculation with LPS. A time interval of at least 12 h between inoculations was necessary for prominent respiratory signs to develop. Production of TNF-alpha, but not IL-1, was also dependent on the time interval between inoculations and was tightly correlated with disease. Lung neutrophil infiltration and pathological changes were comparable after combined PRCV-LPS and single LPS inoculations, and were not associated with disease. These data show that exposure to high endotoxin concentrations in swine buildings can precipitate respiratory disease in PRCV-infected pigs, and that TNF-alpha is probably an important mediator of these effects. This is the first in-vivo demonstration of synergy between respiratory viruses and LPS.

Is acne an infection of blocked pilosebaceous follicles? Implications for antimicrobial treatment.

A model is proposed which is based on the assumption that acne is due to infection of functionally blocked pilosebaceous follicles by propionibacteria. Noninflamed lesions, which are first visible during the adrenarche in acne-prone individuals, do not contain propionibacteria. Comedogenesis appears to be independent of bacterial infection and may be driven by high levels of bioactive interleukin-1 alpha derived from ductal hyperkeratinocytes. The stimulus which triggers interleukin-1 alpha production is unknown. Formalin killed Propionibacterium acnes failed to stimulate production of the cytokine by cultured human keratinocytes in vitro. Inflamed lesions are thought to arise from microcomedones, but the initiating events are unknown. Evidence that propionibacteria are involved in the generation of inflammatory lesions is inconclusive. The cellular infiltrate is consistent with a type IV hypersensitivity response to one or more persistent lesional antigens, not necessarily bacterial. The potent adjuvant activity of P. acnes would up-regulate the immune response to any antigen which came into contact with the mononuclear cell infiltrate. Antibiotics are widely used in the treatment of acne, and their effects in selecting a predominantly resistant commensal population are well recognized. Although they reduce numbers of propionibacteria on the skin, other modes of action may contribute to or explain their therapeutic efficacy. At a time when there is global concern that antibiotic resistance rates in common bacterial pathogens may threaten our future ability to control bacterial infections, practices which promote the spread of antibiotic-resistant bacteria must be fully justified. A thorough reappraisal of the role of propionibacteria in acne is overdue. It is likely that further experimental work is needed to confirm or refute that P. acnes is aptly named.

Efficient induction of an antigen-specific, T helper type 1 immune response by interleukin-12-secreting fibroblasts.

To determine whether the paracrine secretion of interleukin (IL)-12 can efficiently convert immune responses characterized by high levels of synthesis of IL-4 and immunoglobulin E (IgE) into T helper 1 (Th1)-dominated responses, 3T3 fibroblasts were stably transfected to secrete IL-12 (480 units/10(6) cells/48 hr). Their effects on the T helper cell-mediated immune response were investigated in ovalbumin (OVA)-primed mice. Free mouse recombinant IL-12 was included as a control group. IL-12-secreting fibroblasts (3T3/IL-12) were more effective than free recombinant IL-12 at increasing OVA-specific interferon-gamma (IFN-gamma) production and decreasing OVA-specific IL-4 production in CD4+ T cells. In addition, injection with 3T3/IL-12 cells significantly increased anti-OVA immunoglobulin G2a (IgG2a) levels and decreased anti-OVA IgE levels in OVA-primed mice. This work suggests that IL-12-secreting fibroblasts can efficiently induce an antigen-specific Th1 response and may be beneficial in the treatment of diseases caused by undesirable T helper 2 (Th2)-dominated responses, including allergic diseases.

Human amniotic fluid lacks interleukin-2 and interleukin-15 but can interact with the β-chain of the interleukin-2 receptor

The present study investigated whether an explanation for the conflicting reports on the interleukin-2 (IL-2) status of amniotic fluid is due to the presence of IL-15 which shares biological activities with IL-2 and utilizes the IL-2 receptor beta-chain. Amniotic fluids from 45 normally progressing pregnancies between 14 and 16 weeks after the last menstrual period were assayed for IL-2 and IL-15 by bioassay and enzyme-linked immunosorbent assay (ELISA). The ability of amniotic fluids to induce cytotoxic T lymphoblastoid line-2 (CTLL-2) cell proliferation was demonstrated to be dependent upon bioassay culture conditions. In serum-free medium each amniotic fluid stimulated CTLL-2 proliferation with a mean level of IL-2-like bioactivity of 14.7 +/- 2.3 ng/ml but amniotic fluids failed to induce CTLL-2 proliferation in serum-supplemented medium. Treatment with neutralizing anti-IL-2 or anti-IL-15 antibodies failed to inhibit amniotic fluid-induced CTLL cell proliferation in serum-free medium, indicating a lack of IL-2 and IL-15 bioactivity. In contrast, treatment with anti-IL-2 receptor beta-chain antibody significantly reduced amniotic fluid-induced proliferation. The lack of IL-2 and IL-15 activity in amniotic fluids was confirmed using ELISA. Although high levels of IL-15 immunoactivity were detected in all samples, specificity controls showed a lack of specific IL-15 immunoactivity in amniotic fluid. Pretreatment of amniotic fluids with 100-500 ng/ml mouse immunoglobulin G abrogated IL-15 immunoactivity, indicating that amniotic fluid contains molecules binding to Fc regions of immunoglobulins and responsible for false ELISA positivity. These studies unequivocally show that amniotic fluid lacks IL-2 and IL-15 but can stimulate CTLL-2 cell proliferation via the IL-2 receptor beta-chain. The absence of IL-2 and IL-15 in normal mid-trimester amniotic fluid suggests that the cytokine profile of human pregnancy appears to be associated with a bias against type 1 cytokines within the feto-placental unit.

Inactivation of interleukin-6 by neutrophil proteases at sites of inflammation. Protective effects of soluble IL-6 receptor chains.

In contrast to the excessively elevated immunochemically detectable concentrations of interleukin-6 (IL-6) in inflammatory exudates, the IL-6 bioactivities are significantly reduced, suggesting an inactivation of IL-6 at sites of inflammation. Since high amounts of proteases are released by invading neutrophils (PMN) in close temporal correlation to elevated IL-6 concentrations at sites of inflammation, this study focused on effects of the PMN-derived proteases elastase (NE), proteinase 3 (PR 3) and cathepsin G (Cat G) on the bioactivity and molecular integrity of IL-6. Here, we demonstrate that these enzymes play a crucial role in the initiation of the degradation and subsequent inactivation of IL-6 at sites of inflammation. Soluble IL-6 receptor subunits elicit a protective effect against the IL-6 inactivation by Cat G, only. Possible consequences of the proteolytical IL-6 inactivation for local inflammatory processes will be discussed.

Downregulation of Antigen-Presenting Cell Functions After Administration of Mitogenic Anti-CD3 Monoclonal Antibodies in Mice

Antibodies against CD3ɛ are widely used as immunosuppressive agents. Although it is generally assumed that these reagents exert their immunomodulatory properties by inducing T-cell deletion and/or inactivation, their precise mechanism of action remains to be elucidated. Using a murine model, we demonstrate in this report that administration of anti-CD3ɛ antibodies causes the migration and maturation of dendritic cells (DC) in vivo, as determined by immunohistochemical analysis. This maturation/migration process was followed by selective loss of splenic DC, which resulted in a selective inhibition of antigen-presenting cell (APC) functions in vitro. Spleen cells from anti-CD3ɛ–treated animals were unable to productively stimulate naive alloreactive T cells and Th1-like clones in response to antigen, while retaining the ability to present antigen to a T-cell hybridoma and Th2 clones. Anti-CD3ɛ treatment was found to induce a selective deficiency in the ability of spleen cells to produce bioactive interleukin-12 in response to CD40 stimulation. APC dysfunction was not observed when nonmitogenic forms of anti-CD3ɛ antibodies were used, suggesting that splenic DC loss was a consequence of in vivo T-cell activation. Nonmitogenic anti-CD3ɛ monoclonal antibodies were found to be less immunosuppressive in vivo, raising the possibility that APC dysfunction contributes to anti-CD3ɛ–induced immunomodulation. Collectively, these data suggest a novel mechanism by which mitogenic anti-CD3ɛ antibodies downregulate immune responses.

Downregulation of Antigen-Presenting Cell Functions After Administration of Mitogenic Anti-CD3 Monoclonal Antibodies in Mice

AbstractAntibodies against CD3ɛ are widely used as immunosuppressive agents. Although it is generally assumed that these reagents exert their immunomodulatory properties by inducing T-cell deletion and/or inactivation, their precise mechanism of action remains to be elucidated. Using a murine model, we demonstrate in this report that administration of anti-CD3ɛ antibodies causes the migration and maturation of dendritic cells (DC) in vivo, as determined by immunohistochemical analysis. This maturation/migration process was followed by selective loss of splenic DC, which resulted in a selective inhibition of antigen-presenting cell (APC) functions in vitro. Spleen cells from anti-CD3ɛ–treated animals were unable to productively stimulate naive alloreactive T cells and Th1-like clones in response to antigen, while retaining the ability to present antigen to a T-cell hybridoma and Th2 clones. Anti-CD3ɛ treatment was found to induce a selective deficiency in the ability of spleen cells to produce bioactive interleukin-12 in response to CD40 stimulation. APC dysfunction was not observed when nonmitogenic forms of anti-CD3ɛ antibodies were used, suggesting that splenic DC loss was a consequence of in vivo T-cell activation. Nonmitogenic anti-CD3ɛ monoclonal antibodies were found to be less immunosuppressive in vivo, raising the possibility that APC dysfunction contributes to anti-CD3ɛ–induced immunomodulation. Collectively, these data suggest a novel mechanism by which mitogenic anti-CD3ɛ antibodies downregulate immune responses.

Evidence for a crucial role of neutrophil-derived serine proteases in the inactivation of interleukin-6 at sites of inflammation

The bioactivity of interleukin-6 (IL-6) was found to be dramatically reduced in fluids from sites of inflammation. Here, we provide evidence that the neutrophil-derived serine proteases elastase, proteinase 3 and cathepsin G are mainly involved in its degradation and subsequent inactivation. The initially hydrolyzed peptide bonds were detected to be Val 11-Ala 12 and Leu 19-Thr 20 (elastase), Phe 78-Asn 79 (cathepsin G) and Ala 145-Ser 146 (proteinase 3). The soluble IL-6 receptor elicits a protective effect against the IL-6 inactivation by cathepsin G only. The inactivation of IL-6 by neutrophil-derived serine proteases might act as a feedback mechanism terminating the IL-6-induced activation of neutrophils.

Regression of colon cancer and induction of antitumor immunity by intratumoral injection of adenovirus expressing interleukin-12.

Interleukin-12 (IL-12) has been shown to possess potent immunoregulatory and antitumoral effects. We have evaluated the anti-oncogenic potential and the mechanisms of the antitumoral effect of in vivo adenovirus-mediated transfer of IL-12 gene in a murine model of colon cancer. AdCMVIL-12 was constructed to permit coordinated production of p40 and p35 subunits of IL-12 gene to obtain the maximum IL-12 bioactivity. Infection of murine colon cancer CT-26 cells in vitro with AdCMVIL-12 resulted in the production of high levels of IL-12. In vivo gene therapy of colon cancer nodules by intratumoral injection of AdCMVIL-12 induced a local increase in IL-12 and interferon-gamma levels and a complete regression of the tumor in 26 of 34 (76%) mice. Tumor disappeared between days 7 and 10 after vector administration. The antitumoral effect was mediated by CD8+ T cells and was associated with the generation of cytotoxic T lymphocytes against colon cancer cells. Animals that eliminated the tumor were protected against a second administration of neoplastic cells. Treatment with AdCMVIL-12 of one tumor nodule also caused regression of established tumors at distant sites. These data demonstrate that AdCMVIL-12 is a useful therapeutic tool for established colon cancer in mice and should be considered for application in humans.

Synergistic antitumor effects of interleukin-12 gene transfer and systemic administration of interleukin-18 in a mouse bladder cancer model.

We introduced the interleukin-12 (IL-12) gene into the mouse bladder cancer cell line (MBT2) to establish sublines that secrete bioactive IL-12. IL-12-secreting MBT2 (MBT2/IL-12) sublines were completely rejected when subcutaneously implanted into immunocompetent syngeneic C3H mice. Although this antitumor effect did not change when IL-12-secreting cells were injected into immunodeficient mice whose CD8(+) T or CD4(+) T cells had been depleted by the corresponding antibody, it was abrogated when natural killer cells were depleted by anti-asialoGM1 antibody. In addition, when parental MBT2 cells mixed with MBT2/IL-12 cells were subcutaneously injected into mice, admixed MBT2/IL-12 inhibited the growth of the parental tumor. Furthermore, this antitumor effect was enhanced by systemic IL-18 administration. This synergism was abrogated when the mice were treated with interferon-gamma-neutralizing antibody in vivo. In conclusion, local secretion of IL-12 led to effective antitumor activity that was enhanced by systemic administration of IL-18. Interferon-gamma plays an important role in the synergism of IL-12 gene transduction and systemic administration of IL-18.

Antigen-specific cytotoxicity and cell number of adoptively transferred T cells are efficiently maintained in vivo by re-stimulation with an antigen/interleukin-2 fusion protein.

In order to maintain in vivo anti-tumor efficacy of antigen (Ag)-specific T cells in adoptive immunotherapy for a prolonged period, we constructed a fusion protein (OVA/IL-2) containing ovalbumin (OVA) as a model tumor Ag, co-valently linked to murine interleukin-2 (IL-2). The OVA/IL-2 protein produced in a baculovirus expression system displayed potent IL-2 bio-activity. Immunization with the OVA/IL-2 protein after adoptive transfer of OVA-specific T cells maintained the OVA-specific cytotoxicity and cell number of adoptively transferred T cells long term in vivo, while a simple mixture of recombinant OVA (rOVA) and rIL-2 did not. The response was dependent on the injection doses and times of the OVA/IL-2 protein. Furthermore, weekly re-stimulation of adoptively transferred OVA-specific T cells with the OVA/IL-2 protein cured 70% of tumor-bearing mice. In contrast, re-stimulation with a mixture of rOVA and rIL-2 could not significantly prolong the survival period of tumor-bearing mice. These studies suggest that the co-valent linkage between IL-2 and antigen confines the effect of IL-2 to antigen-specific T cells, leading to efficient maintenance of the anti-tumor activity of adoptively transferred T cells.

Constitutive Expression of Interleukin-1 Messenger Ribonucleic Acid in Rat Sertoli Cells Is Dependent upon Interaction with Germ Cells

Interleukin-1 (IL-1), a proinflammatory cytokine originally isolated as a product of activated mononuclear phagocytes, consists of two distinct agonist proteins, IL-1α and IL-1β, of which IL-1β is the major inducible IL-1 protein produced by macrophages. We show here that mRNA of IL-1α, but not IL-1β, is constitutively expressed by the intact rat testis and localize the transcript to Sertoli cells as confirmed by a novel squash technique. The expression is developmentally regulated and appears only after postnatal day 20 in the rat testis, corresponding to onset of puberty. IL-1α mRNA shows a stage-dependent expression pattern during the cycle of the seminiferous epithelium. It is low or absent in stage VII, but present in all other stages of the cycle. The same stage-dependent distribution was also observed at the protein level when bioactive IL-1 was measured in extracts of accurately defined one millimeter segments of seminiferous tubules. No IL-1α mRNA was detected in adult rat testes after germ cell depletion by fetal irradiation or cytostatic drug treatment. Because stage VII is the only segment of the seminiferous tubules lacking DNA replication, we propose that IL-1α is involved in this event during mitosis and meiosis of spermatogenesis and that its expression is dependent upon interactions between Sertoli cells and germ cells.