Stimulation Of Immune Cells Research Articles

IL-8 AND MCP-1 SECRETION IS ENHANCED BY THE PEPTIDE-NUCLEIC ACID IMMUNOMODULATOR, PRODUCT R, IN U937 CELLS AND PRIMARY HUMAN MONOCYTES

Product R (Reticulose™) is a peptide–nucleic acid immunomodulator recently shown to enhance the expression of mRNAs encoding pro-inflammatory cytokines. Interleukin 8 (IL-8) and macrophage chemoattractant protein-1 (MCP-1) are pro-inflammatory chemokines involved in immune cell mobilization and stimulation. To determine whether Product R acts by upregulating these chemokines, we assayed its effects on the expression of IL-8 and MCP-1 mRNAs and proteins by human monocytic U937 cells and by adherent peripheral blood mononuclear cells (PBMCs). U937 cells were cultured for 0–21 days in media containing 0–20% Product R or phosphate-buffered saline (PBS). Compared to control cultures, cells cultured in Product R expressed increased amounts of IL-8 and MCP-1 mRNAs, as measured by reverse transcriptase-polymerase chain reaction (RT-PCR). Product R also increased secretion of IL-8 and MCP-1, as measured by enzyme-linked immunosorbent assay (ELISA), and boosted secretion induced by bacterial lipopolysaccharide (LPS), in a time- and dose-dependent manner. In adherent PBMCs, Product R increased IL-8 and MCP-1 secretion, but reduced LPS-induced MCP-1 secretion. While mRNAs encoding the IL-8 receptor, CXCR2, and the MCP-1 receptor, CCR2, were increased in U937 cells cultured in 5–10% Product R, we observed no change in binding of receptor-specific antibodies. These findings suggest that Product R upregulates the expression of IL-8 and MCP-1, which may boost immune system activity in virally-infected patients.

Immune complex stimulation of peripheral blood mononuclear cells result in enhancement of suppression of IL-12 production dependent on soluble serum factors

Immune complexes can induce the production of various cytokines in vitro. Both IL-10 and IL-12 could be induced by addition of heat-aggregated immunoglobulins to mononuclear cells in serum-free cell culture systems. Addition of native serum to the cell cultures influenced the effects on IL-10 and IL-12 in opposite ways. While IL-10 levels were increased in cell cultures with native human serum, IL-12 production was inhibited as compared to cultures with monomeric IgG. Two series of experiments suggested that the effects of immune complexes on IL-12 production depended on the activity of the classical complement pathway in the serum: 1.) Heat-inactivation of serum reverted the inhibitory effect of immune complexes on IL-12 production. 2.) C4 deficient serum behaved as a heat-inactivated normal serum concerning the effects on IL-12 production, and this effect could be reversed by addition of C4. The effects of neutralizing IL-12 had modest effect on immune complex-induced IL-10 production, and the effects of neutralizing IL-10 had no effect on IL-12 production. IL-10 production in the presence of immune complexes could be partially blocked by anti-FcgammaRII antibodies, while the immune complex-mediated effects on IL-12 not changed by blocking FcgammaRII or FcgammaRIII. Opposite and complement-dependent effects on the production of IL-10 and IL-12 can be of importance in cytokine-dependent autoimmune diseases like rheumatoid arthritis or systemic lupus erythematosus, where local or systemic activation of the classical complement pathway participate in the disease processes. Blocking of complement activation or receptors for activated complement components might gain increased attention as potential targets for immune therapies in the light of such cytokine-deviating effects.

Alterations in mitochondria and mtTFA in response to LPS-induced differentiation of B-cells

Stimulation of immune cells results in altered cell function and metabolism, which must be recognized by and coordinated with energy production from mitochondria. Mitochondria contain their own DNA genome encoding 13 polypeptides that combine with nuclear-derived subunits to create functional enzyme complexes of the electron transport chain. Therefore, coordination of mitochondrial and nuclear transcription is necessary to achieve a sustained elevation in mitochondrial ATP production. Pre-B-lymphocytes stimulated with lipopolysaccharide exhibit increased activity levels of the mitochondrial enzymes, succinate dehydrogenase and cytochrome c oxidase. Immunoblot analyses of purified mitochondria indicate an increase in the mitochondrial transcription factor (mtTFA) levels in mitochondria induced by cell stimulation. This increase is consistent with increased mtTFA production in the cytoplasm. In addition, mitochondrial protein extracts indicate an increase in protein binding to a mtTFA–DNA binding site from the mitochondrial genome, subsequent to cell stimulation. These results indicate that mitochondrial activity changes during B-lymphocyte stimulation, and mtTFA may contribute to the coordination of respiration with cellular energy demand.

Supernatants from Macrophages Stimulated with Microcystin‐LR Induce Electrogenic Intestinal Response in Rabbit Ileum

Microcystin-LR is a cyclic heptapeptide hepatotoxin produced by the cyanobacterium Microcystis aeruginosa. This microorganism often forms toxic blooms in freshwater lakes and reservoirs for drinking water supply, producing serious disorders in humans and animals. Some have suggested that certain biological activities of microcystin may depend upon the stimulation of immune cells. Therefore, the aims of this research were to examine electrogenic intestinal secretion, in vitro, caused by the supernatants from macrophages stimulated with microcystin-LR, as well as to investigate the presence of interleukin-1beta and tumour necrosis factor-alpha in these supernatants. We found that the supernatants of macrophages stimulated with microcystin-LR (0.1, 0.3 and 1.0 microg/ml) caused electrogenic intestinal effects (change in short circuit currents (delta SCC)=57.6, 50.8 and 73.3, respectively, versus control=19.6 microA.cm(-2)) in a time-dependent way (microcystin-LR (1.0 microg/ml)=63.2, 108.8, 120.4 and 132.3 microA.cm(-2) at time 0, 40, 50 and 60 min., respectively). In addition, the intestinal secretory activity present in these supernatants was blocked (57%) by the prior treatment of macrophages with dexamethasone. We also demonstrated that microcystin-LR (0.1, 0.3 and 1.0 ,microg/ml) is capable of stimulating the synthesis of tumour necrosis factor-alpha (375.4, 369.0 and 610.8 pg/ml, respectively, versus control=165.0 pg/ml) and interleukin-1beta (198.9, 189.3 and 522.1 pg/ml, respectively, versus control=39.7 pg/ml). These findings demonstrate that microcystin-LR induces the release of interleukin-1beta and tumour necrosis factor-alpha by peritoneal macrophages in vitro, and that the supernatants from these macrophages induce electrogenic secretion in rabbit ileal mucosa.

Pro-inflammatory effects of substance P: new perspectives for the treatment of airway diseases?

Non-adrenergic, non-cholinergic (i.e. NANC) neuronal transmission has been implicated in the pathophysiology of asthma. Attention has focused on both inhibitory NANC transmission mediated by its putative neurotransmitters vasoactive intestinal peptide (VIP) and nitric oxide (NO) and excitatory NANC transmission mediated by the sensory neuropeptides. Studies on rodent airway and non-airway tissue have demonstrated that activation of non-cholinergic excitatory nerves by mechanical or chemical stimuli can generate local axon reflexes that lead to bronchoconstriction and neurogenic inflammation. The sensory neuropeptides substance P (SP) and neurokinin A (NKA) have various effects that could contribute to changes observed in asthmatic airways, including smooth muscle contraction, submucosal gland secretion, vasodilatation, an increase in vascular permeability, stimulation of cholinergic nerves and stimulation of immune cells 1 Bertrand C. Geppetti P. Tachykinin and kinin receptor antagonists: therapeutic perspectives in allergic airway disease. Trends Pharmacol. Sci. 1996; 17: 255-259 Abstract Full Text PDF PubMed Scopus (81) Google Scholar , 2 Joos G.F. et al. Sensory neuropeptides and the human lower airways: present state and future directions. Eur. Respir. J. 1994; 7: 1161-1171 PubMed Google Scholar . Most of these effects have been observed in both human and animal tissue and are mediated by specific tachykinin receptors; stimulation of the NK2 receptor mediates a major part of the tachykinin-induced airway smooth muscle contraction, whereas stimulation of NK1 receptors induces vasodilatation, plasma protein extravasation, mucus secretion and stimulation of inflammatory and immune cells 3 Advenier C. et al. Role of tachykinins as contractile agonists of human airways in asthma. Clin. Exp. Allergy. 1999; 29: 579-584 Crossref PubMed Scopus (31) Google Scholar . The effects of tachykinins on the airways can be terminated by enzymatic degradation, mainly by the action of the ectoenzyme neutral endopeptidase 2 Joos G.F. et al. Sensory neuropeptides and the human lower airways: present state and future directions. Eur. Respir. J. 1994; 7: 1161-1171 PubMed Google Scholar . (2s-cis)-2-(diphenylmethyl)-N-[(2-methoxyphenyl)methyl]-1-azabicyclo[2.2.2]octan-3-amine ((+)-(2s,3s)-3-methoxybenzyl amino)-2-phenylpiperidine N-[N2-[N-[N-[N-[2,3-didehydro-N-methyl-N-[N-[3-(2-pentylphenyl)-propionyl]-l-threonyl]tyrosyl-l-leucynyl]-d-phenylalanyl]-l-allothreonyl]-l-asparaginyl]-l-serine-n-lactone] N2-[(4r)-4-hydroxy-1-(1-methyl-1H-indol-3-yl)carbonyl-l-prolyl]-N-phenylmethyl-3-(2-naphthyl)-l-alaninamide (s)-N-methyl-N[4-(4-acetylamino-4-phenyl piperidino)-2-(3,4-dichlorophenyl)butyl] benzamide

A mouse model of chronic bacterial lesions (a cotton trap) for studying oral bacteria - lymphocyte interactions.

We established a mouse model of chronic bacterial infection (cotton trap) to get a deeper insight into interactions between immune cells and bacterial strains, that are most commonly isolated from periapical processes. We have used flow cytometry to identify the presence of intracellular cytokines of activated T cells collected from cotton traps, previously infected with different strains of bacteria and implanted subcutaneously into the back of the mice. We provide an evidence that anaerobic bacteria (Bacteroides sp.) and nocardiae are more effective in inducing cytotoxic immunity and Th1 response compared to oral streptococci. Differences in immune response against anaerobic bacteria when compared to streptococci are probably dependent on some non-specific immune cell stimulation (e.g. by bacterial cell wall components), nevertheless the role of specific antigen-dependent immune mechanism can not be excluded.

Effectiveness of immunochemotherapy for gastric cancer: A review of the current status

Currently, the most effective treatment of gastric cancer is surgical resection of the tumor with lymphadenectomy, which is the standard treatment in Japan. It is important to determine whether chemotherapy or immunochemotherapy regimens may prolong the survival of patients with gastric cancer. Although gastric cancer cells, in general, have a low sensitivity to chemotherapy and a low immunogenicity related to stimulation of immune competent cells, a new method including biochemical modulation and nonspecific immunopotentiation with biological response modifiers (BRMs) has permitted us to augment the clinical efficacy of immunochemotherapy for gastric cancer. Further, promising new drugs are being developed and await testing for their possible benefit in an adjuvant setting, and recent advances in molecular biology may open a new avenue for tumor antigen-specific immunotherapy for gastric cancer. We will review the current status of immunochemotherapy for gastric cancer and examine its clinical benefit for survival. Semin. Surg. Oncol. 17:139–143, 1999. © 1999 Wiley-Liss, Inc.

Effectiveness of immunochemotherapy for gastric cancer: a review of the current status.

Currently, the most effective treatment of gastric cancer is surgical resection of the tumor with lymphadenectomy, which is the standard treatment in Japan. It is important to determine whether chemotherapy or immunochemotherapy regimens may prolong the survival of patients with gastric cancer. Although gastric cancer cells, in general, have a low sensitivity to chemotherapy and a low immunogenicity related to stimulation of immune competent cells, a new method including biochemical modulation and nonspecific immunopotentiation with biological response modifiers (BRMs) has permitted us to augment the clinical efficacy of immunochemotherapy for gastric cancer. Further, promising new drugs are being developed and await testing for their possible benefit in an adjuvant setting, and recent advances in molecular biology may open a new avenue for tumor antigen-specific immunotherapy for gastric cancer. We will review the current status of immunochemotherapy for gastric cancer and examine its clinical benefit for survival.

Allergens induce apoptosis in lymphocytes from atopic patients

Repeated stimulation of immune cells may induce an “activation-induced cell death” (AICD) program. Allergy is characterized by the cyclic activation of allergen-reactive immune cells. To study the effects of allergen stimulation in cell proliferation and apoptosis in atopic subjects, peripheral blood mononuclear cells (PBL) from 40 atopic patients with positive reactivity to the allergens Olea Europaea (OE) and Lollium Perenne (LP) (20 without immunotherapy and 20 with specific immunotherapy) and 10 normal subjects were cultured with the allergens OE and LP. PBL from atopic patients proliferate more vigorously than cells from normal subjects after culture in vitro with both allergens, although PBL from atopic subjects without immunotherapy proliferate more than PBL from atopic subjects with immunotherapy. The study of cell proliferation shows that in atopic patients PBL mainly exhibit the CD4/CD45RO phenotype. This preferential proliferation is more evident in PBL from atopic patients treated without immunotherapy. Cell culture with specific allergens induces apoptosis in PBL from atopic patients. The percentage of apoptosis increased when atopic patients had been previously treated with immunotherapy. In addition to the observed increase in cell proliferation, apoptosis mainly occurs in the CD45RO cells that support the involvement of these cells in allergy. Furthermore, results obtained in cells from immunized patients suggest that an AICD process may partly at least explain the mechanism of action of allergen immunotherapy.

MECHANISMS OF TOLERANCE TO A TRANSGENIC MINOR TRANSPLANTATION ANTIGEN

937 β-galactosidase can behave as a minor transplantion antigen in C57BL/6 (B6, H-2b) mice as they reject congenic B6 β-galactosidase transgenic (β-gal tg) skin grafts by day 25. Recall immune responses after rejection reveal β-gal specific proliferation and type 1 cytokine production, cytotoxicity of β-gal tg cells and β-gal specific delayed type hypersensitivity (DTH) responses. We induced immunologic tolerance to β-gal tg skin grafts by pretreatment of the B6 recipients with i.v. injection of soluble β-gal. B6 β-gal tg skin graft survival was prolonged for > 5 months, while third party grafts were rejected within 14 days. Recall responses in tolerized mice revealed an absence of β-gal-specific IFNγ, IL-2, IL-4 or IL-5 production (no type 2 immune deviation). Spleen cells obtained from the tolerant animals were unable to mediate CTL activity or DTH responses. In an effort to discern whether clonal deletion, anergy and/or suppression were active mechanisms of the tolerance, we next tested whether we could recover β-gal specific immunity from tolerant animals by in vitro incubation of tolerized immune cells in the presence of exogenous IL-2. Although the cells were unable to respond to β-gal in primary recall responses, incubation with IL-2 resulted in recovery of β-gal specific, IFNγ - producing T cells, thus excluding clonal deletion as an operative mechanism. Furthermore, spleen cells obtained from tolerized mice acted as suppressor cells, in that co-injection of these spleen cells inhibited ovalbumin (OVA) specific DTH responses normally produced by OVA-primed T cells. Finally, β-gal stimulation of immune cells from the tolerant animals induced significant upregulation of TGFβ message. These results suggest that the i.v. immunization resulted in expansion of a population of β-gal specific, TGFβ producing T cells capable of suppressing the pathogenic, anti-skin graft immune response. Taken together these data demonstrate the feasibility of inducing graft tolerance to a transgenic minor antigen and implicate TGFβ mediated suppression and/or anergy as mechanisms of such tolerance. Lessons obtained from this experimental model may translate into approaches for inducing graft tolerance in human organ transplant recipients.

Corticotropin-Releasing Factor Receptor 1 in Mouse Spleen: Expression After Immune Stimulation and Identification of Receptor-Bearing Cells

A specific polyclonal Ab against the N-terminal domain of corticotropin-releasing factor (CRF) receptor, type 1 (CRF-R1), was employed to an immunohistochemical analysis of the spleen from naive mice and mice exposed to an immune challenge. Cell types stained with anti-CRF-R1 Ab were identified by their nuclear shapes and colocalization with the cell type-specific markers ER-MP58, ER-MP20, Moma-1, Moma 2, anti-CD3e mAbs, and anti-Ig Ab. Only a few clusters of CRF-R1+ cells were found in spleen sections of naive mice at sites typical for granulopoietic islands. However, a 17-fold increase in the mean number of CRF-R1+ cells was noted within hours following a challenge of acute systemic inflammation induced by i.p. administration of LPS. The majority of these cells were identified as mature neutrophils. CRF-R1 was shown to mediate suppression of the IL-1beta secretion by these cells. However, at later time points a large number of granulocyte-macrophage precursors was strongly labeled with anti-CRF-R1 Ab. Western blot analysis of splenic membranes from animals treated with LPS revealed a m.w. of approximately 70,000 for CRF-R1. Subcellular staining patterns were suggestive for the predominant localization of CRF-R1 on granule membranes. CRF-R1 mRNA was detected in spleen but not in bone marrow and peripheral blood leukocytes from naive mice. Thus, it was indicated that CRF-R1 was not produced constitutively by mature or immature neutrophils. Its production was rather triggered by inflammatory stimuli.

Cross-Reactivity in Murine Fluoroquinolone Photoallergy: Exclusive Usage of TCR Vβ13 by Immune T Cells That Recognize Fluoroquinolone-Photomodified Cells

AbstractFluoroquinolone antibacterial agents are well known to elicit photosensitivity as an adverse effect, and their cross-reactivity has been clinically documented. The photoallergenicity of fluoroquinolones is mainly derived from their photohaptenic moiety, and photomodification of skin epidermal cells with fluoroquinolones is thought to be an initial step for this photoallergy. Here we have explored, both in vivo and in vitro, T cell responses to fluoroquinolone-photomodified cells, focusing on their photoantigenic cross-reactivity. Cells were derivatized with fluoroquinolones under exposure to UV-A, and fluoroquinolone photoadducts were detected in photomodified cells by immunostaining, flow cytometry, and cell ELISA using fluoroquinolone-specific mAb. T cell-mediated hypersensitivity induced and elicited by s.c. injection of fluoroquinolone-photomodified epidermal cells was cross-reactive among six fluoroquinolones. In addition, lymph node cells from mice sensitized with fluoroquinolone-photomodified cells proliferated well in vitro not only to Langerhans cell-enriched epidermal cells photoderivatized with corresponding fluoroquinolone, but also to those photomodified with any of five other fluoroquinolones, supporting their cross-reactivity. In three fluoroquinolones tested, Th1 populations that expanded after in vitro photoantigenic stimulation of immune lymph node cells expressed the same Vβ13 of TCR. The sensitivity could be transferred by the i.v. administration of this Vβ13+ T cell line into naive recipients, in which a high percentage of Vβ13+ cells infiltrated at the challenge site. These findings suggest that these fluoroquinolones carry the same photoantigenic epitope, which is recognized by Vβ13+ T cells, leading to fluoroquinolone photosensitivity and cross-reactivity.

Abstracts of published articles in Nippon Ganka Gakkai Zasshi (Journal of the Japanese Ophthalmological Society)

Human urinary Tamm-Horsfall glycoprotein (THP) is a pleotropic protein that binds different cytokines and stimulates various immunocompetent cells. It is unclear whether these important functions of THP are altered in renal transplant patients.We purified THPs from normal individuals (N-THP) and renal transplant patients receiving potent immunosuppressants (R-THP). The carbohydrate (CHO) compositions of THPs were probed by lectin-blotting and lectin-binding ELISA. The functions of THP were assessed by immune cell-stimulation as well as C1q, IL-1β, IL-8 and TNF-α-binding assays. The roles of CHO moieties in THPs were analyzed using CHO-degrading enzyme digestion.Compared to that of N-THP, the binding capacity of R-THP to Maackia amurensis, Galanthus nivalis and Datura stamonium decreased, indicating that R-THP contained lesser amount of Siaα(2,3)Gal/GalNAc, mannose residues, and β(1,4)GlcNAc, but not GlcNAc/branched mannose. The binding capacity of R-THP to complement C1q and tumor necrosis factor (TNF)-α was also decreased. The stimulating effect of R-THP on mononuclear cell (MNC) proliferation and polymorphonuclear neutrophil (PMN) phagocytosis was less potent than that of N-THP. We found that the defective MNC-stimulation by R-THP was due to impaired NF-κB p52 nuclear translocation. The cell-stimulating effects of N- and R-THP could be abolished by digesting them with CHO-degrading enzymes, β-galactosidase and neuraminidase. Interestingly, a potent apoptosis-inducing effect of R-THP on MNC and PMN was noted.R-THP is not only modified in glycosylation but bears an apoptosis-inducing capacity on MNC and PMN, leading to an impaired immune function in renal transplant patients.

Spiralin, a mycoplasmal membrane lipoprotein, induces T-cell-independent B-cell blastogenesis and secretion of proinflammatory cytokines.

Mycoplasmas are bacteria which can cause respiratory, arthritic, and urogenital diseases. During the early phase of infection, mycoplasmas usually induce an inflammatory response and a humoral response preferentially directed against their membrane-bound, surface-exposed lipoproteins. In this report, we describe the effects on immune cells of spiralin, a well-characterized mycoplasmal lipoprotein. Purified spiralin stimulated the in vitro proliferation of human peripheral blood mononuclear cells and murine splenocytes. The stimulation pathway was probably different from that followed by Escherichia coli lipopolysaccharide because the effect of spiralin was not abolished by polymyxin B. Comparison of the effects of whole, native spiralin with those induced by proteinase K-digested spiralin or by the C-terminal half of spiralin (peptide p[13.5]T) revealed that the first half of the protein, which contains the lipoylated N terminus, is responsible for the mitogenic activity. In contrast to whole spiralin, proteinase K-digested spiralin did not trigger murine B-cell differentiation and immunoglobulin G and M secretion. Stimulation of human or murine immune cells led to early secretion of proinflammatory cytokines (human tumor necrosis factor alpha and murine interleukin 1 or 6). Spiralin induced the T-cell-independent blastogenesis of murine B cells but did not stimulate T cells. Altogether, our data demonstrate that spiralin possesses potent immunostimulating activity, similar to that reported for lipoproteins of pathogenic gracilicutes (gram-negative eubacteria; e.g., Borrelia burgdorferi OspA and E. coli Braun lipoprotein), and are consistent with the fact that lipoproteins are major antigens during mycoplasma infections.

Gene therapy of cancer

Gene therapy of cancer

Cloning and expression of FomA, the major outer-membrane protein gene from fusobacterium nucleatum T18

The major outer-membrane protein, FomA, of Fusobacterium nucleatum has been associated with porin activity, interbacterial adherence and stimulation of host immune cells. Until now, molecular analysis of FomA has not been possible because previous attempts to clone the fomA gene were not successful. The inability to clone F. nucleatum genes led to speculation that Escherichia coli may not be a suitable host. This report concerns the amplification of the fomA gene of F. nucleatum T18 using oligonucleotide primers containing restriction endonuclease sites that allow cloning of fomA into the E. coli expression vector pMMB67. The resultant plasmid, pXW1, was transformed into E. coli DH5α, providing high-level expression of recombinant FomA (rFomA). Amino acid sequencing of rFomA demonstrated that the FomA signal peptide was correctly processed by E. coli signal peptidase I. rFomA was correctly localized to the outer membrane by the E. coli export pathway. The rFomA protein also displayed the heat-modifiable oligomeric and conformational properties of native FomA (nFomA). This demonstration of rFomA expression, processing, export, and secondary and tertiary structure in E. coli provides support for the feasibility of molecular analysis of the structure and function of FomA and other F. nucleatum proteins using recombinant techniques.

Induction of cytotoxic T lymphocytes to murine mammary tumor cells with a Kd-restricted immunogenic peptide.

A synthetic peptide E474 SFAVATTAL, derived from the sequence of mouse mammary tumor virus envelope protein, was previously shown to bind class I MHC Kd. Immunization of BALB/c mice with E474 in 50% incomplete Freund's adjuvant (IFA) followed by in vitro stimulation of immune cells with E474-coated antigen-presenting cells resulted in peptide-specific cytotoxic T lymphocytes (CTL). Furthermore, anti-E474 CTL lysed mammary tumor cell lines D2F2 and D2A1, derived from a spontaneous tumor that arose in BALB/c pre-neoplastic hyperplastic alveolar nodule (HAN) D2 line. Expression of Kd by D2A1 and D2F2 cells was verified by flow cytometry, and lysis of D2 tumor cells was blocked by monoclonal antibody 31-34-S, which interacted with the peptide-binding region of Kd, supporting the recognition of E474 in Kd by anti-E474 CTL. Immunization of BALB/c mice with E474 before D2F2 tumor challenge resulted in reduced tumor growth. Therefore, E474 is naturally processed and presented by these tumor cells and can induce anti-tumor immunity.

Protection against endotoxic shock and lipopolysaccharide-induced local inflammation by tetracycline: correlation with inhibition of cytokine secretion

Septic shock results from excessive stimulation of host immune cells, particularly monocytes and macrophages, by lipopolysaccharide (LPS) released from gram-negative bacteria. Macrophage-derived cytokines, such as tumor necrosis factor alpha (TNF-alpha) and interleukin-1beta (IL-1 beta), have been identified as central mediators in the pathogenesis of septic shock and the resultant mortality. Therefore, these cytokines were targets for experimental therapy for septic shock. Because of tetracycline's ability to intervene in cellular mechanisms involved in cytokine secretion, we tested the effect of tetracycline on LPS-induced septic shock and inflammatory lesions in mice. Tetracycline was found to protect mice against LPS-induced lethality and to abolish clinical signs of LPS-induced inflammatory lesions. This protection correlates with tetracycline's ability to reduce LPS-induced TNF-alpha levels in serum. Furthermore, tetracycline was found to inhibit LPS-induced TNF-alpha and IL-1 beta secretion, but not cytokine mRNA accumulation, in human monocytes in vitro. The results presented here suggest that tetracycline is a potent drug for LPS-induced pathology and that its mechanism of action involves blockage of posttranscriptional events of cytokine production.

Effect of visible light on some cellular and immune parameters

The biological effect of visible light of low energy density was investigated in this study. The effects of diffuse (DL) and linearly polarized (LPL) light were compared on models in vitro and in vivo. Experiments in vitro were performed on human lymphocytes to study their blast-transformation and rosette-formation abilities. Both DL and LPL increased the number of blast-transformed cells even in a lymphocyte culture without PHA, and reduced rosette-formation of T lymphocytes. LPL had a more pronounced effect. In vivo exposure to DL and LPL of the spleens of tumour-bearing mice caused the appearance of factor(s) in their serum, inhibiting the incorporation in vitro of [3H]-thymidine into the tumour cells obtained from non-exposed animals. In the other series of experiments serum samples were taken from tumorous animals after the exposure of their spleens to LPL. Following the daily administration of these sera to another group of non-exposed tumorous mice a decreasing tendency of the mitotic kinetics of ascites tumour was observed. The application of visible (preferably linearly polarized) light for the stimulation of human immune competent cells, and clinical trials with extracorporeal irradiation of blood for the promotion of natural defences of an immune-repressed organism are suggested.

Manipulation of skin graft rejection in alloimmune mice by anti-VCAM-1:VLA-4 but not anti-ICAM-1:;LFA-1 monoclonal antibodies

C3H mice were immunized by repeated skin grafting with B10.BR tail skin. Ten days after the last immunization mice received 100 μg (intravenously) of a variety of different monoclonal antibodies (mAbs: anti-ICAM-1, -LFA-1, -VCAM-1, -VLA-4), alone or in combination, followed by further B10.BR skin grafts. Control animals received injections of saline only. Skin graft survival was monitored daily in all groups. Further injections of antibody were given every second day until graft rejection occurred. In separate studies lymphoid cells were harvested from various tissues of the grafted mice at 6 and 20 days post grafting. Aliquots of each sample were analysed by polymerase chain reaction for mRNA for different cytokines (interleukins IL-2, IL-4, IL-10 and IFNγ (gamma-interferon) believed to be important in the regulation of graft rejection. In addition, lymphoid cells were restimulated in vitro with irradiated B10.BR or third-party stimulator cells in the presence or absence of monolayers of C3H-derived endothelial cells (EC), in an attempt to mimic the in vivo environment of the interactions of cells engaged in alloreactivity in these mice. Only anti-VCAM-1 caused significant prolongation of graft survival in immune mice, while in contrast only the combination of anti-ICAM-1 and anti-LFA-1 produced enhanced survival in naive animals. In each case increased survival was associated with decreased activation of Th1 cells (diminished IL-2, IFNγ) and increased activation of Th2 cells (increased IL-4, IL-10). Interestingly, when stimulation of immune cells took place on EC monolayers addition of anti-VLA-4 enhanced activation of immune cells, while anti-VCAM-1 caused some inhibition; no significant perturbation of activation occurred in the absence of EC.