Effect of different antisera on the magnitude of the immune response following induction of antibody formation by allogenic macrophages

Data are presented on the effect of different antisera on the antibody-genesis induction by immune allogeneic macrophages. Significant decrease of the immune response was shown after administration of both alloantiserum and antimacrophagal serum during the first two days after the allogeneic macrophages transfer. Injection of these sera at the subsequent days did not significantly influence the entensity of the immune response. Antierythrocytic serum inhibits accumulation of antibody-producing cells when administered at different periods of time after allogeneic macrophages transplantation.     

Direct evidence that functionally impaired CD4+ T cells persist in vivo following induction of peripheral tolerance

A small population of CD4+ OVA-specific TCR transgenic T cells was tracked following the induction of peripheral tolerance by soluble Ag to address whether functionally unresponsive, or anergic T cells, persist in vivo for extended periods of time. Although injection of OVA peptide in the absence of adjuvant caused a transient expansion and deletion of the Ag-specific T cells, a population that showed signs of prior activation persisted in the lymphoid tissues for several months. These surviving OVA-specific T cells had long-lasting, but reversible defects in their ability to proliferate in lymph nodes and secrete IL-2 and TNF-alpha in vivo following an antigenic challenge. These defects were not associated with the production of Th2-type cytokines or the capacity to suppress the clonal expansion of a bystander population of T cells present in the same lymph nodes. Therefore, our results provide direct evidence that a long-lived population of functionally impaired Ag-specific CD4+ T cells is generated in vivo after exposure to soluble Ag.     

Regulation of T helper cell differentiation in vivo by soluble and membrane proteins provided by antigen-presenting cells

The aim of this study was to test whether the nature of the antigen-presenting cell (APC) can influence the Th1/Th2 balance in vivo. Our data show that dendritic cells (DC), pulsed extracorporeally with antigen, induced the development of cells secreting IL-2, IFN-gamma and IL-4 upon antigen rechallenge in vitro. Priming with peritoneal macrophages sensitized cells that produced IL-4 but not IFN-gamma. To identify the factors involved in T helper development, mice were primed with APC with or without treatment with neutralizing antibodies to costimulatory molecules or cytokines. Our results indicate that priming with DC or macrophages is strictly dependent on the CD28-CTLA4/B7 interaction. Of note, CD86 provides the initial signal to induce naive T cells to become IL-4 producers, whereas CD80 is a more neutral differentiation signal. IL-12, released by the DC, appears as a potent and obligatory inducer of differentiation for IFN-gamma-producing cells. IL-6, although produced by both APC populations, is necessary to direct activation of the Th2-type response by macrophages but not by DC.     

Avian macrophages: contribution to cellular microenvironment and changes in effector functions following activation

The capacity of macrophages to generate metabolites and monokines having effector and regulatory functions can result in a major impact on their cellular microenvironment. Macrophage products synthesized in response to bacterial lipopolysaccharide stimulation include reactive oxygen and nitrogen intermediates as well as tumor necrosis factor (TNF). These secreted products of macrophages exhibit bioactivity either locally or systemically. Although the mechanism of action of avian monokines such as TNF-like factor may be similar to their mammalian counterparts, chicken TNF seems to lyse cells of chicken origin and not of mammalian origin. Furthermore, the generation and activity of products such as TNF is directly influenced by environmental stressors such as heat and toxins.     

Efficiency of cationic lipid-mediated transfection of polarized and differentiated airway epithelial cells in vitro and in vivo

Systematic analysis of a large number of different cationic lipids has led to the identification of novel structures (GL-67) and formulations of cationic lipid:plasmid DNA (pDNA) complexes that facilitate high levels of gene expression in lungs of mice. However, despite significant improvement in gene transfer activity, we show here that the efficiency of GL-67-mediated gene transduction of intact airway epithelia is still relatively low. Administration of GL-67:pCF1-CFTR (encoding the cystic fibrosis transmembrane conductance regulator) complexes into the nasal epithelium of cystic fibrosis (CF) transgenic mice resulted only in marginal correction of the ion transport defects. Measurements of nasal potential differences (PD) showed no correction of the sodium (Na+) transport defect, and only partial restitution of the chloride (Cl-) transport defect was achieved in a small proportion of the animals after perfusion of the nasal epithelium with the complexes. Furthermore, in contrast to results obtained following instillation of GL-67:pDNA complexes into the lungs of mice, perfusion of GL-67:pDNA into the nasal epithelium resulted only in a moderate enhancement of gene transduction activity relative to that attained with naked pDNA alone. To determine the basis for this low efficiency of transfection, a series of studies was conducted to identify some of the barriers governing cationic lipid-mediated gene transfer to the airway epithelium. We show here that the transfection activity of GL-67 was affected by the polarization, differentiation, and proliferative state of the cells. Diminished transfection activity was observed with nonmitotic, highly polarized and differentiated airway epithelial cells. This observed reduction in gene expression with nonmitotic cells was determined to be due in part to inefficient nuclear translocation of the pDNA from the cytoplasm. Together these data indicate that much improvement in the ability of cationic lipids to transfect polarized and differentiated airway epithelial cells is a necessary prerequisite for effective cationic lipid-mediated gene therapy of airway diseases such as CF.     

Nitric oxide and immune complexes are involved in the induction of a novel luminol chemiluminescence in cytotoxic macrophages

A chemiluminescence (CL) was observed immediately after the addition of luminol to thioglycollate-elicited ICR mouse peritoneal macrophages (M phi) that had been incubated overnight with recombinant murine interferon-gamma (IFN-gamma) and lipopolysaccharides (LPS). The intensity of this CL was closely correlated with the cytotoxic activity of M phi. NG-monomethyl-L-arginine (L-NMMA), an inhibitor of nitric oxide (NO) synthase, inhibited the induction of this CL, and L-arginine restored the L-NMMA-induced inhibition. These results suggest that NO is directly involved in the induction of CL. However, we found that immune complexes are required for the induction of CL as well as NO.     

L-arginine uptake and metabolism by lung macrophages and neutrophils following intratracheal instillation of silica in vivo

Cold air inhalation and exercise-induced bronchoconstriction (EIB) have both been used as measures of bronchial responsiveness. Both stimuli are often combined in the Nordic climate. The main objective of the present study was to investigate the climatic influence of cold temperatures upon exercise-induced asthma. The secondary aims were: (a) to assess metacholine bronchial hyper-responsiveness and EIB in children with bronchial asthma (n = 32; mean age 10.8 years) compared to children with other chronic lung diseases (CLD) (n = 26, mean age 10.1 years); and (b) to assess the influence of cold air inhalation upon EIB in the two groups of children. Methods used were: (a) the metacholine concentration causing a reduction in FEV1 of 20% (PC20-M), (b) maximum FEV1 fall (delta FEV1) after submaximal treadmill run (EIB test); and (c) delta FEV1 after submaximal treadmill run while inhaling cold (-20 degrees C) dry air (CA-EIB test). Geometric mean PC20-M did not differ significantly between the asthma children (1.28 mg ml-1) and the CLD children (2.90 mg ml-1). In the asthma children, mean delta FEV1 after EIB test was 12.8% vs 21.8% after adding cold air (P < 0.0001), compared to 5.2 and 7.4%, respectively (P = 0.03), in the CLD group. Maximum sensitivity and specificity for the EIB test were 69.8% at a fall in FEV1 of 6.8%; for the CA-EIB test, 72% at a fall in FEV1 of 10.2%; and for metacholine provocation, 56% at a PC20-M of 1.5 mg ml-1. In conclusion, children with bronchial asthma are substantially more sensitive to cold air than children with CLD, and EIB is markedly increased by cold air inhalation in asthmatic children, maintaining the specificity of the EIB test and increasing the sensitivity. The low sensitivity of the EIB test is probably influenced by the use of inhaled steroids. Metacholine inhalation test has less specificity and sensitivity in discriminating asthma from other chronic lung diseases.     

Successful in vivo and ex vivo transfection of pulmonary artery segments in lung isografts

OBJECTIVE: Gene transfer to lung grafts may be useful in ameliorating ischemia-reperfusion injury and rejection. Efficient gene transfection to the whole organ may prove problematic. Proximal pulmonary artery endothelial transfection might provide beneficial downstream effects on the whole graft. The aim of this study was to determine the feasibility of transfecting proximal pulmonary artery segments in lung isografts. METHODS: Male Fischer rats were divided into six groups. In vivo transfection: In group I (n = 7), a proximal segment of the left pulmonary artery was isolated and injected with saline solution by means of a catheter inserted through the right ventricle. After an exposure period of 20 minutes, clamps were removed and blood flow was restored. In group II (n = 7), the isolated arterial segments were injected with adenovirus carrying the Escherichia coli LacZ gene encoding for beta-galactosidase. Ex vivo transfection: In group III (n = 5), arterial segments were injected ex vivo with saline solution and in group IV (n = 5) with the adenovirus construct. In group V (n = 6), arteries were injected with saline solution and in group VI (n = 11) with liposome chloramphenicol acetyl transferase cDNA. In groups I to IV, animals were killed on postoperative day 3 and transgene expression was assessed by Bluo-Gal staining. In groups V and VI, animals were killed on postoperative day 2 and transgene expression was assessed by chloramphenicol acetyl transferase activity assay. RESULTS: Transgene expression was detected grossly and microscopically in endothelial and smooth muscle cells of pulmonary artery segments from all surviving animals of groups II and IV. In group VI, chloramphenicol acetyl transferase activity was significant in all assessed arterial segments. CONCLUSION: Significant transgene expression is observed in proximal pulmonary artery segments after both in vivo and ex vivo exposure.     

Mast cell activation and migration to lymph nodes during induction of an immune response in mice

The mast cell response in skin and lymph nodes was examined during the sensitization phase of dinitrofluorobenzene (DNFB)-induced contact hypersensitivity in mice. Degranulation of 62% of mast cells in DNFB-exposed skin was evident within 30 min of a dual application of DNFB, reaching a peak of 77% at 24 h, and persisting in 42% after 5 d. Abundant expression of macrophage inflammatory protein (MIP)-1alpha and MIP-1beta mRNAs and proteins was observed in keratinocytes, and mast cell degranulation was significantly inhibited after administration of neutralizing antibodies to MIP-1alpha, but not MIP-1beta. During DNFB sensitization, the mast cell density in the skin decreased by half, concurrent with a fivefold expansion of mast cell numbers in draining lymph nodes. Fluorescent-labeled mast cells injected into the skin appeared in draining lymph nodes after application of DNFB, followed by subsequent migration to the spleen. In lymph nodes, mast cells were an abundant and predominant source of MIP-1beta, neutralization of which partially inhibited T lymphocyte recruitment. These results indicate that mast cells contribute to the induction of this primary immune response by activation at and migration from the site of antigen encounter to draining lymph nodes, wherein they mediate T lymphocyte recruitment by production of MIP-1beta.     

Early reduction of immune activation in lymphoid tissue following highly active HIV therapy

OBJECTIVE: To evaluate immune reconstitution within HIV-infected lymphoid tissue during highly active antiretroviral therapy (HAART). DESIGN AND METHODS: In situ cellular responses were studied in sequential tonsillar biopsies in three asymptomatic HIV-infected (CD4 cells greater than 400 x 10(6)/l) antiretroviral treatment-naive volunteers enrolled in a clinical trial to determine the early effect of HAART. Computerized image analysis was used to study immunohistochemically stained sequential tonsil sections for the patterns of local cytokine production, chemokine receptor expression and cellular distribution. Replicate quantitative assessments of samples before and after 4 weeks of therapy were used for the evaluation of drug effects and compared with four uninfected controls. Tonsillar HIV proviral-DNA was determined by fluorescent in situ 5'-nuclease assay. RESULTS: HIV-infected tonsil tissue was characterized by extensive pro-inflammatory and type 1 cytokine expression. A five- to 15-fold elevation of interleukin (IL)-1 alpha, IL-12, IL-2 and interferon (IFN)-gamma protein expression was found compared with controls, and each encompassed a mean of at least 4.5% of the tissue compartment. This was reduced by 20-90% in all individuals after 4 weeks of HAART. In contrast, type 2 cytokine expression (IL-4, IL-10), plus tumour necrosis factor (TNF)-alpha, remained low throughout the study. HAART reduced, by 40%, the expression of HIV co-receptors, CCR5 and CXCR4, which initially were elevated four to six times over the control values. In addition, the myelomonocytic inflammatory proteins, CD68 and calprotectin, diminished by 26-83% after therapy. The HIV RNA was reduced to undetectable levels in plasma by HAART. However, a large pool of tonsil cells (2-7%), remained HIV DNA positive after 4 weeks of therapy. CONCLUSIONS: Although immune activation may be the direct consequence of HIV replication, HAART-associated reconstitution begins with a reduction in inflammatory cytokine production which precedes the elimination of local proviral reservoirs.     

Alternative splicing of the rabbit beta-globin pre-mRNA in vivo after transient transfection of myoblast and myotube cells

The relationship between preferences among alternative 5' splice sites and their sequences was investigated using as model mouse myoblasts and myotubes after transient transfection with the rabbit beta-globin gene. The preferences for the use of two different 5' splice sites, acting with different efficiencies to direct splicing in vivo are reported. The predominant selection of the upstream splice site has been shown in normal mouse myoblasts. In the case of differentiated myotubes the downstream splice was 1.4 times better used. The results indicate that there were differences in the preferences for the use of the two alternative splice sites between non-differentiated and terminally differentiated cells, within the same-cell line.     

Tissue factor expression in mesothelial cells: induction both in vivo and in vitro

Docking algorithms play an important role in the process of rational drug design and in understanding the mechanism of molecular recognition. An important determinant for successful docking is the extent to which the configurational space (including conformational changes) of the ligand/receptor system is searched. Here we describe a new, combinatorial method for flexible docking of peptides to proteins that allows full rotation around all single bonds of the peptide ligand and around those of a large set of receptor side chains. We have simulated the binding of several viral peptides to murine major histocompatibility complex class I H-2Kb. In addition, we have explored the limits of our method by simulating a complex between calmodulin and an 18-residue long helical peptide from calmodulin-dependent protein kinase IIalpha. The calculated peptide conformations generally matched well with the X-ray structures. Essential information about local flexibility and about residues that are responsible for strong binding was obtained. We have frequently observed considerable side-chain flexibility during the simulations, showing the need for a flexible treatment of the receptor. Our method may also be useful whenever the receptor side-chain conformation is not available or uncertain, as illustrated by the docking of an H-2Kb binding nonapeptide to the receptor structure taken from an octapeptide/H-2Kb complex.     

Role of antigen-presenting cells in activation of human T cells by the streptococcal M protein superantigen: requirement for secreted and membrane-associated costimulatory factors

The requirements for T-cell activation by the streptococcal superantigen (SAg), pepsin-extracted M protein from type 5 streptococci (pep M5), were studied by monitoring Ca2+ influx and cell proliferation. Cells from a pep M5-specific T-cell line showed no change in intracellular Ca2+ levels in response to pep M5 when added alone or with freshly isolated autologous antigen-presenting cells (APC). However, after being incubated with pep M5 overnight, the APC secreted soluble factors that together with pep M5 induced a marked increase in intracellular Ca2+ levels in pep M5-specific T cells or freshly isolated, purified T cells. Removal of the SAg from the overnight APC-derived supernatants resulted in loss of the Ca(2+)-mobilizing activity, which was restored within seconds of addition of SAg, suggesting that both the SAg and the soluble factors synergize to induce the Ca2+ influx. Induction of cell proliferation required additional signals inasmuch as the activated APC-derived supernatant failed to synergize with pep M5 to induce the proliferation of purified T cells and required the presence of phorbol myristate acetate for this activity. Metabolically inactive, fixed APC were impaired in their ability to present pep M5 to T cells. Presentation of pep M5 by fixed APC was, however, restored when the APC-derived soluble costimulatory factors were added to the culture. Our data suggest that pep M5-induced activation of T cells is dependent on APC-derived soluble factors and an APC membrane-associated costimulatory molecule(s). These interactions may be important in regulating the in vivo responses to M proteins, could contribute to the severity or progression of infections with Streptococcus pyogenes, and may influence the susceptibility of individuals to its associated nonsuppurative autoimmune sequelae.     

Regulation of hepatitis B virus mRNA expression in a hepatitis B surface antigen transgenic mouse model by IFN-gamma-secreting T cells after DNA-based immunization

The immunotherapeutic effect of DNA-mediated immunization against chronic hepatitis B virus (HBV) infection has been evaluated in transgenic mice expressing the sequences that code for the envelope proteins of HBV in the liver. In this model of HBV chronic carriers, a single i.m. injection of plasmid DNA encoding HBV envelope proteins is sufficient to generate specific immune responses leading to the clearance of the transgene expression product and the control of HBV mRNA. The relative contributions of the T cell subpopulations induced by DNA immunization were examined using adoptive transfer experiments. It was shown that either CD8+ or CD4+ T lymphocytes from immunocompetent DNA-immunized animals were sufficient to control viral gene expression in the livers of the recipient transgenic mice. This effect was mediated by a cytokine-dependent mechanism common to both T cell subpopulations; this mechanism did not require cell lysis, but did involve the production of IFN-gamma by the activated T cells.     

Cytolytic activity of pulmonary and systemic lymphoid cells from C57BL/6 mice following intrapulmonary or intraperitoneal immunization with allogeneic tumor cells

In order to demonstrate whether specific cytotoxic T cells could be induced in lung parenchyma, C57BL/6 mice were immunized by the intrapulmonary route with allogenetic tumor cells (P815). Ten days after administration of 20 x 10(6) allogeneic cells, peak concentrations of cytotoxic cells were found in lung, tracheobronchial lymph node, and spleen. With reduction in immunizing dose, lytic activity disappeared from spleen and lymph node, but persisted in lung. The cytolytic activity was specific for the immunizing alloantigen, was abolished by antitheta serum, and could not be attributed to macrophages. For comparison, C57BL/6 mice were immunized by the intraperitoneal route with 20 x 10(6) P815 cells. The expected cytolytic activity was found in spleen and lymph nodes: however, unexpectedly high levels of cytolytic activity were also found in pulmonary lymphocytes. This activity was confirmed using a wide range of effector to largest cell ratios in the assay system. Quantitative cytolytic assays demonstrated that the maximum rate of cytolysis by pulmonary lymphocytes obtained from mice immunized intraperitoneally exceeded by 10- to 20-fold the rate of cytolysis by pulmonary lymphocytes obtained from mice receiving intrapulmonary immunization. These data demonstrate that cytolytic T-lymphocytes appear in lung parenchyma after either intrapulmonary or intraperitoneal immunization and that the intraperitoneal route is far more efficient than the intrapulmonary route. This cell-mediated immune mechanism potentially is available for host defense of respiratory tissue.