Macrophage activation in vivo and in vitro

Morphology, lysosomal enzyme activity and phagocytic ability were tested in peritoneal macrophage cultures after stimulation in vivo or in vitro with endotoxin, mineral oil or latex particles, and compared to the same parameters in normal peritoneal macrophages. Treatment with latex did not give changes in the parameters tested after in vivo or in vitro stimulation. In all other types of stimulation the cells displayed varying degrees of spreading and changes in granule content. Extensive ruffling of cell membrane was obvious in endotoxin-stimulated cells. The pattern of lysosomal enzyme activity was complex and depended on the means of stimulation. Acid phosphatase showed the greatest increase after both in vivo and in vitro stimulation, N-acetyl-glucosaminidase could not be increased in vitro. Internalization of opsonized red cells mediated by the Fc receptor increased after in vivo stimulation. No such change was observed after in vitro stimulation. Normal peritoneal macrophages do not internalize significantly via the C₃ receptor. In vivo stimulation triggered the capacity to internalize up to 45% of the attached red cells. A similar reaction was obtained in vitro when both endotoxin and FCS were added to the culture medium, but not when endotoxin or FCS were used alone. We conclude that the use of the term activation of macrophages should always be based on quantitative changes in well defined parameters. Changes in one parameter will not necessarily be accompanied by the whole range of biochemical and morphological perturbations. The capacity to ingest via the C₃ receptor may be the most useful parameter.     

Transgenic mice with intracellular immunity to influenza virus

We have generated transgenic mice that express the intracellular anti-influenza virus protein Mx1 under control of an interferon-responsive regulatory element. Upon infection with influenza virus, mice of a high responder line produce Mx1 protein locally at the sites of initial viral replication, exhibit little viral spread, and survive infection. Mice of a low responder line show more extensive viral spread and survive infection only when virus is given at high doses. To survive low dose infections, these mice require injection of interferon along with virus. The results show that influenza viral pathogenesis is determined by a subtle balance between the dose of the infecting virus and the levels of the antiviral host factor Mx1 and that mice can be rendered resistant to a virulent infection by "intracellular immunization" achieved through germline transformation.     

Respiratory epithelial cells in innate immunity to influenza virus infection

Infection by influenza virus leads to respiratory failure characterized by acute lung injury associated with alveolar edema, necrotizing bronchiolitis, and excessive bleeding. Severe reactions to infection that lead to hospitalizations and/or death are frequently attributed to an exuberant host response, with excessive inflammation and damage to the epithelial cells that mediate respiratory gas exchange. The respiratory mucosa serves as a physical and chemical barrier to infection, producing mucus and surfactants, anti-viral mediators, and inflammatory cytokines. The airway epithelial cell layer also serves as the first and overwhelmingly primary target for virus infection and growth. This review details immune events during influenza infection from the viewpoint of the epithelial cells, secretory host defense mechanisms, cell death, and recovery.     

Beta-defensin antibiotic peptides in the innate immunity of the buffalo: in vivo and in vitro studies

Beta-defensin antimicrobial peptides are multifunctional biomolecules, which are a major component of the oxygen-independent microbicidal system of buffalo polymorphonuclear (PMN) cells. They have great potential for use as proteomic biomarkers of host cell responses in the presence of microbial agents. On purifying these peptides by RP-HPLC, four defensin peptides were revealed. The results from testing against Escherichia coli, Staphylococcus aureus, Streptococcus pyogenes, Candida albicans, Rinderpest Virus (RPV) and Newcastle Disease Virus (NDV), showed that the peptides possessed antimicrobial and antiviral activities. Minimum inhibitory concentration (MIC) values varied according to the peptide amounts and the exposure time. Furthermore, an increase in the levels of these cationic antimicrobial peptides was apparent in milk obtained from natural cases of mastitis, as compared to the levels in normal milk. MALDI-TOF-based amino acid sequencing confirmed the expression of two beta-defensins (LAP and BNBD-2) in mastitis milk. A comparison of peptide sequences revealed that buffalo LAP and BNBD-2 share 98.6% and 100% sequence identity, respectively, with those of cattle. In vitro, Bovine Viral Diarrhoea Virus (BVDV) infection was shown to induce the expression of the beta-defensin gene, as evidenced by the PCR amplification of cDNA with specific primers. The determination of the enhanced expression of beta-defensin peptides in mastitis milk and in PMN cells, can be considered as an advanced approach to the assessment of cellular and molecular responses to cell injury. It is hoped that in vitro studies on phagocytes such as PMN cells and other cell lines, will eventually replace the use of animals in elucidating the roles of these cytokines in response to microbe-derived cell damage. It will also be possible to use defensins as biomarkers to correlate failure in host cell defence systems with peptide heterogeneity.     

Macrophage tropism determinants of human immunodeficiency virus type 1 in vivo.

Strains of human immunodeficiency virus type 1 differ in their abilities to infect and replicate in primary human macrophages. Chimeric clones were constructed from a provirus unable to infect macrophages (NLHX) and envelope sequences (V3 loop) of viruses derived without cultivation from brain (YU2 and w1-1c1) or spleen (w2-1b4) tissues. The substituted V3 loop sequences in each case were sufficient to confer upon NLHX the ability to infect macrophages. Furthermore, an envelope domain immediately N terminal to the V3 loop also was found to modulate the level of replication in macrophages. These results demonstrate that an envelope determinant derived directly from patients with AIDS confers HIV-1 tropism for macrophages.     

Dietary phytoestrogens and cancer: in vitro and in vivo studies.

Thirty postmenopausal women (11 omnivores, 10 vegetarians and 9 apparently healthy women with surgically removed breast cancer) were investigated with regard to the association of their urinary excretion of estrogens, lignans and isoflavonoids (all diphenols) with plasma sex hormone binding globulin (SHBG). A statistically significant positive correlation between urinary total diphenol excretion and plasma SHBG was found which remained statistically significant after elimination of the confounding effect of body mass determined by body mass index (BMI). Furthermore we found a statistically significant negative correlation between plasma SHBG and urinary excretion of 16-hydroxyestrone and estriol which also remained significant after eliminating the effect of BMI. Furthermore we observed that enterolactone (Enl) stimulates the synthesis of SHBG by HepG2 liver cancer cells in culture acting synergistically with estradiol and at physiological concentrations. Enl was rapidly conjugated by the liver cells, mainly to its monosulfate. Several lignans and the isoflavonoids daidzein and equol were found to compete with estradiol for binding to the rat uterine type II estrogen binding site (the s.c. bioflavonoid receptor). It is suggested that lignans and isoflavonoids may affect uptake and metabolism of sex hormones by participating in the regulation of plasma SHBG levels and in this way influence their biological activity and that they may inhibit cancer cell growth like some flavonoids by competing with estradiol for the type II estrogen binding sites.     

Contributions of antinucleoprotein IgG to heterosubtypic immunity against influenza virus

Influenza A virus causes recurring seasonal epidemics and occasional influenza pandemics. Because of changes in envelope glycoprotein Ags, neutralizing Abs induced by inactivated vaccines provide limited cross-protection against new viral serotypes. However, prior influenza infection induces heterosubtypic immunity that accelerates viral clearance of a second strain, even if the external proteins are distinct. In mice, cross-protection can also be elicited by systemic immunization with the highly conserved internal nucleoprotein (NP). Both T lymphocytes and Ab contribute to such cross-protection. In this paper, we demonstrate that anti-NP IgG specifically promoted influenza virus clearance in mice by using a mechanism involving both FcRs and CD8(+) cells. Furthermore, anti-NP IgG rescued poor heterosubtypic immunity in B cell-deficient mice, correlating with enhanced NP-specific CD8 T cell responses. Thus, Ab against this conserved Ag has potent antiviral activity both in naive and in influenza-immune subjects. Such antiviral activity was not seen when mice were vaccinated with another internal influenza protein, nonstructural 1. The high conservation of NP Ag and the known longevity of Ab responses suggest that anti-NP IgG may provide a critically needed component of a universal influenza vaccine.     

Interaction of influenza virus NS1 protein and the human homologue of Staufen in vivo and in vitro.

A screening for human proteins capable of interacting with influenza virus NS1 has been carried out using the two-hybrid genetic trap in yeast. A cDNA corresponding to the human homologue of Drosophila melanogaster Staufen protein (hStaufen) was isolated that fulfilled all genetic controls of the two-hybrid protocol. Using a hStaufen cDNA isolated from a lambda human library, the interaction of hStaufen and NS1 proteins was characterised in vivo and in vitro. Co-transfection of NS1 cDNA and a partial cDNA of hStaufen led to the relocalisation of recombinant hStaufen protein from its normal accumulation site in the cytoplasm to the nuclear location of NS1 protein. NS1 and hStaufen proteins could be co-immunoprecipitated from extracts of co-transfected cells and from mixtures of extracts containing either protein, as well as from extracts of influenza virus-infected cells. Furthermore, both proteins co-localised in the ribosomal and polysomal fractions of influenza virus-infected cells. The interaction was also detected in pull-down experiments using a resin containing purified hStaufen and NS1 protein translated in vitro. Deletion mapping of the NS1 gene indicated that a mutant protein containing the N-terminal 81 amino acids is unable to interact with hStaufen, in spite of retaining full RNA-binding capacity. These results are discussed in relation to the possible mechanisms of action of hStaufen and its relevance for influenza virus infection.     

Analysis of the humoral immune response to influenza virus in vitro.

The induction of in vitro primary and secondary humoral immune responses to influenza virus in murine splenic explant culture is described. Anti-influenza antibody synthesized in vitro was detected and quantitated by a radioimmunoassay which utilized influenza coupled to bromoacetylcellulose. Both in vitro primary and secondary responses could be stimulated over a large range of virus doses. In vitro secondary responses were maximal when spleen donors had been immunized by the parenteral route although secondary type responses could be demonstrated as well after primary immunization by pulmonary infection. In vitro stimulation with influenza virus was relatively insensitive to inhibition at high antigen doses. The results are discussed in terms of the response of other antigens in spleen fragment culture.     

Cell replication and aging: in vitro and in vivo studies.

The relationship between human aging and cell replication has been investigated using two complementary approaches: in vitro studies of human fibroblasts derived from young and old volunteer members of the Baltimore Longitudinal Study and in vivo examinations of bone marrow cell populations from young and old mice and rats. Total proliferative capacity measured as either the onset of cell culture senescence or as in vitro life span was significantly diminished in cell cultures derived from old human donors when compared to parallel cultures established from young donors. Acute replicative abilities as measured by percent replicating cells, cell pupulation doubling time, cell number at confluency, and colony size distribution were also significantly decreased in human old cell populations. An in vivo cytogenetic technique for measuring cell replication was developed utilizing the differential staining properties of metaphase chromosomes of cells that have replicated in the presence of bromodeoxyuridine. With this technique, cell cycle times have been derived in vivo as well as in vitro. Preliminary in vivo results in both mice and rats indicate that cell replication is slowed in old animal cell populations. Further research will be directed both in vitro and in vivo at discerning the mechanisms for this impairment of cellular replication with aging.     

Induction of heterosubtypic immunity to influenza virus by intranasal immunization

Recovery from live influenza virus infection is known to induce heterosubtypic immunity. In contrast, immunity induced by inactivated vaccines is predominantly subtype specific. In this study, we investigated the heterosubtypic protective immunity induced by inactivated influenza virus. Intranasal immunization of mice with inactivated influenza virus A/PR8 (H1N1) provided complete protection against the homologous virus and a drift virus within the same subtype, A/WSN (H1N1), but not against the heterosubtypic virus A/Philippines (H3N2). However, coadministration of inactivated virus with cholera toxin as an adjuvant conferred complete heterosubtypic protection, without observed illness, even under conditions of CD4⁺ or CD8⁺ T-cell depletion. Analysis of immune correlates prior to challenge and postchallenge indicated that humoral immune responses with cross-neutralizing activity in lungs and in sera play a major role in conferring protective immunity against heterosubtypic challenge. This study has significant implications for developing broadly cross-reactive vaccines against newly emerging pathogenic influenza viruses.     

In vivo protection against syngeneic Gross virus-induced lymphoma in rats: comparison with in vitro studies of cell-mediated immunity.

Spleen cells at various times after inoculation of W/Fu rats with a syngeneic Gross virus-induced lymphoma, (C58NT)D, were tested for their in vivo activity in adoptive transfer experiments and for their in vitro reactivity in a 4-hr 51Cr release cytotoxicity assay and in a mixed lymphocyte-tumor cell interaction assay. In adoptive transfer, the best protection against tumor growth was observed with immune spleen cells taken at 30 days after tumor cell inoculation (the peak of reactivity in the mixed lymphocyte-tumor cell interaction assay) whereas cells taken at 10 days (the peak reactivity in the 51Cr release cytotoxicity assay) gave only partial protection. The protection detected in the adoptive transfer experiments was specific for (C58NT)D associated antigens, and this correlated well with the specificity observed in the in vitro cell-mediated immunity assays. T cells, but not complement receptor-bearing cells or macrophages, were essential for the protection against tumor growth in vivo, and also for the in vitro reactivity in the 51Cr release cytotoxicity and the mixed lymphocyte-tumor cell interaction assays.     

Enhanced production of rat interleukin-8 by in vitro and in vivo infections with influenza A NWS virus.

We investigated the interleukin-8 (IL-8)-producing activity of influenza A NWS virus in cultured rat kidney NRK-52E cells and a rat influenza model. The production of rat IL-8 increased significantly in the virus-infected cells but not in UV-inactivated virus- or split-product-treated cells. The increase in IL-8 production could be detected in the bronchoalveolar lavage of infected rats. These data suggest that infectious virus has the potential to accelerate the production of IL-8 in cultured cells and in vivo in airway-lining cells.