The Interferon-α/β Responses of Mice to Herpes Simplex Virus Studied at the Blood and Tissue Level In Vitro and In Vivo

Murine mononuclear leucocytes from bone marrow, spleen, lymph node and blood stimulated in vitro by UV-irradiated Herpes simplex type I virus (HSV) produced about equal proportions of IFN-α and -β determined by immunoassay. Thymocytes produced only IFN-α. The frequency of IFN-α/β mRNA containing cells detected by in situ hybridization was highest with bone marrow (15 per 10⁴ cells), followed by spleen (4/10⁴), lymph node (2/10⁴), blood (1/10⁴) and thymus (0.2/10⁴). Such IFN-α/β producing cells (IPCs) were heavily labelled in autoradiographs, each producing about 0.4 U of IFN. After one intravenous injection of UV-irradiated HSV in mice, high levels of IFN-α and -β were present in blood at 3–9 h and little or none at 24 h or later. Frequent cells strongly positive for IFN-α mRNA at in situ hybridization and for IFN-α/β at immunohistochemical staining were found almost exclusively in the marginal zones of spleens. Occasional IPCs were detected in lymph nodes but not in bone marrow, liver and kidneys. The marginal zone IPCs may be the major source of IFN in blood, and high splenic levels of IFN-α/β should have efficient antiviral and immunoregulatory functions.     

Production of Interferon-α/β by Murine Dendritic Cell Lines Stimulated by Virus and Bacteria

The interferon-α and -β (IFN-α/β) producing ability of the two murine dendritic cell (DC) lines D2SC/1 and FSDC was studied. The D2SC/1 cells produced IFN-α and -β when stimulated by herpes simplex virus (HSV), Sendai virus (SV) or by the bacteria Escherichia coli or Staphylococcus aureus Cowan I. Precultivating (priming) D2SC/1 cells with recombinant IFN-β or a combination of IFN-β and granulocyte–macrophage colony-stimulating factor increased production of IFN-α/β induced by HSV or the bacteria, but not by SV. Also, the kinetics of IFN-α/β responses were different for SV compared to HSV and the bacteria, suggesting different induction mechanisms. The FSDC cells differed from the D2SC/1 cells mainly in that predominantly IFN-β was produced, that little or no IFN-α/β production was induced by the bacteria, and that the IFN-α/β responses were most efficiently primed by IFN-γ. Priming the DC lines with tumour necrosis factor-α, interleukin-10 (IL-10) or IL-4 did not affect the IFN-α/β response induced by HSV. The results show that the two DC lines provide a convenient tool to study the induction and control of the IFN-α/β response, as well as the immunoregulatory role of IFN-α/β produced by DC.     

The Cell Surface Phenotype of Human Natural Interferon-α Producing Cells as Determined by Flow Cytometry

The relationship between the so-called natural interferon-α (IFN-α) producing cell (IPC), stimulated to produce IFN-α by herpes simplex virus type 1 (HSV), and of dendritic cells (DC) in peripheral blood leucocytes was investigated. The simultaneous expression of cell surface antigens and intracellular IFN-α in the HSV-stimulated IPC (HSV-IPC) was examined by flow cytometry (FCM). The HSV-IPC were infrequent, <0.3% of the mononuclear leucocytes, and with homogeneous light scatter characteristics. The HSV-IPC were confirmed to lack leucocyte lineage specific markers, and to express CD4, CD36 and HLA-DR. Furthermore, they expressed high levels of CD44, CD45RA and CD45RB, and lower levels of CD40, CD45R0, CD72 and CD83. The HSV-IPC expression of CD13, CD33 and FcεRI were weak but significant, while no CD5, CD11b, CD16, CD64, CD80 or CD86 were detected. Sorted pure HSV-IPC had irregular shaped nuclei, many mitochondria and vesicles, and rugged cell membranes without veils. Sorted HSV-IPC stimulated proliferation of autologous T cells from HSV immune donors. Thus, the HSV-IPC in many respects resemble immature DC, but clearly differ from typical mature DC. However, they may also represent a specialized population of efficient IFN-α producing cells.     

Interferon-γ Production by Human T Cells and Natural Killer Cells In Vitro in Response to Antigens from the Two Intracellular Pathogens Mycobacterium tuberculosis and Leishmania major

Acquired resistance to both mycobacteria and Leishmania is primarily mediated by interferon-γ (IFN-γ), which triggers mechanisms leading to the death of the microorganism in macrophages. In this study, cell activation and IFN-γ production was investigated in human peripheral blood mononuclear cells (PBMC) from individuals previously sensitized to tuberculin and without known exposure to Leishmania parasites. Immune staining for intracellular IFN-γ and surface markers allowed flow cytometric identification of the cellular sources of IFN-γ in cell cultures incubated with purified protein derivative of tuberculin (PPD) and Leishmania antigens. It was found that IFN-γ was produced in response to both PPD and Leishmania stimulant by T cells in the cultures. Activation of IFN-γ producing natural killer (NK) cells was demonstrated only in some cultures, and only with concomitant T cell activation.     

γδ and αβ T Cells are Equally Susceptible to Apoptosis

Gamma/delta TCR bearing T lymphocytes represent a T-cell subset whose functional relevance remains unclear. Nevertheless these T cells may play a role in the early immune reponse against bacteria. Until now the regulatory mechanisms on this response have not been investigated. The study described here evaluated the immunoregulatory effects of Interleukin-10 on γ/δ and α/β TCR-positive T-cell clones and freshly isolated peripheral-blood mononuclear cells (PBMC). IL-10 has been shown previously to inhibit lectin and antigen-induced proliferation and cytokine production by α/β T cells. The results outlined below show that rhIL-10 strongly inhibits lectin-induced production of IFN-γ, TNF-α. IL-2, and to a lesser degree proliferation and IL-4 production of both T-cell subsets. As IL-10 did not inhibit proliferation but at the same time strongly suppressed cytokine production in various experiments, the hypothesis that it could function as a growth factor for human T cells as has been described for murine thymoeytes was tested. The data demonstrate that, although the γ/δ T-cell clones tested do not produce IL-10 they can use it as a growth factor in combination with IL-2, IL-4 or alone. Furthermore, IL-10 has the same properties on human α/β T-cell clones and PBMC. In summary, it is shown that IL-10 has pleiotropic effects on γ/δ and α/β TCR⁺ T cells by inhibiting lectin-induced cytokine production and by acting as a growth factor for these cells alone or in combination with IL-2 or IL-4.     

Sendai Virus-Induced IFN-α Production Analysed by Immunocytochemistry and Computerized Image Analysis

IFN-α production in Sendai virus-stimulated human buffy coat cultures could readily be demonstrated in individual cells at a protein level by the use of a novel immuno-enzymatic staining procedure. A distinctive rounded, juxtanuclear staining pattern was generated in producer cells by the accumulation of the intracellularly synthesized IFN-α in the Golgi stacks. The technology is based upon acquiring a video image of stained monolayers of cells, viewed in a microscope by a colour camera, which then transfers binary images directly into a computer-controlled operating system. The characteristic appearance of the immunocytochemical staining enabled a computerized image-analysis system to measure IFN-α producing cells based on defined criteria set for morphology, intensity, colour and size. The automated system could accurately and reproducibly register a range of 0.1–7.0% of the total cell population as IFN-α producing cells during the kinetic studies of the response. Congruent results were obtained with manual microscopy and image analysis concerning the assessment of the incidence of IFN-α producing cells in the total cell populations. All IFN-α producing cells expressed surface HLA-DR molecules and 95% of these cells belonged to the myelomonocytic lineage. The image analysis system provided, in contrast to conventional microscopy, an opportunity to assess and document differences of signal intensity and cell size of individual IFN-α producing as well as non-producing cells.     

Interferon-γ Enhancement of E-Selectin Expression on Endothelial Cells is Inhibited by Monensin

The expression of E-selectin reaches a maximum 4–6 h after stimulation of human umbilical vein endothelial cells (HUVEC) in vitro with tumour necrosis factor-α (TNF-α) and then declines to basal level within 24 h. If interferon-γ (IFN-γ) is added to the cell culture medium together with TNF-α the surface expression of E-selectin is augmented and prolonged in a synergistic way. The aim of the present study was to investigate if altered protein glycosylation could explain the IFN-γ induced persistent surface expression of E-selectin. SDS–PAGE analysis of HUVEC glycoproteins, metabolically radiolabelled in the carbohydrate portion, indicated that addition of IFN-γ produced an altered protein glycosylation. Lectin blot analysis using the Sambucus nigra agglutinin lectin also indicated differences in protein glycosylation when HUVEC were incubated with IFN-γ/TNF-α compared to TNF-α alone. The kinetics of surface expression of E-selectin were measured using a cell ELISA assay. When HUVEC were incubated with monensin, a potent inhibitor of late Golgi function, together with both TNF-α and IFN-γ, the additive effect of IFN-γ on E-selectin expression was almost abolished. Since monensin is known to affect glycosylation processing, this experiment suggested that the IFN-γ induced change in protein glycosylation might induce the prolonged surface expression of E-selectin. However, when HUVEC were cultured with IFN-γ/TNF-α in the presence of several different inhibitors of N -glycosylation processing, no significant effect of E-selectin expression was observed. Regulation of adhesion molecule expression after activation of endothelial cells is likely to play a pivotal role for the inflammatory response. Further studies are needed to understand the mechanisms underlying this regulation.     

IFN-γ Inhibits Internalization of Soluble Aminated β-1, 3-D-Glucan by Macrophages and Thereby Down-Regulates the Glucan Induced Release of TNF-α: and IL-1β

We have previously shown that soluble aminated β-1, 3-D-glucan (AG) and glucan-derivatized micro-beads (GDM) bind to the specific β-glucan receptor on mouse peritoneal macrophages. Phagocytosis of GDM by macrophages is mediated through the β-glucan receptor. IFN-γ which increases macrophage phagocytic capacity, also increased the phagocytosis of GDM. In the present study we show that IFN-γ inhibits Internalization of AG in macrophages in a dose- and time-dependent manner. The inhibitory effect of IFN-γ was neutralized by treatment of the macrophages with cycloheximide. These results were confirmed by confocal laser scanning microscopy which showed that IFN-7 treated cells incorporated less fluorescein-labelled AG than did untreated cells. IFN-γ did not change the macrophage-binding capacity for AG showing that the inhibitory effect of IFN-γ is not caused by decreased number of β-glucan receptors on the cells. The stimulatory effect of AG on IL-β and TNF-α release from macrophages was reduced by pretreatment of the cells with IFN-γ.
We conclude that the uptake of AG and GDM in macrophages, both mediated through the β-glucan receptor, are differently regulated by IFN-γ. The reduced internalization of AG after IFN-γ treatment of macrophages, is probably responsible for the down-regulation of IL-1 and TNF-α secretion.     

Interferon-γ- and Tumour Necrosis Factor-α-Producing Cells in Humans who are Immune to Cutaneous Leishmaniasis

Individuals infected with Leishmania major usually acquire immunity to cutaneous leishmaniasis. In this study we have investigated peripheral blood mononuclear cells (PBMC) stimulated by Leishmania antigens in two groups of Sudanese individuals, one with a history of cutaneous leishmaniasis and one living in an area without the disease. The production of interferon (IFN)-gamma, tumour necrosis factor (TNF)-alpha and interleukin (IL)-10 was investigated in culture supernatants, and the cellular sources of IFN-gamma and TNF-alpha were identified. Cells from individuals with a history of cutaneous leishmaniasis produced significantly higher levels of IFN-gamma and TNF-alpha than cells from individuals without a history of the disease. Similar levels of IL-10 were found in the two groups. Flow cytometric analysis revealed high numbers of CD3+ cells producing IFN-gamma and TNF-alpha, and only a few CD3+ cells containing IL-10, in the PBMC cultures from the individuals with a history of cutaneous leishmaniasis. Interferon-gamma and TNF-alpha were predominantly produced by CD4+ T cells rather than CD8+ T cells. The results suggest that cellular immunity against cutaneous leishmaniasis is mediated predominantly through antigen-specific CD4+ T cells in individuals with a history of cutaneous leishmaniasis.     

Production of Interleukin-4, Interferon (IFN)-γ and IFN-α in Human Immunodeficiency Virus-1 Infection: An Imbalance of Type 1 and Type 2 Cytokines may Reduce the Synthesis of IFN-α

Interferon-α (IFN-α) is an important molecule in the antiviral response, but cells from HIV-1-infected individuals show a reduced ability to secrete IFN-α. We investigated an association between an imbalance of type 1/type 2 cytokines and the production of IFN-α in HIV-1 infection. We used whole blood culture to study the cytokine production profile, interferon-γ (IFN-γ) and interleukin-4 (IL-4), in response to HIV-1 antigens and to study the Sendai Virus and HSV-1-induced-production of IFN-α in seven HIV-1-infected patients. An impaired synthesis of IFN-α was obtained in patients with a predominant IL-4 production (IL-4 > IFN-γ), and we found a positive correlation between the ex vivo production of IFN-α and the IFN-γ/IL-4 ratio but not with the HIV RNA copy number in plasma. We investigated the role of T-cell-derived cytokines in the in vitro production of IFN-α by PBMC from eight healthy donors, activated with Sendai Virus or HSV-1. Whereas type 2 cytokines (IL-4, IL-13) inhibited virus-induced IFN-α synthesis, on the contrary, type 1 cytokines (IL-2, IFN-γ) enhanced it. A disarray in the T-cell-derived cytokine response may play a role in the defect of IFN-α production in HIV-1-infected individuals. Further investigations are needed to explore this hypothesis.     

Contribution of Intestinal Epithelial Cells to Innate Immunity of the Human Gut – Studies on Polarized Monolayers of Colon Carcinoma Cells

The aim was to establish an in vitro model for studies of innate defence mechanisms of human intestinal epithelium. Ultrastructural characterization and determination of mRNA expression levels for apical glycocalyx and mucous components showed that polarized, tight monolayers of the colon carcinoma cell lines T84 and Caco2 acquire the features of mature- and immature columnar epithelial cells, respectively. Polarized monolayers were challenged with non-pathogenic Gram+ and Gram− bacteria from the apical side and the proinflammatory cytokines interferon-γ (IFN-γ), tumour necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) from the basolateral side. Immune responses were estimated as changes in mRNA expression levels for the mucous component mucin-2 (MUC2), the glycocalyx components carcinoembryonic antigen (CEA), CEA-related cell adhesion molecule-1 (CEACAM1), CEACAM6, CEACAM7 and MUC3, the antimicrobial factors human β-defensin-1 (hBD1), hBD2, hBD3 and lysozyme, the chemokine IL-8 and the cytokines IL-6 and TNF-α. Tight monolayer cells were generally unresponsive to bacterial challenge, but increased their hBD2 levels when challenged with Bacillus megaterium. T84 cells also increased their TNF-α levels upon bacterial challenge. Tight monolayer cells responded to cytokine challenge suggesting awareness of basolateral attack. TNF-α induced significantly increased levels of IL-8 and TNF-α itself in both cell lines suggesting recruitment and activation of immune cells in the underlying mucosa in vivo . Cytokine challenge also increased levels of CEACAM1, which includes two functionally different forms, CEACAM1-L and CEACAM1-S. In T84 cells, IFN-γ was selective for CEACAM1-L while TNF-α upregulated both forms. Increased CEACAM1 expression may influence epithelial function and communication between epithelial cells and intraepithelial lymphocytes.     

Flow Cytometric Analysis of Natural Interferon-α Producing Cells

Herpes simplex virus-infected cells induce high interferon-alpha (IFN-alpha) production in infrequent cells among peripheral blood mononuclear cells (PBMC), designated natural IFN-alpha producing (NIP) cells. The properties of such NIP cells were compared with defined populations of leucocytes by means of flow cytometric analysis and sorting. The NIP cells are characterized as a discrete population of cells with high forward and low to intermediate orthogonal light scattering, similar to that of early progenitors of myeloid and lymphoid cells. However, they appear to lack the stem cell-associated molecule CD34. Furthermore, NIP cells cannot be localized to the myeloid line of cell differentiation, because they do not express the CD33, CD13, CD11b, CD15 or CD14 antigens. Neither do they express CD10 and CD19 antigens which are present in all stages of B-cell differentiation plasma cells excepted, nor CD7 antigens expressed on early T cells. In combination with previous results, our data support the view that the NIP cell is a unique and distinct cell type in peripheral blood, possibly with a physiological role in the defence against certain viral infections.     

Suppression of Experimental Autoimmune Myasthenia Gravis After CD8 Depletion is Associated with Decreased IFN-γ and IL-4

CDS⁺ T cells can perform both Th1 - and Th2-like functions by producing cytokines such as interferonγ (IFN-γ) and interleukin-4 (IL-4), as well as the immune response down-regulating transforming growth factor-β (TGF-β), which are all involved in the development of experimental autoimmune myasthenia gravis (EAMG), a model for human MG. We have reported that depletion of CD8⁺ T cells results in the suppression of EAMG accompanied by the down-regulation of AChR-specific B cell responses and AChR-reactive IFN-γ secreting Th1-like cells. To identify the involvement of IFN-γ, IL-4 and TGF-β in the development of EAMG after CD8⁺ T cell depletion, the expression of mRNA for these cytokines was studied in mononuclear cells from popliteal, inguinal and mesenteric lymph nodes, spleen and thymus by adopting in situ hybridization with complementary DNA oligonucleotide probes. Depletion of CD8⁺ T cells resulted in decreased levels of IFN-7 and IL-4 mRNA expressing cells in different lymphoid organs except thymus, but no change in the numbers of TGF-β mRNA expressing cells. The results imply that the suppression of EAMG after depletion of CD8⁺ T cells is caused by decreasing the effector factors but not by increasing the suppressor factor(s).     

T-Cell Immunity to Acetylcholine Receptor and its Subunits in Lewis Rats over the Course of Experimental Autoimmune Myasthenia Gravis

Lymph nodes, spleen and thymus obtained from Lewis rats were examined over the course of experimental autoimmune myasthenia gravis (EAMG) for the distribution and the number of antigen-reactive CD4⁺ T helper cells which, upon recognition of Torpedo acetylcholine receptor (AChR) or the α, β, γ or δ subunits of Torpedo AChR, responded by secretion of interferon-gamma (IFN-γ). T cells with these specificities were detected in these three immune organs. Numbers were highest in lymph nodes. In spleen and thymus, numbers of antigen-reactive T cells did not differ. T cells reacting against the intact AChR were more frequent than T cells recognizing any of the subunits. The immunogenicity between the four subunits did not differ, with the exception that the α subunit induced a slightly higher T-cell response. No restriction of the T-cell repertoire to the four subunits was detected during early compared to late phases of EAMG. The AChR and subunit-reactive T cells could—via secretion of effector molecules including IFN-γ—play an important role in the initiation and perpetuation of EAMG. and consequently also of human myasthenia gravis. T cells with the same specificities were also detected in control animals injected with adjuvant only, but at much lower numbers which were within the range of T cells recognizing the control antigen myelin basic protein. They could represent naturally occurring autoimmune T cells.     

Interleukin-4 and Interferon-Gamma Production by Leishmania Stimulated Peripheral Blood Mononuclear Cells from Nonexposed Individuals

Interferon-gamma (IFN-γ) and interleukin-4 (IL-4) production by Leishmania reactive peripheral blood mononuclear cells (PBMC) from non-exposed individuals was investigated. IFN-γ was measured in culture supernatants after antigen stimulation. For the measurement of IL-4, antigen stimulated cells were pulsed with PMA and ionomycin before IL-4 release was measured. L. donovani and L. major antigens induced IL-4 production (105–1748pg/ml) in 13 and seven cultures, and IFN-γ production (1.7- > 66IU/ml) in 14 and 11 of 20 cultures, respectively. IL-4 production rose steeply after 6 days of antigen stimulation suggesting a response due to antigen recognition. Both IL-4 and IFN-γ production was abrogated by depletion of CD2⁺ or CD4⁺ but not CD8⁺ cells. CD2⁺ or CD4⁺ but not CD8⁺ enriched cultures produced cytokines as unseparated PBMC. Thus, in non-exposed individuals circulating Leishmania reactive CD4⁺ T cells could be demonstrated. The cells from different individuals showed different patterns of IFN-γ and/or IL-4 production upon antigenic stimulation. In experimental leishmaniasis the early balance between IFN-γ and IL-4 is important for the clinical outcome. Our findings call for studies of the importance of cytokine production by cross-reactive T cells for the outcome of L. donovani infections in humans and show that the method for IL-4 detection is useful for this purpose.     

Naive Human T-Cells become Non-Responsive towards Anti-TNFα (Infliximab) Treatment In vitro if Co-Stimulated through CD28

Tumour necrosis factor α (TNFα) inhibitors are widely used successfully as immunomodulatory agents in various autoimmune diseases. They primarily target the direct pro-inflammatory effect of TNFα. They have also been found to be critical for T-cell viability and activation. In this study we evaluated the effect of infliximab treatment under different in vitro stimulatory conditions of naive human cord blood T-cells and adult peripheral blood mononuclear cells (PBMC). PBMC and negatively selected cord blood naive human T-cells were stimulated with αCD3 with or without αCD28 co-stimulation. The role of in vitro infliximab treatment was evaluated in relation to transforming growth factor-β1 (TGF-β1) under the above different stimulatory conditions. Anti-TNFα treatment with infliximab significantly suppressed proliferation of adult and cord blood T-cells ( P  < 0.013) during suboptimal stimulatory conditions. Infliximab prevented division of naive CD4⁺ and CD8⁺ T-cells and consequently also activation induced cell death, which was induced after three cell divisions. Interleukin (IL)-2 secretion was significantly decreased during infliximab treatment of suboptimally stimulated T-cells ( P  < 0.05) while TGF-β1 levels were unchanged. Strikingly, the anti-proliferative effect of infliximab was overcome by the administration of anti-TGF-β1 or by the addition of exogenous IL-2. Interestingly, CD28 mediated co-stimulation restored the proliferative response in a dose–responsive manner during infliximab treatment. Finally, exogenous TNFα administration during suboptimal stimulation reduced the inhibitory effect of TGF-β1 upon proliferation ( P  < 0.03). These results demonstrate that anti-TNFα treatment is primarily working upon T cells under low stimulatory conditions and probably through TGF-β1.