Enhancement of in vivo cell-mediated immune responses by three distinct cytokines

The ability of recombinant/purified cytokines to augment delayed-type hypersensitivity (DTH) responses was investigated. Suboptimal doses of haptenized SC were treated in vitro with purified or recombinant derived cytokines and tested for their ability to enhance DTH in vivo. With the use of this protocol, it was shown that both human and mouse rIL-6, as well as mouse rTNF-alpha, potentiated DTH in a dose-dependent manner. In accordance with these data, IL-6/TNF-alpha-containing supernatant from long term nonlymphoid cell lines also possessed the ability to augment DTH. By using the same protocol, we have also identified T cell hybridomas that produce DTH-augmenting activity constitutively. The hybridoma-derived factor, termed the T cell enhancing factor (TCEF), was functionally distinguishable from the defined cytokines IL-1 through IL-6, IFN-gamma, and TNF by bioassay. Furthermore, RNA derived from the hybridoma failed to hybridize with cDNA probes specific for IL-1 to IL-6, IFN-gamma, TNF-alpha, and granulocyte-macrophage CSF. Further characterization of the serum-free conditioned media derived from the hybridoma indicated that the TCEF was a soluble acid labile glycoprotein (Mr greater than 30,000). Finally, we investigated the cellular requirements for DTH augmentation by IL-6, TNF-alpha, and TCEF; all are dependent upon the presence of T cells in the immunizing inoculum. We propose that these cytokines play a critical role in the development of DTH responses in vivo.     

Modulation by gamma interferon of antiviral cell-mediated immune responses in vivo.

Mice were infected with lymphocytic choriomeningitis virus and injected once 24 h later with a monoclonal antibody directed against gamma interferon. In comparison with controls, the increase of numbers of CD8+ T cells and the generation of virus-specific cytotoxic T lymphocytes in spleens and virus clearance from organs were diminished, as was the ability of spleen cells to transmit adoptive immunity to infected recipients. The same treatment slightly but consistently lessened rather than augmented the virus titers early in infection, which was also observed in thymusless nu/nu mice. Injection into infected mice of the lymphokine itself in quantities probably higher than are produced endogenously resulted in lower virus titers in spleens but higher titers in livers. The adoptive immunity in infected mice achieved by infusion of immune spleen cells was not altered by treating the recipients with gamma interferon monoclonal antibody. Such treatment did not measurably affect the production of antiviral serum antibodies. We conclude that in lymphocytic choriomeningitis virus-infected mice, gamma interferon is needed for the generation of antivirally active CD8+ T lymphocytes, and furthermore that in this experimental model, direct antiviral effects of the lymphokine elude detection.     

Spectrum of in vivo and in vitro cell-mediated immune responses in coccidioidomycosis.

The cell-mediated immune responses of 12 healthy, coccidioidin skin-test positive subjects (Group I) were compared with those of 15 healthy, coccidioidin skin-test positive persons who had primary asymptomatic coccidiodomycosis, (Group II), 12 patients with active, pulmonary coccidioidomycosis (Group III), four patients with disseminated disease (Group IV), and five patients who had been in clinical remission for 1 year or longer (Group V). Lymphocytes from healthy subjects in Groups I and II responded in vitro to Coccidioides immitis antigen by undergoing an increased DNA synthesis (lymphocyte transformation) and/or by producing macrophage migration inhibitory factor (MIF). In contrast, patients in Groups III and IV failed to respond to Coccidioides antigens in vivo (skin tests) or in vitro (lymphocyte transformation and production of MIF). The responses of subjects in Group V with inactive disease fell in between those of healthy donors in Groups I and II and patients in Groups III and IV. The cellular immune defect, in terms of antigen recognition, appeared to be specific for C. immitis in all but one patient.     

Direct in vitro evidence for different susceptibilities to 4-hydroperoxycyclophosphamide of antigen-primed T cells regulating humoral and cell-mediated immune responses to sheep erythrocytes: a possible explanation for the inverse action of cyclophosphamide on humoral and cell-mediated immune responses

Suppressor T cells of humoral immune responses, effector T cells mediating DTH, suppressor T cells of DTH, and helper T cells of humoral immune responses, all with specificity to SRBC, were produced in mice. The biologic activity was tested in adoptive transfer experiments. In vitro treatment with different doses of 4-hydroperoxycyclophosphamide (4-HPCy) yielded the result that the various activities tested were not uniformly sensitive to the action of this drug: Suppressor T cells of humoral immune responses and effector T cells mediating DTH were resistant to doses of 4-HPCy that eliminated the activities of suppressor T cells of DTH and helper cells of the humoral immune response. These findings help to explain the various effects cyclophosphamide has on the in vivo immune response and may help to form a basis for the rational manipulation of the immune response by drugs that selectively affect different subgroups of immune cells.     

Lymphocyte homing receptors and the immune response in vivo

An important aspect of the developmental maturation of lymphocytes is their capacity to locate and enter lymphoid organs with great rapidity and specificity and to follow certain routes within these organs for the attainment of particular immunological capabilities. It is now known that this “homing response” to lymphoid organs involves specific glycoprotein receptors on the lymphocyte cell surface. The biochemistry of these receptors and their significance in normal and pathological immune responses are discussed.     

Bromovirus movement protein genes play a crucial role in host specificity.

Monocot-adapted brome mosaic virus (BMV) and dicot-adapted cowpea chlorotic mottle virus (CCMV) are closely related bromoviruses with tripartite RNA genomes. Although RNAs 1 and 2 together are sufficient for RNA replication in protoplasts, systemic infection also requires RNA3, which encodes the coat protein and the nonstructural 3a movement protein. We have previously shown with bromoviral reassortants that host specificity determinants in both viruses are encoded by RNA3 as well as by RNA1 and/or RNA2. Here, to test their possible role in host specificity, the 3a movement protein genes were precisely exchanged between BMV and CCMV. The hybrid viruses, but not 3a deletion mutants, systemically infected Nicotiana benthamiana, a permissive host for both parental viruses. The hybrids thus retain basic competence for replication, packaging, cell-to-cell spread, and long-distance (vascular) spread. However, the hybrids failed to systemically infect either barley or cowpea, selective hosts for parental viruses. Thus, the 3a gene and/or its encoded 3a protein contributes to host specificity of both monocot- and dicot-adapted bromoviruses. Tests of inoculated cowpea leaves showed that the spread of the CCMV hybrid containing the BMV 3a gene was blocked at a very early stage of infection. Moreover, the BMV hybrid containing the CCMV 3a gene appeared to spread farther than wt BMV in inoculated cowpea leaves. Several pseudorevertants directing systemic infection in cowpea leaves were obtained from plants inoculated with the CCMV(BMV 3a) hybrid, suggesting that the number of mutations required to adapt the hybrid to dicots is small.     

Tocopherols play a crucial role in low-temperature adaptation and Phloem loading in Arabidopsis

To test whether tocopherols (vitamin E) are essential in the protection against oxidative stress in plants, a series of Arabidopsis thaliana vitamin E (vte) biosynthetic mutants that accumulate different types and levels of tocopherols and pathway intermediates were analyzed under abiotic stress. Surprisingly subtle differences were observed between the tocopherol-deficient vte2 mutant and the wild type during high-light, salinity, and drought stresses. However, vte2, and to a lesser extent vte1, exhibited dramatic phenotypes under low temperature (i.e., increased anthocyanin levels and reduced growth and seed production). That these changes were independent of light level and occurred in the absence of photoinhibition or lipid peroxidation suggests that the mechanisms involved are independent of tocopherol functions in photoprotection. Compared with the wild type, vte1 and vte2 had reduced rates of photoassimilate export as early as 6 h into low-temperature treatment, increased soluble sugar levels by 60 h, and increased starch and reduced photosynthetic electron transport rate by 14 d. The rapid reduction in photoassimilate export in vte2 coincides with callose deposition exclusively in phloem parenchyma transfer cell walls adjacent to the companion cell/sieve element complex. Together, these results indicate that tocopherols have a more limited role in photoprotection than previously assumed but play crucial roles in low-temperature adaptation and phloem loading.     

Defensins play a crucial role in protecting mice against oral Shigella flexneri infection

An earlier study revealed that 4-day-old mice, but not older mice, were infected with invasive Shigella strains. Here we attempted to determine the underlying mechanism that induces inflammation in the intestines of neonate mice after oral Shigella infection. Wild-type BALB/c mice of different ages (7, 14, and 35 days old) were orally administered GFP-expressing Shigella flexneri 5a M90T strain (5 × 10⁹ CFU) and analyzed for colonization 6 h following infection. We found that Shigella localized in the epithelium, lamina propria, and crypt regions of the small intestines of 7-day-old BALB/c mice. Microarray analysis revealed that expression levels of cryptdin and various types of cryptdin-related mRNA (e.g., cryptrs-2, -5, -7, -12 and lysozyme) in the small intestines were significantly lower in 7-day-old than in older mice regardless of Shigella infection status. Interestingly, matrix metalloprotease-7 (matrilysin)-deficient (MAT^−/−) mice of B6 background had more colonies and more severe symptoms of inflammation in the intestines than did wild-type B6 mice after oral Shigella challenge. This suggests that cryptdin-related antimicrobial molecules are indispensable for efficient protection against oral Shigella infection.     

HSCs play a distinct role in different phases of oval cell-mediated liver regeneration

Hepatic stem cell niche plays an important role in hepatic oval cell-mediated liver regeneration. As a component of hepatic stem cell niche, the role of hepatic stellate cells (HSCs) in oval cell proliferation needs further studies. In the present study, we isolated HSCs from rats at indicated time point after partial hepatectomy (PH) in 2-acetylaminofluorene/PH oval cell proliferation model. Conditional medium (CM) from HSCs were collected to detect their effects on proliferation and the mitogen-activated protein kinase pathway activation of two oval cell lines. We found that CM collected from HSCs at early phase of liver regeneration (4 and 9?days group) contained high levels of hepatocyte growth factor (HGF) and stimulated oval cell proliferation via extracellular signal-regulated kinase and p38 pathway. CM collected from HSCs at terminal phase of liver regeneration (12 and 15?days group) contained high levels of transforming growth factor (TGF)-β1, which suppressed DNA synthesis of oval cells. The shift between these two distinct effects depended on the balance between HGF and TGF-β1 secreted by HSCs. Our study demonstrated that HSCs acted as a positive regulator at the early phase and a negative regulator at the terminal phase of the oval cell-mediated liver regeneration. Copyright ? 2012 John Wiley & Sons, Ltd.     

The essential role of chemokines in the selective regulation of lymphocyte homing

Knowledge of lymphocyte migration has become a major issue in our understanding of acquired immunity. The selective migration of na?ve, effector, memory and regulatory T-cells is a multiple step process regulated by a specific arrangement of cytokines, chemokines and adhesion receptors that guide these cells to specific locations. Recent research has outlined two major pathways of lymphocyte trafficking under homeostatic and inflammatory conditions, one concerning tropism to cutaneous tissue and a second one related to mucosal-associated sites. In this article we will outline our present understanding of the role of cytokines and chemokines as regulators of lymphocyte migration through tissues.     

Cytokine polymorphisms play a role in susceptibility to ultraviolet B-induced modulation of immune responses after hepatitis B vaccination

UVB exposure can alter immune responses in experimental animals and humans. In an earlier human volunteer study, we demonstrated that hepatitis B-specific humoral and cellular immunity after vaccination on average were not significantly affected by UVB exposure. However, it is known that individuals differ in their susceptibility to UVB-induced immunomodulation, and it was hypothesized that polymorphisms in specific cytokines may play a role in this susceptibility. In this respect, we previously demonstrated that immune responses after hepatitis B vaccination are influenced by the minor allelic variant of IL-1 beta in the general population. For all volunteers, single nucleotide polymorphisms were determined for the following UV response-related cytokines: IL-1 receptor antagonist (+2018), IL-1 alpha (+4845), IL-1 beta (+3953), TNF-alpha (-308), and TNF-alpha (-238). Exposure to UVB significantly suppressed Ab responses to hepatitis B in individuals with the minor variant for the IL-1 beta polymorphism. Increased minimal erythema dose values (just perceptible), which resulted in higher absolute UVB exposures, were observed in the same individuals. There were no associations observed between UVB-induced immunomodulation and the other cytokine polymorphisms examined. This study indicates that individual susceptibility to UVB radiation needs to be considered when studying the effects of UVB in humans.     

Peripheral nerves do not play a trophic role in limb skeletal morphogenesis

Research was undertaken to test the hypothesis that thalidomide-induced limb defects resulted from damage to the neural crest or peripheral nerves and that normal limb development depends upon either the quality (level specific) or quantity of peripheral nerves. Barriers which were placed into early chick embryos to block brachial plexus-level neural crest cells from reaching the limb resulted in normal limb skeletons. These data agree with previous work in suggesting that skeletal morphology is independent of innervation.     

Myosin II and the Gal-GalNAc lectin play a crucial role in tissue invasion by Entamoeba histolytica

Entamoeba histolytica is the human parasite responsible of amoebiasis, during which highly motile trophozoites invade the intestinal epithelium leading to amoebic colitis, and disseminate via the blood circulation causing liver abscesses. The invasive process, central to the pathogenesis, is known to be driven by parasites motility. To investigate molecules responsible for in vivo motion, we performed a high resolution dynamic imaging analysis using two-photon laser scanning microscopy. Image analysis of the parasites during invasion of Caco-2 cell monolayers, an enterocyte-like model, and hamster liver shows that E. histolytica undergoes non-Brownian motion. However, studies of movements of parasite strains dominant negative for myosin II, a central component of the cytoskeleton, and for Gal-GalNAc lectin, a major adhesion molecule, indicate that myosin II is essential for E. histolytica intercellular motility through intestinal cell monolayers and for its motility in liver. In contrast, the Gal-GalNAc lectin exclusively triggers invasion of the liver. These observations are in agreement with emerging studies that highlight marked differences in the way that cells migrate in vitro in two dimensions versus in vivo in three dimensions. The approach that we have developed should be powerful to identify adhesive complexes required for in vivo cell migration in normal and pathogenic situations and may, thereby, lead to new therapeutic drug, for pathologies based on cell motility and adhesion.     

Integrin functions play a key role in the differentiation of thymocytes in vivo

T cells express a variety of surface proteins as they develop to maturity in the thymus. In addition to the TCR-CD3 complex and the two major coreceptors, CD4 and CD8, other surface proteins expressed include receptors for cytokines, growth factors, counterreceptors, and extracellular matrix molecules. To determine the role of integrin adhesion receptors in T cell development, we have expressed a trans-dominant inhibitor of integrin function in the thymus. This inhibitor leads to a block of adhesion to fibronectin due to reduced activation of integrin receptors. This reduced adhesion leads to a partial block in differentiation from CD4-CD8- cells to CD4+CD8+ cells, after the CD25+ stage, suggesting that integrins are important during Lck-mediated differentiation. Furthermore, the overall production of CD4+ cells is reduced compared with that of CD8+ cells without changes in negative selection, suggesting that integrins may be involved in the determination of the fate of the cell as well. These results demonstrate that integrin receptor function is required for proper thymocyte development in vivo.     

Regulatory role of miR-2909 in cell-mediated immune response

Keeping in view the micromanagement of immune response by micro RNAs, the present study was directed to explore the role of miR-2909 in the differentiation and maturation of T-lymphocytes within the population of normal human peripheral blood mononuclear cells maintained in in vitro culture. The results of such a study revealed that miR-2909 had the inherent capacity to significantly increase Treg (CD4+CD25+Foxp3+) cell population and dominant Th1-type cytokine (especially with decrease in IL-4 level and higher levels of INF-β and INF-γ) profile. Based upon these results, we propose that miR-2909 may modulate native immunity in general and help in providing protective immunity against viral infections in particular. Copyright ? 2012 John Wiley & Sons, Ltd.     

The ORF1 products of tombusviruses play a crucial role in lethal necrosis of virus-infected plants

Hybrids of cymbidium ringspot (CymRSV) and carnation Italian ringspot (CIRV) tombusviruses were used to identify viral symptom determinants responsible for the generalized necrosis in tombusvirus-infected plants. Surprisingly, symptoms of Nicotiana benthamiana infected with CymRSV/CIRV hybrids were distinctly different. It was demonstrated that not all chimeras expressing wild-type (wt) levels of p19 protein caused systemic necrosis as both parents CymRSV and CIRV did. We showed here that hybrids containing chimeric ORF1 were not able to induce lethal necrosis even if the viral replication of these constructs was not altered significantly. However, if a wt p33 (product of ORF1) of CymRSV was provided in trans in transgenic plants expressing p33 and its readthrough product p92, the lethal necrosis characteristic to tombusvirus infection was restored. In addition, the expression of p33 by a potato virus X viral vector in N. benthamiana caused severe chlorosis and occasionally necrosis, indicating the importance of p33 in wt symptoms of tombusviruses. Thus, our results provide evidence that elicitation of the necrotic phenotype requires the presence of the wt p33 in addition to the p19 protein of tombusviruses.     

Dendritic cells amplify T cell-mediated immune responses in the central nervous system

Neuroinflammation often starts with the invasion of T lymphocytes into the CNS leading to recruitment of macrophages and amplification of inflammation. In this study, we show that dendritic cells (DCs) facilitate T-T cell help in the CNS and contribute to the amplification of local neuroinflammation. We adoptively transferred defined amounts of naive TCR-transgenic (TCR) recombination-activating gene-1-deficient T cells into another TCR-transgenic mouse strain expressing different Ag specificity. Following adoptive transfers, we coinjected DCs that presented one or multiple Ags into the brain and followed the activation of T cells with defined specificities simultaneously. Injection of DCs presenting both Ags simultaneously led to significantly higher infiltration of T cells into the brain compared with injection of a mixture of DCs pulsed with two Ags separately. DCs mediated either cooperative or competitive interactions between T cell populations with different specificities depending upon their MHC-restricting element usage. These results suggest that DC-mediated cooperation between brain-infiltrating T cells of different Ag specificities in the CNS plays an important role in regulation of neuroinflammation. This work also implies that blocking Ag-specific responses may block not only the targeted specificities, but may also effectively block their cooperative assistance to other T cells. Therefore, these data justify more attention to Ag-specific therapeutic approaches for neuroinflammation.     

Plasmacytoid dendritic cells play a major role in apoptotic leukocyte-induced immune modulation

Several APCs participate in apoptotic cell-induced immune modulation. Whether plasmacytoid dendritic cells (PDCs) are involved in this process has not yet been characterized. Using a mouse model of allogeneic bone marrow engraftment, we demonstrated that donor bone marrow PDCs are required for both donor apoptotic cell-induced engraftment and regulatory T cell (Treg) increase. We confirmed in naive mice receiving i.v. syngeneic apoptotic cell infusion that PDCs from the spleen induce ex vivo Treg commitment. We showed that PDCs did not interact directly with apoptotic cells. In contrast, in vivo macrophage depletion experiments using clodronate-loaded liposome infusion and coculture experiments with supernatant from macrophages incubated with apoptotic cells showed that PDCs required macrophage-derived soluble factors--including TGF-β--to exert their immunomodulatory functions. Overall, PDCs may be considered as the major APC involved in Treg stimulation/generation in the setting of an immunosuppressive environment obtained by apoptotic cell infusion. These findings show that like other APCs, PDC functions are influenced, at least indirectly, by exposure to blood-borne apoptotic cells. This might correspond with an additional mechanism preventing unwanted immune responses against self-antigens clustered at the cell surface of apoptotic cells occurring during normal cell turnover.     

Social cues can play a role in timing onset of the breeding season of the ewe

Female Suffolk sheep were pinealectomized around the vernal equinox to eliminate the major environmental input to the reproductive system (photoperiod) and then either isolated from, or maintained with, pineal-intact gonad-intact sheep. The ewes were ovariectomized and treated with constant-release oestradiol implants and reproductive state was monitored by measuring serum LH concentrations. Pinealectomized ewes that were isolated from the normal flock showed a 2 1/2-month delay in onset of the seasonal rise in LH values compared with that of pineal-intact controls (18 November vs 5 September). On the other hand, pinealectomized ewes that were maintained with the flock showed an onset of the seasonal rise in LH that was not delayed. These results suggest a timekeeping role for social cues for timing onset of the breeding season in an animal that normally relies on photoperiodic signals for temporal regulation of the seasonal reproductive cycle.