Cell Activation In Mice Research Articles

Arming antitumor T cells

T cells can kill tumors more effectively if a roadblock in their receptor signaling is removed, report Loeser et al. on page 879. Figure 1 Mice that lack cbl-b spontaneously reject UV-induced tumors. Most tumors are poor targets for the immune system as they frequently lack costimulatory molecules and present antigens that are only weakly immunogenic. The few tumor-specific T cells that do get generated are tolerized and fail to see the tumor as a dangerous enemy. Strategies to induce strong antitumor T cell responses—including cytokine treatment to coax tumors into expressing costimulatory proteins or vaccination with host dendritic cells loaded with tumor antigens—have not always worked. Loeser and colleagues decided instead to circumvent tolerogenic mechanisms within the antitumor T cells themselves. Their target was the ubiquitin ligase cbl-b, which inhibits TCR signals by targeting downstream signaling proteins for proteasomal degradation. The group had previously shown that T cells lacking cbl-b are efficiently activated by weak antigens even in the absence of costimulation, suggesting that cbl-b functioned as an internal tolerance enforcer. They now find that these T cells are efficient tumor killers as well. Mice lacking cbl-b were able to spontaneously reject tumors that were either injected into the animals or induced by UV exposure. Cbl-b–deficient CD8+ T cells destroyed tumors when transferred into wild-type animals. These cells developed into memory cells and offered long-lasting protection against tumor recurrence. One pitfall of removing a tolerogenic T cell protein such as cbl-b is the threat of autoimmunity. The cbl-b–deficient mice do show signs of autoimmune damage. However, these side effects are not lethal, and the mice have a normal lifespan. The team now plans to test whether antitumor T cell activity in mice can be mediated by RNAi knock-down of cbl-b, as this strategy may be practical in humans.

Thalidomide suppresses melanoma growth by activating natural killer cells in mice

Although thalidomide (Thd) is being extensively investigated for its effects on cytokine production and T cell costimulation, it is poorly understood whether it is capable of modulating the activities of natural killer (NK) cells. In this study, Thd effects on NK cell activity were examined with a murine model of melanoma, which is mostly rejected by NK cell-dependent mechanism. Administration of Thd significantly (p<0.01 on Day 21) suppressed the growth of subcutaneous B16F1 melanoma. In Thd-treated mice, marked splenomegaly and augmented splenocyte count were observed. Additionally, the percentage of splenic NK1.1+ cells was elevated to approximately 2.5-fold within 10 days after Thd treatment. The expression of interferon inducible protein (IP)-10, interferon (IFN)-gamma, interleukin (IL)-12 and IL-18 was remarkably upregulated. Production of the cytotoxic molecule perforin was also augmented. These data suggest that Thd strongly activates NK cell activity in mice, possibly resulting in enhanced tumor surveillance defense.

Inhibiting Hedgehog: New Insights into a Developmentally Important Signaling Pathway

What transforms a ball of undifferentiated cells into an organism with a nervous system, digestive tract, and other specialized body parts? Among the proteins that play an important role is one with the unlikely name of hedgehog (Hh). When Hh attaches to a transmembrane protein known as patched (Ptch1), it initiates a series of molecular interactions that lead to activation of the transcription factor Gli (for “glioma associated”) and the onset of key events in embryonic differentiation. We know this process involves freeing a second transmembrane protein, smoothened (Smo), from inhibition. But how do Hh and Ptch1 accomplish this? Scientists would like to know because the ability of this signaling pathway to function properly makes the difference between normal development and devastating abnormalities—and because the pathway is also implicated in tumor growth. Maarten F. Bijlsma, Maikel P. Peppelenbosch, and colleagues began their attempt to find out by noting that previous studies show that enzymes used to make cholesterol are involved in the pathway; that Ptch1 and Smo don’t necessarily bond to each other; that Ptch1 looks like other proteins that pump molecules from one side of the cell membrane to the other; and that cholesterol-like molecules can inhibit the pathway. Based on that information, the researchers hypothesized that when—and only when—Ptch1 is unencumbered by Hh, it pumps a cholesterol-like molecule into the extracellular space, where it inhibits Smo. To test this, the researchers developed an experimental system made up of fibroblasts modified to luminesce when Gli is active (called reporter cells), and to overexpress various combinations of Ptch1, Smo, and Gli. When they mixed reporter cells overexpressing Smo with cells overexpressing Ptch1, Gli activation in the reporter cells was reduced. When they mixed them instead with cells in which Ptch1-producing genes were silenced, Gli activation increased. After performing additional tests to eliminate alternative explanations, the team concluded that Ptch1 inhibits Smo through an intermediary, and that the intermediary molecule can exert its influence between individual cells. The researchers next exposed reporter cells to a medium that had contained Ptch1-overexpressing cells, and found Gli activation to be strongly inhibited. However, when they exposed reporter cells to a serum-free, Ptch-conditioned medium, they found no inhibition. Since serum-free medium doesn’t contain lipoproteins, they concluded that a lipoprotein is involved. Further tests suggested that the lipoprotein acts by helping transport a 3β-hydroxysteroid involved in the pathway inhibition. Interestingly, the researchers noted that certain people with Hh signaling problems have elevated levels of a particular hydroxysteroid, 7-dehydrocholesterol (7-DHC). This led them to test the link between 7-DHC and Gli activity in mouse cells, which in turn led to the conclusion that 7-DHC indeed participates in Ptch1’s inhibition of Smo. But is the Smo-inhibiting molecule actually 7-DHC or a compound derived from 7-DHC? When the researchers exposed medium from 7-DHC-producing mouse cells to ultraviolet radiation—which changes 7-DHC into vitamin D3—or used vitamin D3 in place of 7-DHC, Hh pathway inhibition was even stronger. They also showed that vitamin D3 binds to Smo, and that it inhibits the Hh pathway in live zebrafish embryos. Putting it all together, the researchers concluded that when Hh isn’t present, Ptch1 pumps (pro)-vitamin D3 (i.e., either 7-DHC or vitamin D3) into the extracellular space, where the hydroxysteroid grabs onto Smo, inhibiting Gli activation. When Hh binds to Ptch1, the pump grinds to a halt, Smo is freed of inhibition, and transcription of developmentally important genes kicks in. This new knowledge of the Hh signaling pathway shows how some cells can affect neighboring cells’ development, and it helps explain some of the problems associated with mutations affecting cholesterol biosynthesis. Because the Hh pathway is linked with certain cancers, it also has implications for tumor development. Additional work is now underway to see whether this new understanding of Ptch1’s intercellular inhibition of Smo can be applied to help suppress tumor growth.

Oral Administration of Lactoferrin Increases NK Cell Activity in Mice via Increased Production of IL-18 and Type I IFN in the Small Intestine

Evidence that lactoferrin (LF) influences various immune functions is now accumulating. Recent reports have shown that bovine LF (BoLF) enhances antimicrobial, antiviral, and antitumor immune activities when orally administered. Here, we report that orally administered BoLF increases natural killer (NK) cell populations in peripheral blood and spleen in a dose-dependent manner and enhances interferon-gamma (IFN-gamma) production by NK cells. Using intraperitoneal (i.p.) injection of poly(I:C) to induce NK cell trafficking into the peritoneum, oral BoLF increased NK cell migration. Oral BoLF also produced an immediate increase in the levels of interleukin-18 (IL-18) in the portal circulation. In IL-18 knockout (KO) mice, BoLF did not increase the numbers of NK cells, although NK cell cytotoxic activity and poly(I:C)-induced trafficking activity were both enhanced by oral BoLF, even in IL-18 KO mice. Furthermore, oral BoLF increased the expression of IFN-alpha and IFN-beta in Peyer's patches (PP) and mesenteric lymph nodes (MLN). Oral administration of 2- chloroadenosine selectively depleted the PP cells and blocked the ability of oral BoLF to increase NK cell accumulation in the peritoneum following poly(I:C) i.p. injection. Collectively, these results demonstrate that orally administered BoLF stimulates intestine-associated immune functions, including the production of IL- 18 and type I IFNs and increased NK cell activity.

CD28 in thymocyte development and peripheral T cell activation in mice exposed to suspended particulate matter

The CD28:B7 signaling pathway is very important for the activity of mature peripheral T lymphocytes and thymocyte development. The proper development of thymocytes into mature single positive CD4+and CD8+ T cells is crucial for almost all immune functions. In naturally occurring conditions, T cells maturation in the thymus is influenced by environmental agents. The expression of CD28 and the distribution of CD28(low/high) thymocytes have been examined at various stages of thymocyte development in BALB/c mice exposed to air-suspended particulate matter (ASM). Acute exposure to ASM resulted in the decrease of CD28 expression in the total thymocyte population. The increase of the percentage of CD28(low) and the decrease of CD28(high) thymocytes were observed, which may account for the acceleration of thymocyte development under the conditions of elevated risk resulting from the exposure of animals to environmental xenobiotics. ASM exposure resulted in the increase of the level of proliferation of lymph node T cells induced by anti-CD3 and anti-CD28 monoclonal antibodies activation despite normal expression of CD28 molecule. In contrast, the level of proliferation of spleen T cells was lowered or normal dependently of the concentration of stimuli used for activation. Results of these studies demonstrate that acute exposure of mice to ASM can result in the progression of two contrasting processes in the immune system: upregulation of thymocyte development, which contributes to the maintenance of peripheral T cell pool, and over-activation of lymph node lymphocytes, which may lead to uncontrolled immunostimulation.

Increased antigen presenting cell‐mediated T cell activation in mice and patients without the autoimmune regulator

Patients with autoimmune polyendocrine syndrome type I (APS I)suffer from endocrine and non-endocrine disorders due to mutations in the autoimmune regulator gene (AIRE). Mouse Aire is expressed both in thymic medullary epithelial cells and in peripheral antigen-presenting cells, suggesting a role in both central and peripheral tolerance. We here report that Aire(-/-) dendritic cells (DC) activate naive T cells more efficiently than do Aire(+/+) DC. Expression array analyses of Aire(-/-) DC revealed differential regulation of 68 transcripts, among which, the vascular cell adhesion molecule-1 (VCAM-1) transcript was up-regulated in Aire(-/-) DC. Concurrently, the expression of the VCAM-1 protein was up-regulated on both Aire(-/-) DC and monocytes from APS I patients. Blocking the interaction of VCAM-1 prevented enhanced Aire(-/-) DC stimulation of T cell hybridomas. We determined an increased number of DC in spleen and lymph nodes and of monocytes in the blood from Aire(-/-) mice, and an increased number of blood monocytes in APS I patients. Our findings imply a role for Aire in peripheral DC regulation of T cell activation, and suggest that Aire participates in peripheral tolerance.

Prostaglandin E2 Induces Exocytosis and Eicosanoid Generation by Human Mast Cells through an Il-4-Dependent Mechanism

RATIONALE: Prostaglandin (PG) E2 has both pro- and anti-inflammatory functions, and signals through at least four distinct receptor subtypes. Although PGE2 inhibits the activation of human mast cells (hMCs) through stimulation of adenylyl cyclase and cAMP, hMCs also express EP3 receptors which are reported to amplify mast cell activation in mice.

Chronic TNF-α exposure impairs TCR-signaling via TNF-RII but not TNF-RI

Chronic exposure to TNF-α has been shown to impair T cell-activation in mice and in humans. In the present study, we investigated a possible role of TNF-RII in this long-term effect of TNF-α. Chronic TNF-α exposure led to suppression of subsequent TCR stimulation (e.g., TCR/CD28-induced proliferation, cytokine production (IFN-γ, TNF-α)) but left TCR independent restimulation unaffected. Activation of T cells during TNF-α exposure was required for the inhibitory effect on TCR stimulation. In contrast to the mouse model, the inhibitory effect of long-term TNF-α exposure was mediated via TNF-RII but not TNF-receptor I, and surface expression of the TCR/CD3 complex remained unchanged. Chronic TNF-RII triggering downregulated T cell activation at an early level, as TCR-induced calcium flux and IL-2 mRNA expression were impaired after preculture in the presence of anti-TNF-RII mAbs. Furthermore, chronic TNF-RII-stimulation specifically downregulated store operated calcium channels, which contribute to sustained TCR-induced calcium influx.

Mycophenolate mofetil does not suppress the graft-versus-leukemia effect or the activity of lymphokine-activated killer (LAK) cells in a murine model.

Graft-versus-leukemia (GVL) effect is an essential component in the course of allogeneic stem cell transplantation (SCT). However, both prevention and treatment of established graft-versus-host disease (GVHD), including with drugs such as cyclosporine, can suppress GVL effects. Mycophenolate mofetil (MMF) is becoming a standard of care in SCT recipients for better prevention of GVHD as well as for promoting stem cell engraftment. To evaluate the effect of MMF, an immunosuppressive drug increasingly used for prevention of GVHD, on disease recurrence following SCT in a preclinical animal model. Since GVL effects may be also induced by alloreactive natural killer (NK) cells, the goal was to investigate the effects of MMF on the activity of lymphokine-activated killer (LAK) cells. MMF was administered by daily intraperitoneal injection starting at day 1 post-SCT. Cytotoxic LAK activity was measured by 5-h 35S-release assay, and GVL was tested by the appearance of BCL1 leukemia in a semi-mismatched (C57BL/6 donors to [BALB/c x C57BL/6] F1 recipients) murine model. A dosage regimen of 28-200 mg/kg per day MMF had no negative effect on either cytotoxic LAK activity or GVL (as measured by finding of leukemic cells in recipient spleen by PCR or the appearance of clinical leukemia with adoptive transfer). These results suggest that MMF does not impair GVL effects or reduce LAK cell activity in mice.

Combinational clustering of receptors following stimulation by bacterial products determines lipopolysaccharide responses.

The innate immune system has the capacity to recognize a wide range of pathogens based on conserved PAMPs (pathogen-associated molecular patterns). In the case of bacterial LPS (lipopolysaccharide) recognition, the best studied PAMP, it has been shown that the innate immune system employs at least three cell-surface receptors: CD14, TLR4 (Toll-like receptor 4) and MD-2 protein. CD14 binds LPS from Enterobacteriaceae and then transfers it to MD-2, leading to TLR4 aggregation and signal transduction. LPS analogues such as lipid IVa seem to act as LPS antagonists in human cells, but exhibit LPS mimetic activity in mouse cells. Although TLR4 has been shown to be involved in this species-specific discrimination, the mechanism by which this is achieved has not been elucidated. The questions that remain are how the innate immune system can discriminate between LPS from different bacteria as well as different LPS analogues, and whether or not the structure of LPS affects its interaction with the CD14-TLR4-MD-2 cluster. Is it possible that the 'shape' of LPS induces the formation of different receptor clusters, and thus a different immune response? In the present study, we demonstrate using biochemical as well as fluorescence-imaging techniques that different LPS analogues trigger the recruitment of different receptors within microdomains. The composition of each receptor cluster as well as the number of TLR4 molecules that are recruited within the cluster seem to determine whether an immune response will be induced or inhibited.

Chronic cyclosporine-induced hypertension is coupled to calcineurin-mediated inhibition of t cell activation in mice

P-298 Key Words: Cyclosporine, T Cell Activation, Radiotelemetry

A Bax/Bak-independent Mitochondrial Death Pathway Triggered by Drosophila Grim GH3 Domain in Mammalian Cells

Grim encodes a protein required for programmed cell death in Drosophila, whose proapoptotic activity is conserved in mammalian cells. Two proapoptotic domains are relevant for Grim killing function; the N-terminal region, which induces apoptosis by disrupting inhibitor of apoptosis protein (IAP) blockage of caspase activity, and the internal GH3 domain, which triggers a mitochondrial pathway. We explored the role of these two domains in heterologous killing of mammalian cells by Grim. The GH3 domain is essential for Grim proapoptotic activity in mouse cells, whereas the N-terminal domain is dispensable. The GH3 domain is required and sufficient for Grim targeting to mitochondria and for cytochrome c release in a caspase- and N-terminal-independent, IAP-insensitive manner. These Grim GH3 activities do not require Bax or Bak function, revealing GH3 activity as the first proapoptotic stimulus able to trigger the mitochondrial death pathway in mammalian cells in the absence of multidomain proapoptotic Bcl-2 proteins.

Effect of chinese softshell turtle egg powder on immune functions in mice

The effects of whole Chinese soft-shell turtle egg powder (WTE) on immune functions is unknown. Female ICR 6-week-old mice (n=80) were administered for 30 days with 1.125 g·kg−1 WTE. Spleen and thymus were weighed and spleen lymphocyte proliferation response to concanavalin A (Con A), plaque forming cell(PFC) assay, phagocytosis assay and NK cell activity detection were performed on day 30. Compared to vehicle control, WTE increased the relative weight of spleen (p<0.05). Mice treated with WTE showed increased Con A stimulated lymphocyte proliferation (p<0.05) and PFC (p<0.01). Carbon clearance capacity (p<0.05) and NK cell activity (p<0.01) were also enhanced while a mild improvement was seen in phagocytosis capacity of peritoneal cavity macrophages in WTE treated mice. In conclusion, our data provides evidence of the enhanced effects of WTE in cell-mediated immunity, humoral immunity and NK cell activity in mice.

The threshold pattern of calcineurin-dependent gene expression is altered by loss of the endogenous inhibitor calcipressin.

Calcineurin links calcium signaling to transcriptional responses in the immune, nervous and cardiovascular systems. To determine the function of the calcipressins, a family of putative calcineurin inhibitors, we assessed the calcineurin-dependent process of T cell activation in mice engineered to lack the gene encoding calcipressin 1 (Csp1). Csp1 regulated calcineurin in vivo, and genes triggered in an immune response had unique transactivation thresholds for T cell receptor stimulation. In the absence of Csp1, the apparent transactivation thresholds for all these genes were shifted because of enhanced calcineurin activity. This unbridled calcineurin activity drove Fas ligand expression, which normally requires high T cell receptor stimulation and results in the premature death of T helper type 1 cells. Thus, calcipressins modulate the pattern of calcineurin-dependent transcription, and may influence calcineurin activity beyond calcium to integrate a broad array of signals into the cellular response.

Regulation of plasma substance P and skin mast cells by odorants.

Mast cells stimulate inflammation and itch sensation in the skin by releasing various mediators when they are activated. Stress exacerbates some skin diseases. We have reported that inhalation of certain odorants modulates immune reactions in the skin. The possible usage of odorants in the regulation of skin inflammation and itch sensation was to be examined. Female volunteers were subjected to interview stress with or without odorant inhalation. Mice were immobilized while inhaling odorants. Toluidene blue-stained sections were analyzed for activated mast cells. Plasma substance P level was determined by enzyme-linked immunoassay. Interview stress induced plasma substance P only in volunteers who did not inhale odorants containing 2% 1,3-dimethoxy-5-methyl benzene (DMMB). Immobilization stress induced mast cell activation in mice and the activation was blocked by exposure to DMMB. Stress causes mast cell activation via an increase in substance P. The effect of stress is suppressed by inhalation of DMMB.

Stress-induced dura vascular permeability does not develop in mast cell-deficient and neurokinin-1 receptor knockout mice

Migraine headaches are often precipitated by stress and seem to involve neurogenic inflammation (NI) of the dura mater associated with the sensation of throbbing pain. Trigeminal nerve stimulation had been reported to activate rat dura mast cells and increase vascular permeability, effects inhibited by neonatal pretreatment with capsaicin implicating sensory neuropeptides, such as substance P (SP). The aim of the present study was to investigate NI, assessed by extravasation of 99-Technetium-gluceptate ( 99Tc-G), as well as the role of mast cells, SP and its receptor (NK-1R) in dura mater of mice in response to acute stress. Restraint stress for thirty min significantly increased 99Tc-G extravasation in the dura mater of C57BL mice. This effect was absent in W/W v mast cell-deficient mice and NK-1 receptor knockout mice (NK-1R−/−), but was unaltered in SP knockout mice (SP−/−). Acute restraint stress also resulted in increased dura mast cell activation in C57BL mice, but not in NK-1R−/− mice. These data demonstrate for the first time that acute stress triggers NI and mast cell activation in mouse dura mater through the activation of NK-1 receptors. The fact that SP−/− mice had intact vascular permeability response to stress indicates that some other NK-1 receptor agonist may substitute for SP. These results may help explain initial events in pathogenesis of stress-induced migraines.

Enhanced Recovery of NK Cell Activity in Mice under Restraint Stress by the Administration of a Biological Response Modifier Derived from the Mycelia of the Basidiomycete Tricholoma matsutake

Some types of stressor act on the immune system via the network comprising the endocrine-immune-nervous systems, and are reportedly responsible for the onset of diseases as well as giving impetus to their advance. It is important for the maintenance and promotion of health to cope with stress-induced changes in immunocompetence. Therefore, we studied the effects of administration of a novel biological response modifier (CM6271) derived from the mycelia of the basidiomycete Tricholoma matsutake on the NK cell activity in mice under restraint stress, in order to evaluate its potential to modulate immune responsiveness in stress-loaded individuals. (1) When C57BL/6 mice were restrained in 50-ml tubes for more than 6 h, splenic NK cell activity decreased significantly, but recovered gradually after the mice were released. The extent of the reduction of activity and the degree of recovery depended on the duration of the restraint. (2) The oral administration of CM6271 caused a significant acceleration of the recovery of the activity. This effect was dependent on the timing of administration and the dose given. (3) The administration of CM6271 had no clear effect on the blood levels of ACTH, corticosterone or lipid peroxide levels in the liver. These findings suggest that CM6271 promotes recovery from the decrease in NK cell activity induced by restraint stress.

NK cells mediate increase of phagocytic activity but not of proinflammatory cytokine (interleukin-6 [IL-6], tumor necrosis factor alpha, and IL-12) production elicited in splenic macrophages by tilorone treatment of mice during acute systemic candidiasis.

The participation of NK cells in the activation of splenic macrophages or in resistance to systemic candidiasis is still a matter of debate. We had previously reported that there is a correlation between natural killer cell activation and resistance to systemic candidiasis. In those experiments we had used tilorone to boost NK cell activity in mice. Here we show a mechanism elicited by tilorone in splenic macrophages which could explain their effect on mouse survival during acute disseminated Candida albicans infection. The results demonstrate that tilorone treatment elicits, by a direct effect, the production of proinflammatory cytokines (interleukin-6 [IL-6], tumor necrosis factor alpha [TNF-alpha], and IL-12) by splenic macrophages. In addition, it increases the capacity of splenic macrophages to phagocytize C. albicans through activation of NK cells. We also demonstrate that the presence of NK cells is essential for maintaining a basal level of phagocytic activity, which characterizes splenic macrophages of naïve control mice. The results demonstrate that it is possible to identify two phenotypically and functionally peculiar cell populations among splenic macrophages: (i). cells of the "stimulator/secretor phenotype," which show high levels of major histocompatibility complex (MHC) class II surface expression, are poorly phagocytic, and synthesize the proinflammatory cytokines IL-6, TNF-alpha, and IL-12, and (ii). cells of the "phagocytic phenotype," which express low levels of MHC class II molecules, are highly phagocytic, and do not secrete proinflammatory cytokines.

Natural killer cell activation in mice and men: different triggers for similar weapons?

The signaling pathways that regulate B and T lymphocytes are remarkably conserved between humans and mice. However, recent evidence suggests that the pathways regulating natural killer (NK) cell activation may actually differ between these two species. We discuss the controversies in the field and propose that this divergence could be deceptive: despite some clear differences between human and mouse NK cell receptors, the many ways of activating NK cells and their functions may well be conserved.

Inhibition of class II trans-activator function by HIV-1 tat in mouse cells is independent of competition for binding to cyclin T1

The Tat trans-activator protein from HIV-1 inhibits the function of the class II trans-activator protein (CIITA), resulting in reduced MHC class II gene transcription in human cells. Tat does so by competing with CIITA for binding to cyclin T1, a component of the transcriptional elongation complex PTEFb. Since Tat does not functionally interact with mouse cyclin T1, we decided to examine the ability of Tat to inhibit CIITA in mouse cells. We found that Tat inhibited CIITA activity in mouse cells though this inhibition was independent of cyclin T1. The inhibition required the transcriptional activation domain of CIITA, but did not involve alterations in MHC class II promoter occupancy. Although Tat blocked the interaction between CIITA protein and human cyclin T1, it had no effect on the binding between CIITA and mouse cyclin T1. Therefore, Tat can inhibit the ability of CIITA to activate transcription of MHC class II genes in mouse cells by a mechanism that appears to be distinct from that proposed for human cells.