Recognition of Listeria monocytogenes infection by natural killer cells: Towards a complete picture by experimental studies in rats.

The study of cellular immune responses in animal disease models demands detailed knowledge of development, function, and regulation of immune cells, including natural killer (NK) cells. Listeria monocytogenes (LM) bacterium has been explored in a large area of research fields, including the host pathogen interaction. Although the importance role of NK cells in controlling the first phase of LM burden has been investigated, the interaction between NK cells and infected cells in details are far from being comprehended. From in vivo and in vitro experiments, we can drive several important pieces of knowledge that hopefully contribute to illuminating the intercommunication between LM-infected cells and NK cells. Experimental studies performed in rats revealed that certain NK cell ligands are influenced in LM-infected cells. These ligands include both classical- and non-classical MHC class I molecules and C-type lectin related (Clr) molecules that are ligands for Ly49- and NKR-P1 receptors respectively. Interaction between these receptors:ligands during LM infection, demonstrated stimulation of rat NK cells. Hence, these studies provided additional knowledge to the mechanisms NK cells utilise to recognise and respond to LM infection outlined in the current review.

Listeria monocytogenes infection enhances the interaction between rat non-classical MHC-Ib molecule and Ly49 receptors.

Murine NK cell Ly49 receptors, functionally analogous to KIRs in humans recognize MHC class I molecules and play a key role in controlling NK cell function. We have previously shown that the paired activating Ly49s4 and inhibitory Ly49i4 receptors recognize undefined non-classical MHC-Ib ligands from the RT1-CE region in rats. Here, the RT1-CE16 gene of the RT1d haplotype was stably transfected into the mouse RAW macrophage cell line, termed RAW-CE16d cells. Combining RAW-CE16d cells with Ly49 expressing reporter cells demonstrated Ly49i4 and Ly49s4 specificity for CE16d. The Ly49s4/i4:CE16d interaction was confirmed by specific MHC-I blocking monoclonal Abs. Further, we used our in vitro model to study the effect of Listeria monocytogenes (LM) on CE16d after infection. LM infection and IFN-γ stimulation both led to enhanced CE16d expression on the surface of transfected RAW-CE16d cells. Interestingly, the reporter cells displayed increased response to LM-infected RAW-CE16d cells compared with IFN-γ-treated RAW-CE16d cells, suggesting a fundamental difference between these stimuli in supporting enhanced Ly49 recognition of CE16d. Collectively, our data show that Ly49s4 and Ly49i4 recognize the non-classical RT1-CE16d molecule, which in turn is up-regulated during LM infection and thereby may contribute to NK-mediated responses against infected cells.

Listeria monocytogenes infection differentially affects expression of ligands for NK cells and NK cell responses, depending on the cell type infected.

The pivotal role of NK cells in viral infection is extensively studied, whereas the role of NK cells in bacterial infection has been poorly investigated. Here, we have examined how Listeria monocytogenes (LM) affects expression of ligands for NK cell receptors and subsequent NK cell responses, depending on the type of cell infected. LM infected rat cell lines derived from different tissues were coincubated with splenic NK cells, and NK cell proliferation and IFN-γ production were measured. In addition, expression of ligands for the NK cell receptors Ly49 and NK cell receptor protein 1 (NKR-P1), MHC class I and C-type lectin-related molecules, respectively, was assessed. Infected pleural R2 cells, but not epithelium-derived colon carcinoma cell line CC531 cells, induced proliferation of NK cells. Reporter cells expressing the inhibitory NKR-P1G receptor or the activating NKR-P1F receptor were less stimulated under incubation with infected CC531 cells versus uninfected CC531 controls, suggesting that the ligand(s) in question were down-regulated by infection. Conversely, LM infection of R2 cells did not affect reporter cell stimulation compared with uninfected R2 controls. We characterized a rat monocyte cell line, termed RmW cells. In contrast to LM infected R2 cells that up-regulate MHC class I molecules, RmW cells displayed unchanged MHC class I expression following infection. In line with MHC class I expression, more NK cells produced a higher amount of IFN-γ against infected R2 cells compared with RmW cells. Together, L. monocytogenes infection may variously regulate cellular ligands for NK cells, depending on the cell type infected, affecting the outcome of NK cell responses.

Regulation of CCN1 (Cyr61) in a porcine model of intestinal ischemia/reperfusion.

Intestinal ischemia is a serious condition that may lead to both local and systemic inflammatory responses. Restoration of blood supply (reperfusion) to ischemic tissues often increases the extent of the tissue injury. Cysteine-rich angiogenic inducer 61 (Cyr61)/CCN1 is an extracellular matrix-associated signaling protein that has diverse functions. CCN1 is highly expressed at sites of inflammation and wound repair, and may modify cell responses. This study aimed to investigate regulation and cellular distribution of CCN1 in intestinal ischemia/reperfusion (I/R) injury in pigs. After intestinal I/R, increased expression of CCN1 was detected by quantitative RT-PCR, Western blot analysis and immunohistochemistry compared with non-ischemic intestine. Immunoflorescence staining revealed that CCN1 was mainly up-regulated in intestinal mucosa after intestinal I/R. Microvillus epithelial cells and vascular endothelial cells were strongly positive for CCN1 in intestinal I/R, while natural killer cells and/or subsets of neutrophils were only modestly positive for CCN1. Furthermore, blood samples taken from the portal and caval veins during ischemia and after reperfusion showed no change of the CCN1 levels, indicating that CCN1 was locally regulated. In conclusion, these observations show, for the first time, that the CCN1 molecule is up-regulated in response to intestinal I/R in a local manner.

Calcium intervention ameliorates experimental model of multiple sclerosis.

OBJECTIVE: Multiple sclerosis (MS) is the most common inflammatory disease of the CNS. Experimental autoimmune encephalomyelitis (EAE) is a widely used model for MS. In the present research, our aim was to test the therapeutic efficacy of Calcium (Ca) in an experimental model of MS. METHODS: In this study the experiment was done on C57BL/6 mice. EAE was induced using 200 µg of the MOG35-55 peptide emulsified in CFA and injected subcutaneously on day 0 over two flank areas. In addition, 250 ng of pertussis toxin was injected on days 0 and 2. In the treatment group, 30 mg/kg Ca was administered intraperitoneally four times at regular 48 hour intervals. The mice were sacrificed 21 days after EAE induction and blood samples were taken from their hearts. The brains of mice were removed for histological analysis and their isolated splenocytes were cultured. RESULTS: Our results showed that treatment with Ca caused a significant reduction in the severity of the EAE. Histological analysis indicated that there was no plaque in brain sections of Ca treated group of mice whereas 4 ± 1 plaques were detected in brain sections of controls. The density of mononuclear infiltration in the CNS of Ca treated mice was lower than in controls. The serum level of Nitric Oxide in the treatment group was lower than in the control group but was not significant. Moreover, the levels of IFN-γ in cell culture supernatant of splenocytes in treated mice were significantly lower than in the control group. CONCLUSION: The data indicates that Ca intervention can effectively attenuate EAE progression.

Natural killer cells and their role in rheumatoid arthritis: friend or foe?

Rheumatoid arthritis (RA) is a long-term disease that leads to inflammation of the joints and surrounding tissues. Natural killer (NK) cells are an important part of the innate immune system and are responsible for the first line of defense against pathogens during the initial immune challenge before the adaptive immune system eventually eliminates the infectious burden. NK cells have the capacity to damage normal cells or through interaction with other cells such as dendritic cells, macrophages, and T cells cause autoimmune diseases, such as RA. NK cells isolated from the joints of patients with RA suggest that they may play a role in this disease. However, the involvement of NK cells in RA pathology is not fully elucidated. Both protective and detrimental roles of NK cells in RA have recently been reported. A better understanding of NK cells' role in RA might help to develop new therapeutic strategies for treatment of the RA or other autoimmune diseases. We have decided in this paper to focus on the NK cell biology, and attempt to bring the interested readership of this Journal up to date on the NK cell, specifically its possible relation to RA.

The role of natural killer cells in the defense against Listeria monocytogenes lessons from a rat model.

Ly49 receptors in rodents, like killer cell immunoglobulin-like receptors in humans, regulate natural killer (NK) cell activity. Although inhibitory Ly49 receptors clearly recognize classical major histocompatibility complex class I (MHC-I) molecules, the role for the activating Ly49 receptors has been less well understood. Here, we discuss recent data from a rat model for listeriosis. Rats depleted of NK cells, or more specifically the Ly49 receptor-bearing cells, showed increased bacterial loads in their spleen. Athymic nude rats with no functional T cells but increased numbers of Ly49-expressing NK cells were more resistant to infection, indicating a central role of NK cells in early immune defense against Listeria in this species. Listeria infection of macrophages or enteric epithelial cells led to upregulation of MHC-I, including nonclassical (Ib) molecules not regularly recognized by T cells. We have shown that activating Ly49 receptors are more efficiently stimulated when binding to upregulated class Ib antigens on infected cells. From this we postulate that activating Ly49 receptors may have a sentinel function in the early immune response against Listeria in detecting diseased cells 'flagged' by increased MHC-Ib expression.

Healing of long-term frozen orthotopic bone allografts is not affected by MHC differences between donor and recipient.

BACKGROUND: The use of bone grafting in orthopaedic surgery has increased dramatically in recent years. However, the degree to which immune responses are important for the survival of the allograft is not fully understood. In particular it remains unclear whether differences in the major histocompatibility complex (MHC) influence incorporation of bone allografts and their subsequent biologic performance. QUESTIONS/PURPOSES: Therefore, we asked whether isolated mismatch for MHC antigens of deep frozen bone allografts in the long-term causes (1) immune reactions, and whether these reactions have any effect on (2) morphologic features of the graft, (3) radiographic graft healing, and (4) graft strength. METHODS: We used an established orthotopic tibial segment transplantation technique that allows determination of mechanical strength, histologic evaluation, and immune responses. Tibial segments that had been deep-frozen at -80°C for 1 year were transplanted into 24 PVG (RT1 (c)) rats from either 12 syngeneic donors or 12 MHC congenic donors PVG.1U (RT1 (u)). We determined immune responses using an indirect Coombs reaction and determined graft healing radiographically and mechanically after 6 months. RESULTS: We detected no alloantibody production to graft MHC-I antigens, and found no differences between syngeneic and MHC mismatched grafts in terms of remodeling with host bone, graft healing, and mechanical strength. CONCLUSIONS: Mismatches for MHC antigens do not seem to play a decisive role in healing of long-term, deep-frozen bone allografts.

The rat NK cell receptors Ly49s4 and Ly49i4 recognize nonclassical MHC-I molecules on Listeria monocytogenes-infected macrophages.

Ly49 receptors in rodents, like KIRs in humans, regulate NK cell activity. Although inhibitory Ly49 receptors clearly recognize MHC-I molecules, ligands for the activating Ly49 receptors are less well defined. Here, we show that the activating Ly49s4 and the inhibitory Ly49i4 receptors recognize nonclassical MHC-I molecules on the rat macrophage cell line R2 (RT1(d)). Listeria infection of R2 macrophages led to increased expression of classical and nonclassical MHC-I molecules. Coincubation of these infected cells with reporter cells expressing Ly49i4 or Ly49s4 increased the reporter cell responses. These responses were blocked by mAb OX18 (anti-MHC-I) and AAS1 (anti-nonclassical MHC-I). IFN-γ treatment of normal R2 cells also increased the MHC-I expression and enhanced the reporter cell responses. These results suggest that activating and inhibitory Ly49 receptors monitor MHC-I expression on Listeria-infected cells.

Listeria monocytogenes infection affects a subset of Ly49-expressing NK cells in the rat.

NK cells are protective against certain bacterial and viral infections, and their production of IFN-γ is important for the early innate immune defence against L. monocytogenes. We have previously shown that depletion of NK cells in rats leads to increased bacterial burden upon L. monocytogenes infection, and that a subset of NK cells encompassing the majority of Ly49 receptors (Ly49s3+ NK cells) contributed to this effect. In this study, we have further investigated how the Ly49s3+ NK cell subset is affected by L. monocytogenes infection. We observed an increased percentage of Ly49s3+ NK cells in the spleen and a reduction in the bone marrow within the first 48 hrs of L. monocytogenes infection. Concomitantly, we observed increased expression levels of the inflammatory chemokine receptors CCR5 and CXCR3 by Ly49s3+ bone marrow NK cells, as compared to Ly49s3- NK cells, suggesting involvement of Ly49s3+ NK cells in the early phase of infection. However, NK cell production of IFN-γ was independent of Ly49 receptor expression. Furthermore, we observed increased expression levels of MHC class I molecules on both macrophages and NK cells during the first 48 hrs of infection, paralleled by a reduction in the surface expression of Ly49s3 on NK cells. In conclusion, L. monocytogenes infection modulates the tissue distribution of Ly49s3+ NK cells, and induces increased MHC class I expression and hence reduced surface expression of Ly49 receptors on NK cells. These changes indicate that L. monocytogenes infection may have multiple effects on NK cells in vivo, and suggests the involvement of Ly49-expressing NK cells in the immune responses towards L. monocytogenes.

The activating rat Ly49s5 receptor responds to increased levels of MHC class Ib molecules on Listeria monocytogenes-infected enteric epithelial cells.

We have investigated whether rat Ly49 receptors can monitor Listeria-infected intestinal epithelial cells through altered expression of MHC class I molecules. The rat colon carcinoma epithelial cell line CC531 infected with Listeria expressed higher levels of both classical and nonclassical MHC-I molecules. Reporter cells expressing the activating Ly49s5 receptor displayed increased stimulatory responses when incubated with Listeria-infected CC531 cells in vitro, which could be blocked with mAb 8G10 specific for nonclassical MHC-I molecules of the RT1(u) haplotype, but not with mAb OX18 reacting with classical MHC-I molecules in this haplotype. Similar responses were observed against IFN-γ-treated cells that also upregulated their expression of MHC-I molecules. Thus, the Ly49s5 receptor can respond to increased levels of nonclassical MHC-I molecules induced on target cells by either bacterial infection or cytokine stimulation. We furthermore found that splenic NK and NKT cells produced IFN-γ in response to Listeria-infected CC531 cells, and that this was not limited to Ly49-expressing cells, since similar levels of IFN-γ production were observed in Ly49(+) and Ly49(-) NK cell subsets. Therefore, NK cells may recognize Listeria-infected cells through both MHC-I-dependent and -independent innate immune receptor systems.

Short-term treatment with COX-2 inhibitors does not impair fracture healing.

BACKGROUND: The effects of cyclooxygenase (COX) inhibition on fracture healing are insufficiently documented, and the aim of this study was to evaluate the effects of nonspecific and specific COX-2 inhibition in the early phase of fracture healing. METHODS: Thirty rats were randomized in three groups. A diaphyseal fracture was performed and stabilized by intramedullary nailing. In group A parecoxib in a dose of 1 mg/kg body weight/day was given prior to surgery and daily for seven days; in group B diclofenac 2 mg/kg body weight/day was given; and in group C the same amount of saline was given. Blood samples were harvested at 7 and 30 days postoperatively and analyzed for active medications. At 30 days the rats were sacrificed, and the fractures were examined for bone mineralization and tested mechanically. RESULTS: The fractures healed by the production of callus. Plasma concentrations at seven days of medication revealed therapeutic levels of parecoxib, valdecoxib, and diclofenac. There were no significant differences in bone mineralization or mechanical characteristics between the three groups at 30 days postfracture. CONCLUSION: This study indicates that nonspecific or specific COX-2 inhibitors in therapeutic doses during seven days after fracture do not significantly influence bone healing.

Impact of freezing on immunology and incorporation of bone allograft.

With an increasing clinical use of deep frozen allograft for bone reconstruction, it is important to understand the immunological and biological events of allograft incorporation. In this study, we have investigated the impact of deep freezing on immunology and biopotency for incorporation of bone allografts. Deep frozen bone grafts matched or mismatched for major histoscompatibilty complex (MHC) were implanted in an 8-mm segmental defect in the tibia in rats. The construct was stabilized with intramedullary nailing. The immune response was evaluated by determination of serum antibody against the grafts MHC molecules at day 1 and after 2 and 4 months. Incorporation of the graft was compared with fresh syngeneic grafts and assessed with the use of conventional radiography, biomechanical testing and measurement of bone mineral content and density after 4 months. The analyses revealed no antibody responses in the rats that received grafts from donors differing at histocompatibility loci, and at 4 months the frozen grafts showed an overall reconstruction that was not significantly different from the fresh grafts. This study indicates that in the long run there are no significant consequences; either immunological or biomechanical, of the use of deep frozen allogenous bone as compared to fresh autogenous bone grafts in this animal model.

Review article: bone transplantation and immune response.

Bone is the second most common transplant tissue after blood, with the iliac crest autologous graft being most used. Bone transplantation induces osteogenesis to repair bone defects. Despite being the most efficient, autogenous bone requires an additional incision and its supply may be inadequate. Deep-frozen allogeneic bone can be an alternative, but is at risk of microbiological contamination, transmission of unrecognised germs, delayed incorporation, and cellular and humoral immune reactions. Synthetic graft substitutes combine scaffolding properties with biological elements to stimulate cell proliferation and differentiation and eventually osteogenesis. However, they generally lack osteoinductive or osteogenic properties and have various effects on bone healing. We present an overview of bone grafts and graft substitutes in clinical use, and the immune responses to allogeneic bone.

Impact of MHC mismatch and freezing on bone graft incorporation: an experimental study in rats.

Cortical bone graft failure develops for poorly defined reasons, and the effects of the immune responses on the incorporation of an allograft are less clear. In a rat model of tibial allotransplantation, we have studied biometric and histological changes of the graft and the humoral immune response against it. We have also compared fresh with prefrozen grafts to study putative effects of freezing on the healing of the graft and the immune response against it. Fresh and frozen cortical bone grafts matched or mismatched for major histocompatibility complex antigens (syngeneic and allogeneic grafts) were implanted in an 8-mm segmental defect in the tibia. The construct was stabilized with intramedullary nailing. Incorporation of the graft was assessed with use of conventional radiography, micro computed tomography (CT(, biomechanical testing and histological examination. The immune response was evaluated by monitoring distribution of leukocytes in the blood and by measuring antibodies in a tailor-made fluorescence activating cell scanning (FACS( analysis. We found that the fresh syngeneic grafts were well integrated after 8 weeks with intact bone cells. In the fresh allogeneic grafts, all cells were dead with radiological signs of resorption, and mechanical testing indicated failure of incorporation. The frozen grafts showed poorer overall reconstruction than the fresh syngeneic grafts, but the incorporation was better than the fresh allogeneic grafts. A measurable alloantibody response was only detected after fresh allografting. The combined results suggest that freezing of bone allograft impedes the antibody response against major histocompatibility complex (MHC( antigens and improves incorporation, but frozen allografts still perform poorer than do frozen syngeneic grafts.

A one-step real-time PCR assay for detection of DQA1*05, DQB1*02 and DQB1*0302 to aid diagnosis of celiac disease.

Celiac disease is an autoimmune disorder that develops after dietary exposure of the small intestine to gluten peptides in cereals. Celiac disease has a strong genetic component associated with HLA-DQ2 and HLA-DQ8, and testing for absence of these genetic markers is useful when serological tests and biopsies are indeterminate, as it renders celiac disease highly unlikely. We have developed a new real-time PCR assay, using sequence-specific primers (PCR-SSP) and TaqMan probes, for detection of DQB1*05, DQB1*02 (coding for DQ2) and DQB1*0302 (coding for DQ8). PCR amplification and detection of DQ2 and DQ8 was accurately and unambiguously performed from genomic DNA isolated from cell lines and human DNA. Amplification was scored digitally, without laboratory manipulation of amplified PCR products and with a higher accuracy than PCR-SSP. This assay should increase accuracy and throughput, and reduce risks of contamination in laboratories where testing for HLA DQ2 and DQ8 is performed as part of diagnosis of celiac disease.

Effect of hyperglycemic condition on proteoglycan secretion in cultured human endothelial cells.

BACKGROUND: Proteoglycans (PGs) are important constituents of the plasma membrane and of the basement membrane supporting the endothelial cell layer. Changes in the amounts or the structures of PGs in the endothelium may affect important functions such as turnover of lipoproteins, filtration properties, and regulation of chemokines during inflammation, which are all relevant in diabetes. AIM OF THE STUDY: The purpose of this study was to investigate if hyperglycemic conditions would affect the biosynthesis and secretion of PGs in cultured primary human endothelial cells. METHODS: Primary human umbilical cord vein endothelial cells were established and cultured in vitro. The cells were cultured either in medium with low glucose (LG) (1 g/l) or high glucose (HG) (4.5 g/l). From day 3-4 cells were labeled with (35)S-sulfate for 24 h. (35)S-Labeled macromolecules (medium) were purified by gel chromatography, and isolated macromolecules were analyzed by gel chromatography after different types of treatment, electrophoresis, and immunoprecipitation. RESULTS: Lower levels of secreted PGs were found in human endothelial cells exposed to HG. The major part of the PGs released was of the heparan sulfate (HS) type, and immunoprecipitation experiments showed that one such PG was syndecan-1. However, there was no difference in the ratio between HS and chondroitin sulfate (CS) under the different experimental conditions. Further, the PGs expressed neither differ with regard to molecular size of the glycosaminoglycan (GAG) chains, nor were their polyanionic properties affected by the different experimental conditions. CONCLUSION: The results obtained suggest that treatment of primary human endothelial cells with hyperglycemia leads to a decrease in PG secretion in primary cultures of human endothelial cells.

Lipopolysaccharide impairs fracture healing: an experimental study in rats.

BACKGROUND: It has been shown that trauma causes translocation of lipopolysaccharide (LPS) endotoxins from the gut. LPS has been identified as a major bacterial bone resorbing factor. The effects of LPS on bone healing are therefore of clinical interest, as trauma involving fractures followed by sepsis is a clinical scenario. We investigated the effects of systemic and local administration of LPS on the healing of femoral fractures in rats. ANIMALS AND METHODS: In 3 groups, each consisting of 9 rats, a mid-diaphyseal osteotomy/fracture of the femoral bone was performed and then nailed. In one group of animals, LPS was applied intraperitoneally (systemically), and in another group, LPS was applied locally at the fracture site. The third group served as a control. The animals were killed after 6 weeks, and the mechanical characteristics of the healing osteotomies were evaluated. RESULTS: We found that LPS induced a hypertrophic and immature callus, as evaluated by bone mineral content and density. In the rats given LPS intraperitoneally, the mechanical strength characteristics were reduced, as evaluated by bending moment, rigidity, and energy absorption. INTERPRETATION: The rats given LPS intraperitoneally reflect a clinical situation with fracture trauma and endotoxinemia. Our findings indicate that endotoxinemia may impair the fracture healing processes.