Spleen Lymphocyte Apoptosis Research Articles

Effects of anti-stress agents on the growth performance and immune function in broiler chickens with vaccination-induced stress

ABSTRACT The aim of this study was to evaluate the effects of anti-stress agents on the growth performance and immune function of broilers under immune stress conditions induced by vaccination. A total of 128, 1-day-old Arbor Acres broilers were randomly divided into four groups. Group normal control (NC) was the control group. Group vaccination control (VC), T 0.5%, and T 1% were the treatment groups, which were nasally vaccinated with two doses of the Newcastle disease virus (NDV) vaccine. The chicks in groups T 0.5% and T 1% were fed conventional diets containing 0.5% and 1% anti-stress agents. Thereafter, these broilers were slaughtered on 1, 7, 14, and 21 days post-vaccination. The results indicated that anti-stress agents could significantly reduce serum adrenocorticotropic hormone (ACTH) (P < 0.01) and cortisol (CORT) (P < 0.05) levels, and improve the growth performance (P < 0.05) and immune function of broilers (P < 0.05); However, the levels of malondialdehyde (MDA) (P < 0.05) were decreased, and the decreased total antioxidant capacity (T-AOC) (P < 0.01) levels mediated by vaccination were markedly improved. In addition, anti-stress agents could attenuate apoptosis in spleen lymphocytes (P < 0.01) by upregulating the ratio of Bcl-2 to BAX (P < 0.01) and downregulating the expression of caspase-3 and -9 (P < 0.01), which might be attributed to the inhibition of the enzymatic activities of caspase-3 and -9 (P < 0.05). In conclusion, anti-stress agents may improve growth performance and immune function in broilers under immune-stress conditions. RESEARCH HIGHLIGHTS Investigation of effects and mechanism of immune stress induced by vaccination. Beneficial effect of anti-stress agents on growth performance, immune function, oxidative stress, and regulation of lymphocyte apoptosis. Demonstration of the effects of apoptosis on immune function in the organism.

Microwave-Assisted Enzymatic Extraction of Flavonoids from Armeniaca mume Sieb. Blossom and Their Immunomodulating Effect in Mice with DSS-Induced Colitis.

Armeniaca mume Sieb. blossom is among the traditional Chinese edible flowers, and it is widely used in the food and pharmaceutical industries. Flavonoids are among the most abundant bioactive compounds in A. mume Sieb. blossom. However, the research on the extraction of flavonoids from A. mume Sieb. blossom and their immunomodulating function is insufficient. In this study, we developed a microwave-assisted enzymatic extraction of flavonoids from A. mume Sieb. blossom (FAMB) and explored their immunomodulating effect on mice with dextran sulfate sodium salt-induced colitis. The results showed that the optimum parameters for microwave-assisted enzymatic extraction of FAMB were as follows: cellulase: 2.0%; microwave power: 200 W; microwave action time: 5 min; and enzymatic hydrolysis time: 50 min. FAMB significantly promoted the lymphocyte proliferation and natural killer (NK) cell killing activity in colitis mice, and increased the concentrations of TNF-α, IFN-γ, and IL-2 in serum. FAMB also significantly reduced the apoptosis of spleen lymphocytes in these mice. These results demonstrated that the microwave-assisted enzymatic method could significantly improve the yield and efficacy extraction of FAMB. FAMB showed a good immunomodulation effect on colitis mice.

Protective Role of a New Polysaccharide Extracted from Lonicera japonica Thunb in Mice with Ulcerative Colitis Induced by Dextran Sulphate Sodium.

Lonicera japonica Thunb is a traditional Chinese herbal medicine for treating intestinal inflammation. The extraction method of Lonicera japonica Thunb polysaccharide (LJP) has been developed previously by our research group. In this study, a Fourier transform infrared spectrometer (FT-IR) was used to perform a qualitative analysis of LJP and a precolumn derivatization high-performance liquid chromatography (HPLC) ((Palo Alto, CA, USA) method was used to explore the monosaccharide composition of LJP. Then, we studied the effect of LJP on the intestinal flora and immune functions of dextran sulfate sodium- (DSS-) induced colitis ulcerative mouse models. The results showed that LJP was consisted of 6 types of monosaccharides and had the characteristic absorption of typical polysaccharides. LJP can increase significantly the weight, organ index, serum cytokines (interleukin, tumor necrosis factor, and interferon-γ), secretory immunoglobulin A (SIgA) concentration, and natural killer (NK) cell and cytotoxic lymphocyte (CTL) activities in DSS-treated mice. The results of intestinal flora showed that a high dose (150 mg kg−1) of LJP had the best effects on improving the intestinal probiotics (Bifidobacterium and Lactobacilli) and antagonizing the pathogenic bacteria (Escherichia coli and Enterococcus). In addition, the measurement results of the spleen lymphocyte apoptosis confirmed from another perspective that LJP had protective effects of immune cells for DSS-treated mice.

The potential use of fish collagen as a new functional materials due to its good immune-compatibility

As a marine-derived biological macromolecule, fish collagen is attracting more and more attention for its potential application in the biomedical field. Currently, there is no systematic evaluation of immuno-compatibility of fish collagen in vivo. In the present study, 200 mg/kg hydrolyzed tilapia fish collagen (HTC) was injected subcutaneously into mice for 21 consecutive days. To evaluate the effects of HTC on spleen lymphocytes proliferation, cell counting assay (CCK-8) was performed. Flow cytometry was applied to study the effect of HTC on the apoptosis of spleen lymphocytes. Mouse serum immunoglobulin G (IgG) and immunoglobulin M (IgM) were determined by Enzyme-linked immunosorbent assay (ELISA). The results showed no significant difference for the proliferation and apoptosis of spleen lymphocytes between the HTC group and the control group (subcutaneously injected with 0.9% NaCl for 21 consecutive days). And there was no significant difference for serum IgG and IgM levels between the control group and HTC group. The above results showed that the hydrolyzed tilapia fish collagen possesses good immune-compatibility, which can potentially be used as biocompatible implanted biomaterials in vivo.

Effects of induced stress from the live LaSota Newcastle disease vaccination on the growth performance and immune function in broiler chickens

This study was conducted to assess the growth performance and immunological effects of vaccination-induced stress on broilers. The chickens were administered 0, 2, 4, 8, and 16 doses of live LaSota Newcastle disease (ND) vaccine and slaughtered on the 1st, 7th, 14th, and 21st day post vaccination. The results showed that the serum antibody titers after Newcastle disease virus (NDV) vaccination were elevated at day 7 post vaccination, peaked at day 14, then declined by day 21. Interestingly, the antibody titers peaked at 2 doses, and no further dose-dependent titer increases were observed. This study demonstrated that vaccination-induced stress increased serum adrenocorticotropic hormone and cortisol, affected growth performance (average daily gain, average daily feed intake, and feed conversion ratio), and triggered apoptosis in spleen lymphocytes by downregulating the ratio of Bcl-2 to BAX and upregulating the gene expressions of caspase-3 and -9, which was concordant with the activation of the enzymatic activities of caspase-3 and -9. This study suggests that NDV vaccine doses in broilers must be controlled judiciously because increasing the number of doses resulted in increased lymphocyte apoptosis while the peak of the antibody titer and optimal growth performance were achieved at a low number of doses (2 doses).

Suppression of lymphocyte apoptosis in spleen by CXCL13 after porcine circovirus type 2 infection and regulatory mechanism of CXCL13 expression in pigs

Porcine circovirus-associated disease (PCVAD) is one of the most serious infectious diseases in pigs worldwide. The primary causative agent of PCVAD is porcine circovirus type 2 (PCV2), which can cause lymphoid depletion and immunosuppression in pigs. Our previous study demonstrated that Laiwu (LW) pigs, a Chinese indigenous pig breed, have stronger resistance to PCV2 infection than Yorkshire × Landrace (YL) pigs. In this study, we found that the YL pigs showed more severe lymphocyte apoptosis and higher viral load in the spleen tissue than LW pigs. To illustrate the differential gene expression between healthy and infected spleens, transcriptome profiling of spleen tissues from PCV2-infected and control YL pigs was compared by RNA sequencing. A total of 90 differentially expressed genes (DEGs) was identified, including CD207, RSAD2, OAS1, OAS2, MX2, ADRB3, CXCL13, CCR1, and ADRA2C, which were significantly enriched in gene ontology (GO) terms related to the defense response to virus and cell–cell signaling, and another nine DEGs, KLF11, HGF, PTGES3, MAP3K11, XDH, CYCS, ACTC1, HSPH1, and RYR2, which were enriched in GO terms related to regulation of cell proliferation or apoptosis. Among these DEGs, the CXCL13 gene, which can suppress lymphocyte apoptosis during PCV2 infection, was significantly down-regulated in response to PCV2 infection in YL but not in LW pigs. By analysis of the regulatory elements in the promoter and 3′-untranslated region (3′-UTR) of porcine CXCL13, we found that the single nucleotide polymorphism (SNP) −1014 G (LW) > A (YL) and the Sus scrofa microRNA-296-5p (ssc-miR-296-5p) participated in regulating CXCL13 expression during the response to PCV2 infection.

Farnesyltransferase inhibitor FTI-277 inhibits PD-L1 expression on septic spleen lymphocytes and promotes spleen lymphocyte activation.

Farnesyltransferase inhibitors have been tested in clinical trials for the treatment of tumours. In sepsis, the binding of programmed death 1 (PD-1) to programmed death ligand 1 (PD-L1) promotes lymphocyte apoptosis and decreases cytokine expression, thus affecting survival rates. The PD-1/PD-L1 pathway plays an important role in chronic viral infection, bacterial infection and sepsis. However, the precise immunosuppressive and anti-inflammatory functions of this pathway remain poorly understood. In our previous study, the induction of sepsis by caecal ligation and puncture (CLP) resulted in increased farnesyltransferase activity and farnesylated protein levels in the spleen relative to sham treatment. However, the effect of inhibition of farnesyltransferase activity on overall survival rates in patients with sepsis and the specific signalling pathway involved remain to be investigated. In this study, mice with CLP-induced sepsis were treated with farnesyltransferase inhibitor (FTI-277), and PD-L1 expression on septic spleen lymphocytes was examined. Flow cytometric analysis revealed that PD-L1 is expressed constitutively on lymphocytes and that PD-L1 protein expression was up-regulated strongly following CLP. FTI-277 down-regulated PD-L1 mRNA and protein expression on septic spleen lymphocytes in a dose-dependent manner. This effect was associated closely with nuclear factor kappa B (NF-κB). In addition, the significant damping effect of FTI-277 on the PD-L1 signal promoted interferon (IFN)-γ secretion, interleukin (IL)-2 production and splenocyte proliferation in response to anti-CD3+ CD28+ antibodies in mice. Furthermore, FTI-277 reduced spleen lymphocyte apoptosis in septic mice. Therefore, FTI-277 regulates spleen lymphocyte activity via the PD-L1 signalling pathway, with significant anti-inflammatory effects attributable to suppression of the NF-κB pathway. Farnesyltransferase represents a valuable therapeutic target for the treatment of sepsis.

Deep anesthesia worsens outcome of rats with inflammatory responses.

We tested the hypothesis that deep anesthesia with sevoflurane, known as a potent immunomodulator, for 4h would worsen the 24-h outcomes of rats through modulation of the inflammatory responses. Forty-nine male Wistar rats, administered low dose of lipopolysaccharide (0.5mg/kg) intravenously to elicit moderate inflammatory responses mimicked mild surgical stress, underwent one minimum alveolar concentration (MAC) or 2 MAC sevoflurane anesthesia for 4h. The 24-h survival rate, arterial blood gases, plasma interleukin (IL)-6 and tumor necrosis factor (TNF)-α concentrations, and rate of T lymphocyte apoptosis in spleen were evaluated. We further examined the effects of hypotension and TNF-α discharge on the survival rate. The survival rate in 2 MAC group was significantly lower accompanied with decreased base excess and increased level of cytokines (IL-6, TNF-α) compared to 1 MAC group. The apoptosis rate did not differ between the two groups. Neither norepinephrine infusion to restore hypotension nor administration of anti-TNF-α antibody improved the outcome in the 2 MAC group. Deep anesthesia with sevoflurane even for a short-term period augments the release of inflammatory cytokines evoked by inflammatory insults like surgical stress, impairs the acid-base balance, and subsequently deteriorates the outcomes.

Study of curcumin up-regulating mitofusion2 for inhibiting lymphocytes apoptosis of septic mice

Objective To investigate the effect and mechanism of curcumin oninhibiting lymphocyte apoptosis of septic mice. Methods Forty-five male BALB/c mice of clean grade were randomly (raudom number)divided into three groups: sham operation group (n=15), sepsis model group (n=15) and curcumin treatment group (n=15). The experiment was repeated 3 times. Mice in sepsis group were made to be sepsis models by cecal ligation and puncture (CLP) and lavaged the stomach with normal saline (NS) once a day for a week before operation.In curcumin group, mice dealt with CLP were given curcumin once a day for a week in dose of 200 mg/kg before operation. Mice in sham group were only dealt with open abdominal without ligation and puncture, and equal volumes NS was given instead for a week before surgery. The mortality of mice in each group were calculated 24 h after operation and all the survived mice were sacrificed for getting spleen lymphocytes. Apoptosis of lymphocytes were detected by flow cytometry and TUNEL; mitochondrial membrane potential was detected by flow cytometry; Mitofusion2 (Mfn2), Bax (pro-apoptotic protein, promotion of cell death), Bcl-2 (B-cell lymphoma-2, anti-apoptotic protein, prevention of cell death) were detected by Western blot.Data were analyzed by SPSS 18.0. Chisquare test was used to analyzed the counting data, and the measurement data were analyzed by using One-way analysis of variance (ANOVA). Results The mortality rate of mice in sepsis group was higher than that in sham group (46.7%vs. 0.00%, P<0.01), curcumin obviously reduced the death rate compared with sepsis group (40.0%vs. 46.7%, P=0.01). Sepsis group demonstrated a significant increase in lymphocytes apoptosis compared with sham group [(17.1±1.67)%vs. (9.43±1.06)%, P<0.01] , which was dramatically reduced by cureumin dealt with [(12.70±1.25)%vs. (17.1±1.67)%, P=0.012]. Meanwhile, the reduction rate of mitochondrial membrane potentials in sepsis group was more noticeable than that in sham group [(45.13±4.14)%vs. (21.63±1.62), P<0.01], which was significantly decreased by curcumin protection [(29.67±2.15)%vs. (45.13±4.14)%, P=0.001]. The expressions of Bax in cytoplasm in sepsis group significantly decreased compared with sham group (P<0.01) and the expression of Bax in plastosome increased (P=0.001). Pretreatment with curcumin obviously up-regulated (P<0.01) the levels of Bax in cytoplasm and down-regulated (P=0.02) the levels in plastosome. Compared with sham group, the expression of Mfn2 (P=0.015) and Bcl-2 (P=0.01) in sepsis group reduced, and when pretreatment with curcumin, the Mfn2(P=0.002) and Bcl-2(P=0.001) protein levels significantly increased. Conclusions Cureumin can obviously raise the protein level of Mfn2, promote the mitochondria fusion and then suppress the spleen lymphocyte apoptosis of septic mice. Key words: Curcumin; Sepsis; Mice; Lymphocyte; Apoptosis; Mitochondria; Mitofusion2; Mitochondria fusion

Moderate physical training attenuates perinatal low-protein-induced spleen lymphocyte apoptosis in endotoxemic adult offspring rats.

To evaluate the effects of a moderate physical training (T) on the blood and splenic lymphocytes subsets and the rate of apoptosis in adult offspring submitted to perinatal low-protein (LP) diet. Male Wistar rats were divided according to their mother's diet: control (C, 17 % casein) and undernourished (LP, 8 % casein). At the 60th day, pups were submitted to moderate physical training (8 weeks, 5 days week(-1), 60 min day(-1), at 70 % of VO2max). After T period, pups received an injection of lipopolysaccharide (LPS). B, NK, and TCD3+ lymphocytes subsets were analyzed by flow cytometry. Spleen lymphocytes apoptosis was evaluated by DNA fragmentation, phosphatidylserine externalization (PSE), and mitochondrial transmembrane depolarization (MTD) using a flow cytometer. Plasma TNF-α concentrations were analyzed by ELISA. LP + LPS pups showed a higher percentage of blood B, CD4+, and NK and a reduction in TCD3+, CD8+ than C pups. The percentage of NK and CD3+ was restored in LP + T + LPS pups. In the spleen, T normalized the percentage of NK in LP + LPS pups. LP + LPS pups showed a higher percentage of cells with PSE and MTD than C + LPS pups that was attenuated by T. The concentration of TNF-α was higher in LP + LPS than C + LPS, but it was attenuated in LP + T + LPS pups. Moderate physical training was able to revert the effects of perinatal LP diet on circulation lymphocytes subsets and attenuated splenic lymphocytes apoptosis and plasma TNF-α concentrations.

Inhibitive effect on apoptosis in splenic lymphocytes of mice pretreated with Lingzhi (Ganoderma Lucidum) spores

ObjectiveTo investigate how the pretreatment of mice with Ganoderma spores affected the apoptosis of their splenic lymphocytes induced by dexamethasone after 19 days treatment. MethodsSixty Kunming mice were randomly divided into six groups: blank control groupdrenched with normal saline; a drug control group drenched with 150 mg/mL Ganoderma spores; a model group treated with saline; a low dose group with 50 mg/mL Ganoderma spores; a moderate dose group with 100 mg/mL Ganoderma spores; and a high dose group with 150 mg/mL Ganoderma spores. The effect of Ganoderma spores on apoptosis in spleen lymphocytes was analyzed. All groups were treated for 19 days. On day 20, the model group and the 3 treatment groups were intraperitoneally injected dexamethasone to induce apoptosis. Splenic index and apoptosis indes were employed to measure cell apoptosis. ResultsThe results showed that Ganoderma spores reduced the splenic index to different degrees in each group and the best effect was seen in the high dose group (P<0.05). Terminal dexynucleotidyl transferase (TdT)-mediated 2′-Deoxyuridine 5′-Triphosphate nick end labeling staining revealed that the apoptotic index in all groups administered Ganoderma spores differed significantly from the model group, and a dose-response was observed. Flow cytometric analysis indicated that spleen lymphocyte apoptosis in the model group was extensive. Each dose of Ganoderma spores inhibited dexamethasone-induced apoptosis in spleen lymphocytes, and a dose-response was observed as well. The highest dose of Ganoderma spores decreased Malondialdehyde content in serum induced by dexamethasone (P<0.05). ConclusionThe findings imply that the pretreatment of the mice with Ganoderma spores could reduce the apoptosis rate induced by dexamethasone in their splenic lymphocytes.

Abstract T P221: Effect of CD4 And CD8 T Lymphocytes on Infarct Volume Following Experimental Stroke in Male Splenectomized Mice

We have previously shown profound splenic activation by 6 hours following experimental stroke, resulting in rapid and widespread increased production of inflammatory factors by basal and activated splenocytes in male mice. Our lab and others have also shown a subsequent extensive loss of lymphocytes and increased lymphocyte apoptosis in spleen 12 hours after experimental stroke. Lastly, we and others have shown that splenic immunocytes can contribute to increased brain inflammation and infarct volume. However, it is unclear which splenic immunocyte populations may contribute to the evolving ischemic brain injury. Using a mouse splenectomy model and adoptive cell transfer protocols, we evaluated the effect of different splenic immunocyte populations, specifically CD4 and CD8 T lymphocytes, on infarct volume. Male C57BL/6J mice were splenectomized 14 days before experimental stroke and treated 24 hours before experimental stroke with either vehicle (saline), CD4 T lymphocytes, or CD8 T lymphocytes obtained from GFP reporter mice. Each mouse (n=8-10 per group) then underwent 60 minutes of middle cerebral artery occlusion via intraluminal filament. All mice were euthanized and brains collected at 96 hours of reperfusion. Infarct volume (% corrected contralateral structure) was determined by image analysis of coronal brain slices stained with 2,3,5-triphenyltetrazolium chloride. Adoptive transfer of GFP positive CD4 (cortex, 44±3%; striatum, 50±2%) or CD8 (cortex, 48±2%; striatum, 53±3%) T lymphocytes in splenectomized male mice did not alter infarct volume compared to vehicle-treated (cortex, 38±4%; striatum, 42±5%) splenectomized males (cortex, p=0.140; striatum, p=0.143). Although adding back CD4 or CD8 T lymphocytes did not appear to alter infarct volume, these immunocyte populations are comprised of both inflammatory and regulatory subtypes. Thus, future studies will evaluate the effect of different CD4 and CD8 subpopulations on ischemic brain outcomes. This study was supported by National Institutes of Health grant NS076013.

The pathological changes and apoptosis of spleen lymphocytes in mice induced by electromagnetic pulses

To study the effects of electromagnetic pulses (EMP) on pathological changes and apoptosis of spleen lymphocytes in mice. The male BALB/c mice (18 ∼ 22 g) were sham-exposed or exposed to EMP at 200 kV/m for 400 pulses a day for 7 days. On the 1st, 3rd, 7th, 14th 28th days after exposure the mice were killed. The weight of mice, the pathological change and the weight of mouse spleens were observed, the spleen indexes were calculated. The lymphocytes extracted from spleens were counted. The apoptosis and cell cycle of the lymphocyte were detected by flow cytometry, and the migration of the lymphocyte was measured by transwell assay. No pathological changes were found on the first day after exposure. However, the expanded sinusoid and the changed structure of spleen corpuscle on the 3rd day after exposure were observed. There was no difference of spleen indexes between the sham group and the exposure group on the 1st and 14th day after exposure. On the 3rd and 7th days after exposure, the spleen indexes of exposure group were significantly higher than those of sham-exposure group (P < 0.05). On the 28th day after exposure, the spleen indexes of exposure group was significantly lower than those of sham-exposure group (P < 0.05). The number of spleen lymphocytes on the 3rd and 7th days after exposure in exposure group increased significantly, compared with sham-exposure group (P < 0.05). But there were no differences of apoptotic cells and cellular cycle between the exposure group and sham-exposure group (P > 0.05). The ability of migration of the exposure group was significantly higher than that of sham-exposure group (P < 0.05). than the sham group (P < 0.05). The spleen of the male mouse is one of the target organs of EMP. After exposure to EMP, the number of spleen lymphocytes increased. But there were no differences of cell apoptotic cells and cell cycle between the sham group and the exposure group, due to the enhanced migration of lymphocytes induced by EMP.

DEPLETION OF NK CELLS IN A MURINE POLYTRAUMA MODEL IS ASSOCIATED WITH IMPROVED OUTCOME AND A MODULATION OF THE INFLAMMATORY RESPONSE

Sepsis and associated diseases such as systemic inflammatory response syndrome and multiple organ dysfunction syndrome represent common posttraumatic complications on intensive care units induced by a variety of body defense mechanisms. Natural killer (NK) cells are part of the innate immune system. They are thought to play an important role in the development of such syndromes by interplay with other immune cell types and subsequent activation of the inflammatory cascade. To test this hypothesis, NK cells were depleted by administration of antimouse asialo-GM1 antibody in a murine polytrauma model consisting of femur fracture, hemorrhagic shock, and subsequent sepsis. Mortality and immune parameters such as cytokine expression in lung and liver, lymphocyte phenotyping, lymphocyte apoptosis, and organ pathology were determined 96 h after sepsis induction. Survival values showed 50% in the control sepsis group and 100% after NK cell depletion. Thus, NK cell depletion resulted in 50% mortality reduction. Furthermore, we found reductions in the inflammatory response, represented by IL-6 expression in liver, and a decrease in infiltrating neutrophils in the liver and lung. In addition, lymphocyte apoptosis in spleen was decreased by depletion of NK cells. Taken together, these data demonstrate that NK cells contribute to the pathogenetic pathways in a murine polytrauma model. One main mechanism of action seems to be the induction of systemic inflammatory events. Thus, depletion of NK cells results in attenuated inflammation and an overall improvement in outcome. Therefore, NK cells can be considered as important targets for therapeutic strategies.

BOTH GRAM-NEGATIVE AND GRAM-POSITIVE EXPERIMENTAL PNEUMONIA INDUCE PROFOUND LYMPHOCYTE BUT NOT RESPIRATORY EPITHELIAL CELL APOPTOSIS

To determine whether and by which pathway (via the death receptor or mitochondrial mediated pathway) lymphocyte apoptosis occurs in pneumonia and to determine if increased bronchial epithelial cell apoptosis occurs in pneumonia. Prospective randomized study in a university research laboratory. Male C57BL/6 mice (n = 30). Animals received an intratracheal injection of Streptococcus pneumoniae or Pseudomonas aeruginosa to induce gram-positive or gram-negative pneumonia, respectively and were killed 24, 30, or 48 h later. Presence of pneumonia was confirmed via gross visual examination of lungs and by histology. Lymphocyte apoptosis in spleen and thymus was analyzed by flow cytometry for active caspases 3, 8, and 9 and by immunohistochemical (IHC) staining for active caspase 3 and DNA strand breaks. Respiratory epithelial cell apoptosis was assessed by IHC. Histologically, pneumonia was present in all bacteria-treated animals but none in sham-treated mice. Extensive lymphocyte apoptosis in spleen and thymus was documented by characteristic morphological changes on hematoxylin and eosin staining and by IHC staining in both S. pneumonia and P. aeruginosa infection. Flow cytometry confirmed IHC and showed apoptotic lymphocytes positive for active caspases 3, 8, and 9 in both thymi and spleens in both infections. In contrast to the extensive lymphocyte apoptosis, only rare scattered apoptotic changes were seen in respiratory epithelial or endothelial cells in pneumonia due to either organism. Increased lymphocyte but not bronchial cell apoptosis occurs in both gram-positive and gram-negative pneumonia and probably involves both the extrinsic and intrinsic pathway.

Nitric Oxide Induces Apoptosis in Spleen Lymphocytes from MRL/Ipr Mice

Nitric Oxide Induces Apoptosis in Spleen Lymphocytes from MRL/Ipr Mice

Nitric Oxide Induces Apoptosis in Spleen Lymphocytes from MRL/lpr Mice

Background MRL/MpJ -Tnfrsf6 lpr(MRL/ lpr) mice, a murine model of systemic lupus erythematosus (SLE), have defective expression of Fas, substantially reducing signaling for apoptosis via this mechanism. However, it is known that MRL//pr mice have increased spontaneous apoptosis of leukocytes. These conflicting observations have stimulated interest in apoptosis in this SLE model. MRL/ lpr mice overproduce nitric oxide (NO) as autoimmune disease progresses. In vitro administration of NO may induce or decrease apoptosis depending on the cell type. Therefore, we hypothesized that NO induces MRL/ lpr spleen lymphocyte apoptosis independent of Fas receptor engagement. Methods Percentages of apoptotic spleen lymphocytes from MRL/ lpr and BALB/cJ mice were determined ex vivo after in vivo treatment with NG-monomethyl-l-arginine (NMMA), a nitric oxide synthase (NOS) inhibitor. After culture in varying concentrations of a slow-acting NO donor, the following were determined in spleen lymphocytes: (1) levels of apoptosis, (2) the effect of phorbol myristate acid (PMA) on levels of NO-induced apoptosis, and (3) protein kinase C (PKC) activity. Results Spleen lymphocytes from MRL/ lpr mice with active disease had increased levels of ex vivo apoptosis when compared with BALB/cJ controls. This increase was reduced by pharmacologic inhibition of NOS in MRL/ lpr but not in BALB/cJ mice. Exogenous administration of NO in vitro reduced PKC activity and induced apoptosis in MRL/ lpr spleen lymphocytes, an effect that could be reduced via coadministration of PMA in vitro. Conclusion These results suggest that NO plays a role in spleen lymphocyte apoptosis in MRL/ lpr mice, possibly via inhibition of PKC, despite a Fas defect.

Nitric Oxide Induces Apoptosis in Spleen Lymphocytes from MRL/Ipr Mice

BackgroundMRL/MpJ-Tnfrsf6Ipr(MRL/Ipr) mice, a murine model of systemic lupus erythematosus (SLE), have defective expression of Fas, substantially reducing signaling for apoptosis via this mechanism. However, it is known that MRL/Ipr mice...

Nitric Oxide Induces Apoptosis in Spleen Lymphocytes from MRL/lpr Mice

Nitric Oxide Induces Apoptosis in Spleen Lymphocytes from MRL/lpr Mice

Calcium and Ca-dependent phospholipid signalling systems are involved in spleen lymphocyte apoptosis

Calcium and Ca-dependent phospholipid signalling systems are involved in spleen lymphocyte apoptosis