Reexamination of the blood lymphocyte transformation test in the diagnosis of chronic beryllium disease

The T cell response to beryllium, measured in bronchoalveolar lavage by the lymphocyte transformation test (LTT), is a critical diagnostic test for discriminating between chronic beryllium disease (CBD) and other granulomatous diseases. We examined the sensitivity, reproducibility, and methods of a less invasive, peripheral blood LTT in 17 patients with CBD and in 18 beryllium-exposed control subjects. Ninety-four percent of CBD cases (16/17) had abnormal blood LTT results, and all 18 beryllium-exposed control subjects had normal blood LTT results. Split samples for 10 beryllium disease cases and eight control subjects demonstrated that the blood LTT was reproducible between two separate laboratories. The LTT was equally sensitive with 10% and 20% serum in the culture medium. We conclude that an abnormal blood LTT can be used to diagnose CBD in patients with compatible lung pathology.     

Murine peritoneal macrophage gangliosides inhibit lymphocyte proliferation

Gangliosides have been shown to act as immunoregulatory agents by altering proliferative responses of lymphocytes to both antigens and mitogens. Most early studies have utilized brain gangliosides and have required high concentrations. The role of endogenous gangliosides from macrophages has remained unexplored. In this study, thioglycolate-elicited murine peritoneal macrophage gangliosides were purified and added to cultures of murine lymphocytes. Nanogram amounts caused a profound inhibition of LPS-induced DNA synthesis of splenocytes and of purified B lymphocytes, without demonstrable cellular toxicity. No effect was seen using asialo-GM1. This effect was present across a wide range of lipopolysaccharide (LPS) doses. Nanogram amounts of macrophage gangliosides also inhibited concanavalin A (ConA)-mediated lymphocyte proliferation. Inhibition of LPS-induced mitogenesis was present even if gangliosides were removed from the extracellular environment after 15-60 min of incubation prior to the addition of LPS. This inhibition was reversible with incubation of ganglioside pre-treated lymphocytes in medium containing serum. These inhibitory properties of macrophage gangliosides are distinct from those found in studies using brain gangliosides, and support a potential role for macrophage gangliosides as negative modulators of lymphocyte proliferation.     

Immunology of chronic beryllium disease

PURPOSE OF REVIEW: This review discusses the immunology of chronic beryllium disease. It addresses the importance of the interaction between class II molecules and the T cells that recognize beryllium, along with the subsequent immune response that results in sensitization and disease, and genetic factors leading to variation in this response. RECENT FINDINGS: HLA-DPB1 with a glutamic acid at amino acid position 69 (Glu69) confers increased risk of beryllium sensitization and is not specific for chronic beryllium disease. The degree of negative surface charge of the molecule may increase risk of chronic beryllium disease but not sensitization. In the absence of Glu69, HLA-DRB1 alleles may function in beryllium presentation, increasing the risk of chronic beryllium disease. The T-cell response as assessed by the beryllium lymphocyte proliferation test is dependent on central memory T-cells, while Th1 cytokine secretion leading to granulomatous inflammation and chronic beryllium disease is dependent on the activity of effector memory T cells. Polymorphisms in cytokine genes, such as the TGF-beta1 gene, also affect the risk of chronic beryllium disease and more severe disease. SUMMARY: The current diagnostic criteria for sensitization and chronic beryllium disease rely on the beryllium lymphocyte proliferation test. By understanding the novel immunologic mechanisms and genetic factors associated with sensitization and chronic beryllium disease, we may improve our ability to detect beryllium health effects with new diagnostics, and hopefully refine therapies for disease.     

T cell recognition in chronic beryllium disease

Chronic beryllium disease (CBD) is a granulomatous lung disorder caused by beryllium exposure in the workplace and is characterized by the accumulation of beryllium-specific CD4(+) T cells. Depending on genetic susceptibility and the nature of the exposure, CBD occurs in up to 20% of exposed workers. Genetic susceptibility has been associated with particular HLA-DP alleles, especially those possessing a negatively charged glutamic acid residue at the 69th position of the beta-chain. The mechanism for this association lies in the ability of these HLA-DP molecules to bind and present beryllium to pathogenic CD4(+) T cells. Large numbers of effector memory, beryllium-specific CD4(+) T cells are recruited to the lung of these subjects and secrete Th1-type cytokines upon beryllium recognition. The presence of circulating beryllium-specific CD4(+) T cells directly correlates with the severity of lymphocytic alveolitis. With the presence of a known antigenic stimulus, CBD serves as an important model of immune-mediated, organ destruction. Thus, our findings in CBD have important implications for studies in autoimmune diseases, in particular those with an unknown inciting antigen and an inaccessible target organ.     

Modulation of murine lymphocyte and macrophage proliferation by parenteral zinc

The effects of a single i.p. injection of zinc (0.7, 1.3, 4.0 or 12.0 mg/kg), 24 h prior to sacrifice, on lymphocyte blastogenesis as well as lymphocyte and macrophage progenitor cell proliferation were examined using cells from adult BALB/c mice. Splenic lymphocyte blastogenesis in response to T cell mitogens decreased for mice receiving the highest zinc dosage while responses to B cell mitogens were initially depressed, subsequently increased, and finally declined sharply as the LD50 was approached. Splenic B cell colony formation decreased linearly in relation to zinc dosage with a 50% suppression of colony formation observed at approximately 8.0 mg/kg. In contrast, bone marrow granulocyte-macrophage colonies were enhanced at higher dosages (greater than or equal to 2.5 mg/kg) of zinc. These results indicate that zinc exposure at dosages less than the LD50 can influence lymphocyte blastogenesis and clonal expansion of both B cell and macrophage progenitors.     

Comparison of lymphocyte transformation and macrophage migration inhibition tests in the detection of beryllium hypersensitivity.

In seven patients with chronic beryllium disease (Be) the Be lymphocyte transformation test was positive in 100%, independent of steroid therapy, and was reproducible. The Be macrophage migration inhibition test was only positive in four of seven patients (57%) not on steroids, and was not reproducible. In 72 potentially exposed healthy beryllium workers the lymphocyte transformation test was negative in all subjects. The macrophage test was positive in four of 78 and again the results were not reproducible. The workers with positive results showed no differences in age, type or duration of employment from those with negative results and showed no evidence of disease. In addition, the macrophage test was positive in two of 45 non-exposed control subjects. We also confirmed the above advantages of the lymphocyte transformation technique by using tuberculin antigen (PPD). The PPD lymphocyte transformation test gave positive results in approximately 60% of healthy beryllium workers, but the PPD macrophage test was only positive in 7%. We conclude that the Be lymphocyte transformation test is the most sensitive and reproducible and advocate its use in the diagnosis of disease and in monitoring the health of potentially exposed workers.     

TNF polymorphism and bronchoalveolar lavage cell TNF-alpha levels in chronic beryllium disease and beryllium sensitization

BACKGROUND: Beryllium stimulates TNF-alpha from chronic beryllium disease (CBD) bronchoalveolar lavage (BAL) cells. OBJECTIVE: We sought to relate TNF polymorphisms to beryllium-stimulated TNF-alpha production, to the development of CBD, and to the risk of more severe CBD over time. METHODS: We recruited 147 patients with CBD, 112 beryllium-sensitized subjects, and 323 control subjects; genotyped 5 TNF promoter polymorphisms; and measured beryllium-stimulated and unstimulated BAL cell TNF-alpha production from a subset of subjects. RESULTS: Beryllium-stimulated, but not beryllium-unstimulated, BAL cell TNF-alpha production was significantly increased in patients with CBD compared with that seen in those only sensitized (P = .0002). Those subjects with the TNF -857T allele and the only haplotype (haplotype 4) containing this allele demonstrated significantly lower unstimulated BAL cell TNF-alpha production compared with that seen in noncarriers (P = .009). Patients with CBD alone and combined with sensitized subjects carrying the TNF haplotype 1 compared with those without this haplotype had significantly increased beryllium-stimulated BAL cell TNF-alpha levels (P = .02). We found no significant association between patients with CBD, sensitized subjects, and control subjects with any of the TNF promoter polymorphisms or haplotypes. A greater decrease in Pao(2) at maximum exercise was noted in patients with CBD with the -1031C allele (P = .03) and with haplotypes other than the TNF haplotype 1 (P = .01), 3 (from 5) of which contain the -1031C allele. CONCLUSIONS: The -857T allele and haplotype 1 are associated with BAL cell TNF-alpha production, indicating a potential role of TNF promoter variants in regulation of TNF production in sensitized subjects and patients with CBD. CLINICAL IMPLICATIONS: TNF promoter variants are not risk factors for CBD or sensitization.     

Cyclic AMP inhibits macrophage suppressor function and enhances lymphocyte proliferation.

The effects of increasing the level of cyclic AMP (cAMP) activity on lymphocyte proliferation in the rat mixed lymphocyte reaction (MLR) were investigated. Dibutyryl cAMP (dbcAMP) and prostaglandin E2 (PGE2) produced a dose-dependent reduction in proliferation in the lymph node (LN) MLR, but produced a substantial increase in proliferation in the spleen MLR at the lower concentrations used (10(-5)-10(-4) M dbcAMP; 10(-7)-10(-6) M PGE2). Enhancement of proliferation was dependent on the presence of macrophages and was probably due to inhibition of macrophage activation. This was based on the following findings: (1) spleen MLR proliferation was lower than that in the LN MLR; (2) depletion of spleen macrophages increased proliferation in the spleen MLR and addition of these macrophages to the LN MLR reduced proliferation; (3) macrophage depletion from the spleen MLR abolished the proliferation-enhancing effect of dbcAMP. In conclusion, cAMP enhances lymphocyte proliferation in this system, apparently as a consequence of suppressing the inhibitory influence of macrophages.     

Purified podophyllotoxin (CPH-86) inhibits lymphocyte proliferation but augments macrophage proliferation

Purified podophyllotoxin (CPH-86) is an inhibitor of microtubular aggregation used in the treatment of cancer, psoriasis and rheumatoid arthritis. To better understand its immunopharmacology we examined its effects on human lymphocytes and monocytes and guinea pig macrophages. CPH-86 inhibits mitogen-induced human lymphocyte proliferation and macrophage growth factor-stimulated macrophage proliferation with ID50s of approximately 10(-7) M. The effect of CPH-86 on lymphocytes in conjunction with mitogen is nonlethal, evident during the early but not the late phases of proliferation, and associated with early increases in cyclic AMP levels. In contrast to these obviously inhibitory effects, CPH-86 (10(-7) M) alone induces IL-1 by human monocytes and, with mitogen, it induces IL-2 production by human lymphocytes. It directly stimulates macrophage proliferation and potentiates the effects of low doses of macrophage growth factor to do so. The latter effects may be mediated by colony stimulating factor production. The effects of CPH-86 are not mediated by inhibition of prostaglandin synthesis. The stimulation of monokine and lymphokine production by CPH-86 may represent positive features of its action and may be immunotherapeutic.     

慢性HIV/AIDS患者T淋巴细胞凋亡与疾病进展关系的研究

目的 探讨慢性未经抗病毒治疗的HIV/AIDS患者T淋巴细胞及各亚群凋亡与疾病进展的相关性.方法 以36例慢性未经抗病毒治疗的HIV/AIDS患者为研究对象,根据CD4细胞计数分为3组:<200个/μl组,200～350个/μl组,>350个/μl组,同时选取16例健康志愿者作对照,分离外周血单核细胞(PBMC)后,采用CD45RO及CD27标记T细胞亚群,Annexin V标记细胞凋亡,用流式细胞仪检测各项指标.其中4例患者及4例健康志愿者的PBMC在体外培养,比较分析体外培养0、3、6、12、24 h不同时间点T细胞凋亡的变化情况.结果 (1)HIV/AIDS患者CD4~+、CD8~+T细胞及各亚群上Annexin V表达百分比均显著高于健康人(P<0.05),但3组患者之间比较差异无统计学意义(P>0.05);(2)HIV/AIDS患者CD4~+、CD8~+T细胞及各业群上Annexin V表达百分比与CD4~+T细胞计数及病毒载显均无显著相关性(P>0.05);(3)随着体外细胞培养时间的延长,HIV/AIDS患者CD4~+T细胞的凋亡及死亡细胞百分比均显著高于健康人(P<0.05),并且HIV/AIDS患者CD4~+T细胞较CD8~+T细胞更易发生凋亡和死亡.结论 HIV/AIDS患者的T细胞凋亡水平显著高于健康人,并且CD4~+T细胞较CD8~+T细胞更易发生凋亡和死亡,但是T细胞凋亡水平与HIV的疾病进展程度并没有相关性.     

Immunomodulatory action of chronic exercise on macrophage and lymphocyte cytokine production in mice

This study was designed to evaluate the effects of 8-week voluntary running exercise on cytokine production of macrophages and lymphocytes. Seven-week-old-male BALB/c inbred mice were divided into two groups: a group given voluntary exercise (exercise group, n=32), and the other, a non-exercise group (control group, n=32). Exercise consisted of spontaneous running in wheels for 3 days per week over 8 weeks. The levels of nitric oxide (NO2-) and interleukin (IL)-1 beta production by lipopolysaccharide (LPS)-stimulated peritoneal macrophages from the exercise group was significantly higher than that in the control group (P < 0.05-P < 0.01). In the exercise group, stimulation indices by concanavalin A (Con A) was significantly higher than they were in the control group (P < 0.05-P < 0.001). When compared with the control group, the exercise group showed a significant (P < 0.05) increase in the splenic lymphocyte production of IL-2 stimulated by Con A (449.5 +/- 28.2 and 853.7 +/- 116.0 pg 4 x 10(5) cells(-1) 48 h(-1) for the control group and the exercise group, respectively). IL-4 production of splenocytes stimulated by Con A in the exercise group (37.6 +/- 5.1 pg 4 x 10(5) cells(-1) 48 h(-1)) was higher than that in the control group (30.9 +/- 3.9 pg 4 x 10(5) cells(-1) 48 h(-1)); however, the difference was not statistically significant. These results suggest that 8-week voluntary running exercise effectively enhanced macrophage and lymphocyte functions in mice.     

Spontaneous lymphocyte proliferation and depressed cellular immunity in Hodgkin''s disease

Patients with Hodgkin's disease have increased numbers of spontaneously proliferating circulating lymphocytes. In order to test the relationship between this phenomenon and the in vitro mitogenic response, both spontaneous lymphocyte proliferation and the response to stimulation with phytohaemagglutinin were quantified. In addition, the proliferating lymphocytes were identified autoradiographically and characterized for the presence of lymphocyte surface markers and monocyte markers. Spontaneous thymidine incorporation by lymphocytes from patients with Hodgkin's disease was increased compared with normal controls, and the phytohaemagglutinin response was depressed. A significant inverse relationship was demonstrated between the spontaneous thymidine incorporation on day 0 and the phytohaemagglutinin response on day 3 (P is less than 0.01). The activated lymphocytes were heterogeneous with respect to both morphology and surface markers. These data suggest that the circulating proliferating lymphocytes in Hodgkin's disease are reactive cells and that the quantitative depression in cellular immunity demonstrable in these patients may be related to this chronic reactivity.     

Enhanced lymphocyte longevity and absence of proliferation and lymphocyte apoptosis in Quilty effects of human heart allografts.

"Quilty effect" (QE) is a common and problematic observation in endomyocardial biopsy specimens from patients after cardiac transplantation. The origin, fate, and significance of QE cellular elements are unknown. Twenty-six paraffin-embedded endomyocardial biopsy specimens with QE (five QE As and twenty-one QE Bs) from twenty-two cardiac allografts were studied by immunohistochemistry for expression of Bcl-2, Fas antigen, proliferating cell nuclear antigen (PCNA), perforin, T cells (UCHL-1), macrophages (CD68), and apoptosis by in situ terminal deoxyribonucleotide transferase (TdT)-mediated dUTP nick end labeling (TUNEL). Approximately 50% of the lymphocytes present, mainly in the deeper region of 20 of 21 QE Bs and all 5 QE As, expressed Bcl-2 in a pseudo-nodular pattern surrounding high endothelial venules. Fas expression was detected in lymphocytes in 20 of 21 QE Bs and 5 QE As in a similar pattern to Bcl-2. However, endothelial cells and macrophages were Bcl-2 negative, whereas both cell types were Fas positive. Perforin was negative in nearly all lymphocytes. TUNEL staining revealed that lymphocytes in QEs did not undergo apoptosis; however, TUNEL positivity was observed in approximately 70% of endothelial cells and macrophages and certain adjacent cardiac myocytes in 20 of 21 QE Bs and 5 QE As. One large QE B with a germinal center was noted. Germinal center cells expressed PCNA intensely but were negative for Bcl-2, Fas, and TUNEL. Cells surrounding the germinal center expressed abundant Bcl-2. The following conclusions were drawn. 1) Apoptosis does not occur in lymphocytes in QE where enhanced Bcl-2 (apoptosis inhibitor) and Fas antigen (apoptosis inducer) are expressed. 2) PCNA negativity indicates that QE lymphocytes may not proliferate, and perforin negativity indicates that they may not exhibit perforin-based cytotoxicity. We propose that there may be a relationship between the longevity of lymphocytes in QE and the absence of apoptosis.     

Molecular mechanisms involved in macrophage survival, proliferation, activation or apoptosis

Macrophages play a critical role during the immune response. Like other cells of the immune system, macrophages are produced in large amounts and most of them die through apoptosis. Macrophages survive in the presence of soluble factors, such as IFN-g, or extracellular matrix proteins like decorin. The mechanism toward survival requires the blocking of proliferation at the G₁/S boundary of the cell cycle that is mediated by the cyclin-dependent kinase (cdk) inhibitor, p27^kip and the induction of a cdk inhibitor, p21^waf1.     

Diminished production of anti-inflammatory mediators during neutrophil apoptosis and macrophage phagocytosis in chronic granulomatous disease (CGD)

Genetic defects in the phagocyte nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase enzyme system result in chronic granulomatous disease (CGD). In addition to recurrent, life-threatening infections, patients with CGD frequently present with sterile inflammatory complications, suggesting that NADPH-oxidase deficiency predisposes to these responses in the absence of persistent microbial infection. The mechanisms involved in the aberrant, inflammatory process are unknown. In this study, we have shown that neutrophils isolated from CGD patients, which are more resistant to spontaneous apoptosis in vitro, also produce significantly less of the anti-inflammatory mediator cyclopentenone prostaglandin D(2) (PGD(2)). In addition, during phagocytosis of opsonized and nonopsonized apoptotic targets, CGD macrophages are severely compromised in their ability to produce PGD(2) and transforming growth factor-beta (TGF-beta). We suggest that delayed apoptosis of inflammatory cells, such as neutrophils and deficient production of the anti-inflammatory mediators PGD(2) and TGF-beta during macrophage clearance of apoptotic debris and invading pathogens, contributes to persistence of inflammation in CGD.     

Neutrophil apoptosis: impact of granulocyte macrophage colony stimulating factor on cell survival and viability in chronic kidney disease and hemodialysis patients

Introduction

Altered neutrophil apoptosis might be responsible for recurrent bacterial infections encountered in hemodialysis (HD) and chronic kidney disease (CKD) patients. This work was designed to assess the neutrophil apoptotic activity and the impact of implementation of granulocyte macrophage colony stimulating factor (GM-CSF), as a survival factor, on neutrophil apoptosis among these patients.

Material and methods

Twenty-five patients on regular HD along with 34 CKD patients on conservative treatment, as well as 15 healthy controls, were investigated for apoptotic rate via assessment of neutrophil expression of Annexin-V by flow cytometry, before and after 20 h culture in absence and presence of GM-CSF. Neutrophil viability was determined using light microscopy. The preservation of neutrophil activation in these patients was analyzed by flow cytometric CD18 neutrophil expression. Chronic inflammatory state was evaluated by estimating C-reactive protein (CRP) and soluble intercellular adhesion molecule-1 (sICAM-1). Obtained data were statistically analyzed.

Results

Compared to controls, both HD and CKD groups had a significant increase of Annexin-V and CD18 expression and significant decrease in neutrophil viability. Culture of their neutrophils with GM-CSF showed significant decrease of apoptosis accompanied by improvement of neutrophil viability compared to their cultured cells without GM-CSF. These patients also showed significant elevation of CRP and sICAM-1.

Conclusions

Granulocyte macrophage colony stimulating factor demonstrated an evident impact on improving in vitro neutrophil survival and viability in HD and CKD patients. Therefore, this may represent promising preventive and/or therapeutic strategies against infection frequently observed in these patients and causing morbidity and mortality.     

Self-presentation of beryllium by BAL CD4+ T cells: T cell-T cell interactions and their potential role in chronic beryllium disease

Chronic beryllium disease (CBD) is characterized pathologically by granulomatous inflammation in the lung, composed of a large core of epithelioid cells surrounded by a dense shell of CD4+ T cells. Using beryllium-specific CD4+ T cell lines derived from the bronchoalveolar lavage (BAL) fluid of CBD patients, we show that purified CD4+ T cells produced significant amounts of IFN-gamma and TNF-alpha upon exposure to beryllium in the absence of antigen-presenting cells (APC). However, unlike BAL T cells stimulated by beryllium in the presence of APC, self-presentation by BAL T cells did not induce detectable IL-2 production, and in its absence these activated T cells die from programmed cell death. Resting BAL CD4+ T cells constitutively express high levels of HLA-DP, lymphocyte function-associated antigen 1 (LFA-1) and ICAM-3. When stimulated with beryllium/APC, the adhesion molecule ICAM-1 was up-regulated, as well as several costimulation molecules including CD28, OX-40 (CD134), 4-1-BB (CD137) and B7-1 (CD80). Notably, CD28 was not up-regulated during self-presentation by BAL T cells, and these cells do not express OX-40L, suggesting that lack of appropriate costimulation was responsible for programmed cell death observed upon beryllium self-presentation. Restricting anti-MHC class II mAb completely eliminated beryllium-induced T cell proliferation during self-presentation and significantly reduced IFN-gamma and TNF-alpha production. Our data demonstrate for the first time that self-presentation by BAL T cells in response to beryllium can occur ex vivo, in the absence of professional APC, with a specific dependence on T cell-expressed MHC class II molecules and exogenous IL-2 for survival.