Effect of synthetic colloids on major histocompatibility complex class II expression

STUDY OBJECTIVE: Synthetic colloids are used for perioperative fluid management. We hypothesized that their use may be associated with changes in major histocompatibility complex (MHC) class II expression. This could affect patients' morbidity and mortality during clinical intervention. SETTING: University research laboratory. SUBJECTS: Whole blood samples from healthy volunteers. INTERVENTIONS: Whole blood samples from healthy volunteers (n = 6) were incubated with different concentrations of hydroxyethyl starch (HES) from maize and potato (pHES), dextran, and polygelin (gelatine) for 24 hours with or without 100 U/mL human interferon gamma (IFN-gamma; stimulus for MHC class II expression). The expression of human leukocyte antigen (HLA): HLA-DR, HLA-DQ, and HLA-DP was detected simultaneously by a fluoresceinisothiocyanate (FITC)-labeled antibody and analyzed by flow cytometry on lymphocytes and monocytes. MEASUREMENTS AND MAIN RESULTS: Hydroxyethyl starch, pHES, and dextran induced a significant increase in HLA expression. The induction of MHC class II was independent of the structure (50 mg/mL: control, 8.7+/-1.4%; HES, 28 +/- 9.7%; pHES, 29.8 +/- 11.7%; and dextran, 50.2 +/- 8.1%). In contrast, polygelin increased HLA expression only at the highest concentration of gelatine (5 mg/mL, 7.8 +/- 1%; 50 mg/mL, 7.6 +/- 0.8%; 100 mg/mL, 7.3 +/- 1%; 200 mg/mL,16.2 +/- 2.3%). The addition of IFN-gamma decreased HLA expression in presence of highest concentration of HES and dextran. CONCLUSION: In an ex vivo laboratory setting, we demonstrate that high concentrations of plasma expanders are associated with increased HLA expression on lymphocytes and monocytes. However, further in vivo studies are necessary to demonstrate the clinical significance of this observation.     

The effect of steroids on the regulation of major histocompatibility complex-class II expression on nonlymphoid tissue

The induction of MHC-class II antigens on human nonlymphoid tissues plays an essential role during the inhibition and augmentation of the immune response. Steroids have long been shown to possess strong immunosuppressive properties and successful steroid treatment has been associated with the absence of MHC-class II antigens on grated tissues. In this study we more specifically investigated the effect of steroids on the regulation of MHC-class II expression on nonlymphoid tissue. First, the influence of prednisolone on the induction process of the MHC-class II antigens on nonlymphoid tissue was determined. For this purpose vascular endothelial cells, kidney epithelial cells, fibroblasts, and a human colon tumor cell line were incubated with rIFN-gamma or primary MLC supernatant in the absence or presence of different concentrations of prednisolone. It was demonstrated that the induction process of MHC-class II antigens on these cell types was not affected by the drug at the different concentrations tested (1, 10, and 100 micrograms/ml). Next, the effect of prednisolone on the production of MHC-class II inducing factors was investigated. The drug was added at initiation of culture to primary MLC and the MHC-class II-inducing capacity of the supernatants was determined on day 7. Prednisolone at concentrations of 0.5-100 micrograms/ml clearly inhibited the overall production of factors responsible for MHC-class II induction on nonimmunological cells. The drug inhibited the production of IFN-gamma as well as non-IFN-gamma MHC-class II-inducing mediators. At a concentration of 1 microgram/ml the production of IFN-gamma and non-IFN-gamma MHC-class II-inducing mediators was reduced by, respectively, 85 per cent and 75 per cent. At a concentration of 10 micrograms/ml the production of both IFN-gamma and non-IFN-gamma mediators was almost completely inhibited. It is concluded that steroids downregulate the expression of MHC-class II antigens on nonlymphoid tissue by the inhibition of the production of MHC-class II-inducing mediators. However, once these mediators are present, the induction process itself is not affected by the drug.     

Impact of class II major histocompatibility complex antigen expression on the immunogenic potential of isolated rat vascular endothelial cells

We have investigated the immunogenic potential of rat heart vascular endothelial cells by their ability to induce an accelerated rejection of a relevant heart allograft, and related the immunogenic potential to the expression of class II major histocompatibility complex (MHC) antigens on the endothelial cell surface. Only 12% of freshly isolated rat vascular endothelial cells express class II antigens in serum-free medium, and the level of expression is low as judged by immunoperoxidase staining and/or the ability of endothelial cells to bind staphylococci to the cell surface after treatment with monoclonal antibodies to the class II molecule. On the other hand, 99% of the endothelial cells under the same conditions express class I, and the level of expression is high. The class II antigen expression of vascular endothelial cells can be upregulated to more than 98% by recombinant gamma-interferon in vitro--and, concomitantly, the level of expression becomes high, even on the cell surface. Treatment with gamma-interferon did not substantially alter the level of class I expression. The endothelial cells expressing class II antigens weakly, are also weakly immunogenic in vivo: 10(7) endothelial cells are required to reduce the graft survival by 50% of that of the unprimed host. On the contrary, the endothelial cells of the same lineage induced to express class II antigens by gamma-interferon in vitro are highly immunogenic in vivo, as immunogenic as freshly-isolated spleen dendritic cells: only 10(4) endothelial cells are required to induce a 50% reduction of graft survival. These observations demonstrate for the first time that rat vascular endothelial cells are immunogenic in a primary transplantation response in vivo--and, moreover, that the immunogenic capacity of the endothelial cells is directly proportional to the extent of class II MHC antigen expression on the cell surface.     

Cytomegalovirus-induced regulation of major histocompatibility complex class I antigen expression in human aortic smooth muscle cells.

Cardiac allograft vasculopathy (accelerated transplant atherosclerosis) is considered by most to involve a chronic allogeneic immune response to one or more constituents in the coronary vascular wall. Recent evidence suggests that there is an association between cytomegalovirus infection and the development of cardiac allograft vasculopathy (CAV). To determine whether CMV directly infects and/or potentially influences immunogenicity of vascular tissue, human umbilical vein (HU-VECs) or human aortic (HAECs) endothelial cells and human aortic smooth muscle cells (HASMCs) were isolated, cultured, and infected with CMV strain AD 169. Infection was detected using an immunoperoxidase-labeled monoclonal antibody to CMV immediate-early antigen (L-14). The presence and relative quantity of MHC class I and II antigens were determined flow cytometrically using monoclonal antibodies to monomorphic class I and class II HLA determinants. Gamma interferon was used as a positive control stimulant for the upregulation of MHC determinants. Both pooled HUVECs as well as 2 cell lines of HAECs served as targets for CMV infection though less than 10% of the cells were infected despite inocula of 10 pfu/cell. Infection of the pooled HUVECs resulted in no significant changes in the cell surface density of either MHC class I or II determinants. In contrast, HASMCs were excellent targets for CMV infection with virtually 100% of cells infected. CMV infection of 2 distinct HASMC cultures resulted in an increase of 254 +/- 158 relative fluorescence units (RFUs) in MHC class I antigen expression, as assessed by fluorescence intensity, in a variable portion of the HASMCs. A second population of cells exhibited a decrease of 73 +/- 16 RFUs in MHC class I antigen expression. No significant change in MHC class II antigen expression was noted. These results demonstrate that while HUVECs and HAECs are targets of CMV infection, human aortic smooth muscle cells can more readily be infected by CMV. Furthermore, CMV can regulate smooth muscle cell MHC class I expression, hence potentially altering immunogenicity. A pathophysiologic link between cardiac allograft vasculopathy and CMV disease can therefore be hypothesized.     

Donor major histocompatibility complex class I expression determines the outcome of prenatal transplantation

Purpose

The failure of in utero transplantation in immune-competent recipients suggests the existence of a fetal immune barrier. The importance of donor major histocompatibility complex (MHC) class I expression in the induction of prenatal tolerance remains undefined. We hypothesized that donor cell MHC class I expression facilitates engraftment in prenatal allogeneic recipients rather than promoting immune rejection.

Methods

B6.Ly5.2 (class I⁺) or B6.TAP^−/− (class I⁻) murine fetal liver cells were transplanted into age-matched allogeneic fetal recipients. Survival to weaning and subsequent growth was assessed. Engraftment rates and peripheral blood chimerism levels were measured serially.

Results

The presence or absence of class I expression did not affect survival or growth of recipients and no graft-vs-host disease developed. Allogeneic recipients of B6.Ly5.2 cells exhibited significantly higher levels of donor hematopoietic chimerism when compared to recipients of B6.TAP^−/− cells (27% + 10% vs 11% + 8%; P = .004) that deteriorated further over time.

Conclusions

Donor class I MHC antigen expression is essential for stable long-term engraftment and maintenance of donor-specific tolerance. Further studies are needed to better characterize the role of the fetal innate immune system in prenatal allotransplantation.     

Constitutive expression of major histocompatibility complex class II antigens in pulmonary epithelium and endothelium varies among different species

We have observed high constitutive levels of class II antigen expression on porcine and human coronary endothelium, but not on the endothelium of rats and mice. This study examines whether a similar interspecies difference exists in the expression of class II molecules on pulmonary epithelium and endothelium. Lung tissues from na?ve human, porcine, and rodent sources were stained with the monoclonal antibody ISCR3 and examined by light microscopy. Immunoperoxidase staining of class II molecules was observed on human and porcine pulmonary epithelium and endothelium, but was absent in rats and mice. By using an antibody with cross-species reactivity, we demonstrated that na?ve swine pulmonary epithelium and endothelium, unlike those of rodent species, express basal levels of class II antigens in a manner similar to that observed in human lung tissue. These interspecies differences may explain experimental differences observed between murine and large-animal constructs.     

Donor class I and class II major histocompatibility complex antigen expression following liver allografting in rejecting and nonrejecting rat strain combinations

Orthotopic liver allografts in the nonrejecting DA-to-PVG strain combination and in the DA-to-LEW strain combination were studied at various times after transplantation for donor class I and class II MHC expression using immunohistological techniques and quantitative analyses. DA-to-DA isografts were also studied. In the isografts, weak class I induction on hepatocytes and biliary epithelium was noted from day 5, and this persisted to day 15, the last time point examined. In DA-to-PVG allografts, class I induction also appeared on hepatocytes and biliary epithelium from day 5, but was more intense than in the isografts. Nevertheless, the induction was patchy within most grafts, and in some grafts was not prominent. Quantitative absorption analyses demonstrated that the maximum increase in donor class I expression was only 3-fold over the normal liver. In the strong DA-to-LEW combination, class I induction on hepatocytes seemed to appear earlier, beginning at day 3, and was more uniform and intense than in the DA-to-PVG model from day 5. In the isografts, there was no induction of class II antigens on hepatocytes or biliary epithelium at any stage, but from days 5 to 15 there was a marked increase in the number of isolated, class II-positive cells in the hepatic lobule, probably representing class II induction in the Kupffer cells of the isografts. In DA-to-PVG allografts, biliary epithelium became class II-positive from day 5, and this persisted to day 30, the last time point examined. Weak but definite class II induction was seen on some hepatocytes from day 5 through day 30. However, the majority of hepatocytes remained class II-negative. By day 30, there was virtually no donor class II staining the sinusoids, but isolated class II-positive cells of recipient type were seen, the pattern suggesting a replacement of the graft Kupffer cells by recipient Kupffer cells at this stage. By quantitative absorption analysis, donor class II expression in the grafts increased approximately 5-fold. In DA-to-LEW allografts, class II induction was not noticeably different from that seen in the DA-to-PVG model, except that induction of class II antigens on the Kupffer cells possibly appeared earlier in this strain combination.     

In vivo suppression of major histocompatibility complex class II expression on porcine vascular endothelial cells by an HMG-CoA reductase inhibitor

BACKGROUND: Vascular endothelial cells of man and pig, but not rodents, strongly express major histocompatibility complex (MHC) class II antigens in vivo, probably via the inducible promoter IV of the class II transactivator. There is abundant in vitro evidence that MHC class II positive vascular endothelial cells can activate T cells. Peripheral antigen presentation by endothelial cells is potentially important for organ-specific immunity, for allograft rejection, and possibly for immune responsiveness in general. Given the reported effects of statins on promoter IV of the class II transactivator, we evaluated in vivo expression of MHC class II antigens in pigs treated with atorvastatin calcium. METHODS: Pigs were given 3 mg/kg/day of atorvastatin orally daily for 16 days, and then killed 24 hr after the last dose. Heart, kidney, and liver were removed for immunohistological and quantitative absorption analysis. RESULTS: Double-labeling studies using immunofluorescence on frozen section for Factor VIII and MHC class II showed a marked suppression of MHC class II on vascular endothelial cells in all 4 treated pigs, in comparison with untreated pigs. This was confirmed using immunoperoxidase techniques on frozen sections. Quantitative absorption analysis showed up to 25-fold reduction in MHC class II expression. CONCLUSIONS: Statins substantially suppress endothelial cell MHC class II expression in vivo. This is likely to inhibit organ-specific immune responses, and possibly also general immune responsiveness. In a transplantation setting, in addition to other regulatory effects on the recipients immune system, statins might reduce the long-term capacity of the donor organ to activate rejection mechanisms.     

Successful allogeneic transplantation of rat islets expressing cytokine-induced major histocompatibility complex class II antigen

Cultured neonatal rat (F344, RT1(1v1)) islets that were devoid of MHC class II (OX6) antigen and antigen-presenting cells were treated with recombinant murine interferon (IFN-gamma) and/or recombinant murine tumor necrosis factor (TNF-alpha) in vitro. The IFN-gamma and TNF-alpha resulted in some disruption of the integrity of the islets by 7 days of culture, but the combination resulted in disaggregation of the islets within 7-8 days. Insulin release into the medium and secretion in response to glucose were adversely affected by the cytokines. The IFN-gamma resulted in expression of class II antigen on about 10% of the endocrine cells after 3-4 days in culture. This effect of IFN-gamma was potentiated by TNF-alpha resulting in 27% of the cells expressing class II antigen. Islets treated with IFN-gamma and TNF-alpha alone or in combination were not rejected in a subsequent transplant underneath the kidney capsule of WF rats (RT1u). We conclude that expression of class II antigen alone is not sufficient to initiate an allogeneic rejection response, but that cytokine-mediated destruction of endocrine cells could be the basis of immune-mediated islet-cell loss in islet-allograft rejection or autoimmune diabetes.     

Major histocompatibility complex class I and class II expression by myocytes in cardiac biopsies posttransplantation

A total of 85 cardiac biopsies from patients 23-265 days posttransplant were studied for the correlation of the rejection grade score with the level of major histocompatibility complex (MHC) class I and class II expression on cardiac myocytes and endothelial cells, the quantitative level of leukocytic infiltrate, and the immunophenotype of the leukocytes. Results indicate a lack of absolute correlation between rejection grade scores and levels of MHC antigen expression. Further, a lack of absolute correlation was also seen with quantitation of leukocytic infiltrates and relative levels of MHC antigen expression. Of great interest was our preliminary finding that as early as 4 weeks prior to a rejection episode scored by routine histological criteria as grade 4, cardiac biopsy from the patient demonstrated high levels of MHC class I and class II expression. Similar increases of MHC antigen expression prior to an increase in histological rejection score grades were also noted in serial biopsies of 2 other patients. These data suggest that it may be quite useful to examine levels of MHC antigens on cardiac biopsies posttransplantation as an additional parameter for monitoring of cardiac rejection episodes.     

Increased expression of class I major histocompatibility complex antigens on hepatocytes in rejecting human liver allografts

We studied hepatocellular expression of major histocompatibility (MHC) antigens in 43 serial liver transplant biopsies from 22 patients (42 percutaneous, 1 autopsy specimen), 4 normal liver biopsies, and 8 percutaneous biopsies of diseased livers from non-liver-transplant patients. Frozen tissue sections were stained by an indirect immunofluorescence technique using monoclonal antibodies (MCAb) that recognize nonpolymorphic human class I or class II MHC determinants. Ethidium bromide was used to stain nuclei and rhodamine-conjugated anti-basement-membrane antibodies to delineate epithelial and vascular structures. HLA-DR antigens recognized by MCAb OKIa1 and I2 were not detected on hepatocytes but were detected on the bile duct epithelium in 7 of 27 transplant biopsies, including 5 with acute rejection and 1 with chronic liver disease that later progressed to chronic rejection. HLA-A, B, C antigens recognized by MCAb 34/28 intensely stained cells lining the liver sinusoids but were negative on hepatocytes in 4 normal liver biopsies and 7 of 8 non-transplant biopsies. Expression of class I MHC antigens on hepatocyte membranes was increased in 17 of 21 (81%) biopsies from patients with acute rejection, in 4 of 4 with chronic transplant liver disease, but in only 3 of 18 (17%) biopsies from patients with no rejection (chi square = 8.62, P less than 0.01). Our observations demonstrate increased expression of MHC class I antigens in association with acute rejection in human orthotopic liver transplantation. Histologic resolution of the rejection episode is generally followed by a decrease in hepatocyte class I antigen expression. Further analysis of this response may have value in assessing the severity of the rejection and effectiveness of treatment.     

The major histocompatibility complex of the rat

Immunogenetic studies in the rat began with the investigation of blood group antigens (1-7) and evolved into the investigation of histocompatibility antigens and transplantation phenomena (8-17). In the course of this work, several nomenclature systems evolved that were eventually reconciled in a series of comparison studies (18-24). The biennial Workshops on Alloantigenic Systems in the Rat held under the aegis of the Transplantation Society (24) have provided the forum for the continued evolution of this field. In addition, several reviews (24-31) and books (32-35) have provided periodic summaries of different aspects of rat immunogenetics. This review will focus on the structure of the major histocompatibility complex (MHC) of the rat from the serological, biochemical, and molecular points of view.     

Decreased expression of class II major histocompatibility complex (MHC) molecules on monocytes is found in open-heart surgery related immunosuppression

The expression of class II major histocompatibility complex (MHC) molecules on monocytes and the relative proportion of immunoregulatory lymphocyte subpopulations were studied from the blood of eight patients undergoing a cardiac operation. The aim of the study was to reveal mechanisms responsible for depression of cellular immunity and B-lymphocyte activation associated with open-heart surgery. A decreased staining of HLA-DR and even more of HLA-DQ class II MHC molecules was found on the monocyte surface in samples taken 2 and 7 days after operation when compared to preoperative values. The relative proportion of monocytes in mononuclear cells increased after surgery, but within lymphocyte subpopulations only a decrease in total T cells and an increased number of activated (HLA-DR positive) T cells were found, whereas the rations between various T cells subsets remained relatively stable.