Increase In MHC Research Articles

Heterotopic Rat Heart Transplantation (Lewis to F344): Early ICAM-1 Expression After 8 Hours of Cold Ischemia

Primary graft dysfunction is a still poorly understood complication after cardiac transplantation. Ischemia/reperfusion injury contributes to different disorders resulting in impaired graft function. In a heterotopic rat heart transplantation model we extended graft ischemic time up to 8 hours. Using immunohistochemistry we detected an up to 4-fold increase in intracellular adhesion molecule-1 (ICAM-1) expression during 4 hours of reperfusion, independent of ischemic time (30-minute ischemia: 7.65 +/- 2.15 without reperfusion, 19.46 +/- 4.6 after 4-hour reperfusion; 240-minute ischemia: 5.6 +/- 1.99 and 22.3 +/- 3.77; 480-minute ischemia: 3.7 +/- 1.56 and 13.1 +/- 2.2). Eight-hour ischemic allografts had an increase in CD8-positive cells (1.37 +/- 0.5 and 2.3 +/- 0.77) and a significant increase in MHC II expression (11.48 +/- 2.1 and 18.27 +/- 1.34) during 4 hours of reperfusion. We hypothesize that these findings reflect an early inflammatory reaction in the allograft possibly triggered by oxidative stress. During therapeutic interventions, both of these pathways must be considered.

Estrogen Selectively Promotes the Differentiation of Dendritic Cells with Characteristics of Langerhans Cells

The steroid hormone estrogen regulates the differentiation, survival, or function of diverse immune cells. Previously, we found that physiological amounts of 17beta-estradiol act via estrogen receptors (ER) to promote the GM-CSF-mediated differentiation of dendritic cells (DC) from murine bone marrow progenitors in ex vivo cultures. Of the two major subsets of CD11c(+) DC that develop in these cultures, estrogen is preferentially required for the differentiation of a CD11b(int)Ly6C(-) population, although it also promotes increased numbers of a CD11b(high)Ly6C(+) population. Although both DC subsets express ERalpha, only the CD11b(high)Ly6C(+) DC express ERbeta, perhaps providing a foundation for the differential regulation of these two DC types by estrogen. The two DC populations exhibit distinct phenotypes in terms of capacity for costimulatory molecule and MHC expression, and Ag internalization, which predict functional differences. The CD11b(int)Ly6C(-) population shows the greatest increase in MHC and CD86 expression after LPS activation. Most notably, the estrogen-dependent CD11b(int)Ly6C(-) DC express langerin (CD207) and contain Birbeck granules characteristic of Langerhans cells. These data show that estrogen promotes a DC population with the unique features of epidermal Langerhans cells and suggest that differentiation of Langerhans cells in vivo will be dependent upon local estrogen levels and ER-mediated signaling events in skin.

Expression of the MHC class II transactivator (CIITA) type IV promoter in B lymphocytes and regulation by IFN-γ

The MHC class II transactivator (CIITA), the master regulator of MHC class II (MHC II) expression, is a co-activator that controls MHC II transcription. Human B lymphocytes express MHC II constitutively due to persistent activity of CIITA promoter III (pIII), one of the four potential promoters (pI–pIV) of this gene. Although increases in MHC II expression in B cells in response to cytokines have been observed and induction of MHC II and CIITA by IFN-γ has been studied in a number of different cell types, the specific effects of IFN-γ on CIITA expression in B cells have not been studied. To investigate the regulation of CIITA expression by IFN-γ in B cells, RT-PCR, in vivo and in vitro protein/DNA binding studies, and functional promoter analyses were performed. Both MHC II and CIITA type IV-specific RNAs increased in human B lymphocytes in response to IFN-γ treatment. CIITA promoter analysis confirmed that pIV is IFN-γ inducible in B cells and that the GAS and IRF-E sites are necessary for full induction. DNA binding of IRF-1 and IRF-2, members of the IFN regulatory factor family, was up-regulated in B cells in response to IFN-γ and increased the activity of CIITA pIV. In vivo genomic footprint analysis demonstrated proteins binding at the GAS, IRF-E and E box sites of CIITA pIV. Although CIITA pIII is considered to be the hematopoietic-specific promoter of CIITA, these findings demonstrate that pIV is active in B lymphocytes and potentially contributes to the expression of CIITA and MHC II in these cells.

IL-13 Regulates the Immune Response to Inhaled Antigens

The large inhibitory effect of IL-13 blockers on the asthma phenotype prompted us to ask whether IL-13 would play a role in regulating the allergic immune response in addition to its documented effects on structural pulmonary cells. Because IL-13 does not interact with murine T or B cells, but with monocytes, macrophages, and dendritic cells (DCs), we examined the role of IL-13 in the activation of pulmonary macrophages and DCs and in the priming of an immune response to a harmless, inhaled Ag. We found that a majority of cells called "alveolar or interstitial macrophages" express CD11c at high levels (CD11c(high)) and are a mixture of at least two cell types as follows: 1) cells of a mixed phenotype expressing DC and macrophage markers (CD11c, CD205, and F4/80) but little MHC class II (MHC II); and 2) DC-like cells expressing CD11c, CD205, MHC II, and costimulatory molecules. Endogenous IL-13 was necessary to induce and sustain the increase in MHC II and CD40 expression by pulmonary CD11c(high) cells, demonstrated by giving an IL-13 inhibitor as a measure of prevention or reversal to allergen-primed and -challenged mice. Conversely, IL-13 given by inhalation to naive mice increased the expression of MHC II and costimulatory molecules by CD11c(high) cells in an IL-4Ralpha-dependent manner. We found that exogenous IL-13 exaggerated the immune and inflammatory responses to an inhaled, harmless Ag, whereas endogenous IL-13 was necessary for the priming of naive mice with an inhaled, harmless Ag. These data indicate that blockade of IL-13 may have therapeutic potential for controlling the immune response to inhaled Ags.

Human BDCA-1-Positive Blood Dendritic Cells Differentiate into Phenotypically Distinct Immature and Mature Populations in the Absence of Exogenous Maturational Stimuli: Differentiation Failure in HIV Infection

Current immunological opinion holds that myeloid dendritic cell (mDC) precursors migrate from the blood to the tissues, where they differentiate into immature dermal- and Langerhans-type dendritic cells (DC). Tissue DC require appropriate signals from pathogens or inflammatory cytokines to mature and migrate to secondary lymphoid tissue. We show that purified blood mDC cultured in vitro with GM-CSF and IL-4, but in the absence of added exogenous maturation stimuli, rapidly differentiate into two maturational and phenotypically distinct populations. The major population resembles immature dermal DC, being positive for CD11b, CD1a, and DC-specific ICAM-3-grabbing nonintegrin. They express moderate levels of MHC class II and low levels of costimulatory molecules. The second population is CD11b(-/low) and lacks CD1a and DC-specific ICAM-3-grabbing nonintegrin but expresses high levels of MHC class II and costimulatory molecules. Expression of CCR7 on the CD11b(-/low) population and absence on the CD11b(+) cells further supports the view that these cells are mature and immature, respectively. Differentiation into mature and immature populations was not blocked by polymyxin B, an inhibitor of LPS. Neither population labeled for Langerin, E-cadherin, or CCR6 molecules expressed by Langerhans cells. Stimulation of 48-h cultured DC with LPS, CD40L, or poly(I:C) caused little increase in MHC or costimulatory molecule expression in the CD11b(-/low) DC but caused up-regulated expression in the CD11b(+) cells. In HIV-infected individuals, there was a marked decrease in the viability of cultured blood mDC, a failure to differentiate into the two populations described for normal donors, and an impaired ability to stimulate T cell proliferation.

Phosphotidylserine modulates the phenotype of mouse dentritic cells

Abstract Introduction: Dentritic cells (DCs) are the most potent professional antigen presenters and are required to initiate adaptive immunity. Immature DCs (iDC) capture antigen efficiently, but express low levels of MHC II and costimulatory molecules. DCs mature in response to danger signals, migrate into secondary lymphoid organs and activate T cells. Maturation is characterized by increase in MHC II and costimulatory molecule expression. Apoptotic cells inhibit DCs maturation. Phosphotidylserine (PS) is an anionic phospholipid that is limited to the inner leaflet of plasma membranes in live cells and externalized upon apoptosis. We examined the effects of PS on DCs maturation. Methods: Bone marrow derived mouse DCs were generated in the presence of granulocyte macrophage stimulating factor (GM-CSF), exposed to PS or phosphotidylcholine (PC) containing liposomes on day 5, and treated with lipopolisaccharide (LPS) on day 6. On day 7, DCs were analyzed by flow cytometry. Results: DCs exposed to media or PC and treated with LPS to induce maturation expressed significantly high levels of MHCII, CD80, CD86 and CD40 compared to iDCs. DCs exposed to PS expressed significantly less CD40 in response to LPS than control DCs. ∗MHC IICD80CD86CD40iDC17 (± 5.33)43 (± 24.3)14 (± 4.8)8 (± 7.4)mDC54 (± 28.2)100 (± 44.1)70 (± 28.6)∗57 (± 16.5)∗PC-DC55 (± 27.0)105 (± 46.2)73 (± 24.4)63 (± 17.5)∗PS-DC49 (± 29.5)102.6 (± 49.3)56 (± 8.6) † 41 (± 14.2)∗∗Statistically significant (p Conclusions: We demonstrated that PS modulates mouse DC maturation, specifically reducing CD40 expression. Diminished CD40 signaling is likely to lead to reduced IL-12 production and inhibition of Th1 responses. These findings suggest that PS, externalized by apoptotic cells and some tumor cells, could prevent autoimmunity and contribute to immune evasion by tumor.

Augmented Latex‐Specific IGE Antibody Response in BALB/c Mice Upon Concurrent Exposure to Natural Rubber Latex Proteins with Glutaraldehyde

Health care workers are exposed to numerous agents that are known to induce hypersensitivity‐mediated diseases. Yet, little is known regarding the role of coexposure to these agents on the development of hypersensitivity responses. The present studies were conducted to evaluate the immunomodulatory role of dermal exposure to glutaraldehyde (Glut) on the induction of IgE antibodies to natural rubber latex (NRL) proteins. Female BALB/c mice were dermally exposed to Glut (0.05–1 ppm; 0.1–1%) and nonammoniated latex (NAL; 25 µg) 5 days/week for up to 86 days. The NAL alone at concentrations up to 1% did not induce significantly elevated levels of total serum or latex specific IgE. In contrast, both total and NAL‐specific serum IgE were dose‐dependently (p < 0.01 and p < 0.05, respectively) elevated in mice concurrently exposed to 25 µg NAL and increasing concentrations of Glut up to 0.75 ppm. Further testing was performed to investigate the mechanism by which Glut augmented the latex‐specific response. Barrier integrity tests demonstrated that Glut did not induce sufficient disruption of the strateum corneum (less than 1% 3H20 penetration was observed in a guinea pig model) to allow for increased penetration of the latex proteins. However, co‐exposure to 25 µg NAL and 0.75 ppm Glut for 2 days as compared to the vehicle control was shown to induce a 15‐fold increase in MHC II positive Langerhans' cells in the epidermis. Additional experiments confirmed the upregulation of a Th2 response. Upon sacrifice following 86 days of exposure, animals exposed to 25 µg NAL and 0.75 ppm Glut demonstrated a significant increase (p < 0.01) in CD40 + (3.95 ± 0.38 × 106), B220 + (7.67 ± 1.18 × 106), and IgE + B220 + (3.28 ± 0.75 × 106) cells as compared to the vehicle control groups (2.29 ± 0.18 × 106, 3.31 ± 0.18 × 106, and 0.82 ± 0.15 × 106 cells), respectively. These studies demonstrate the potential for mixed exposures in the health care environment to modulate the development of IgE mediated responses to natural rubber latex proteins, underscoring the importance of environmental factors in the development of allergies to foreign antigens.

Vitamin D3 affects differentiation, maturation, and function of human monocyte-derived dendritic cells.

We studied the effects of 1alpha,25-dihydroxyvitamin D3 (1alpha, 25-(OH)2D3) on differentiation, maturation, and functions of dendritic cells (DC) differentiated from human monocytes in vitro in the presence of GM-CSF and IL-4 for 7 days. Recovery and morphology were not affected by 1alpha,25-(OH)2D3 up to 100 nM. DC differentiated in the presence of 10 nM 1alpha,25-(OH)2D3 (D3-DC) showed a marked decrease in the expression of CD1a, while CD14 remained elevated. Mannose receptor and CD32 were significantly increased, and this correlated with an enhancement of endocytic activity. Costimulatory molecules such as CD40 and CD86 were slightly decreased or nonsignificantly affected (CD80 and MHC II). However, after induction of DC maturation with LPS or incubation with CD40 ligand-transfected cells, D3-DC showed marginal increases in MHC I, MHC II, CD80, CD86, CD40, and CD83. The accessory cell function of D3-DC in classical MLR was also inhibited. Moreover, allogeneic T cells stimulated with D3-DC were poor responders in a second MLR to untreated DC from the same or an unrelated donor, thus indicating the onset of a nonspecific hyporesponsivity. In conclusion, our data suggest that 1alpha,25-(OH)2D3 may modulate the immune system, acting at the very first step of the immune response through the inhibition of DC differentiation and maturation into potent APC.

Platelet-Activating Factor Plays a Role in the Mechanism of Major Histocompatibilty Complex in T Lymphocytes

In recent studies, using a swine model of single lung transplantation, we demonstrated that IRI alone increased MHC II expression in the host's peripheral T lymphocytes. The inhibition of increased MHC II expression with TCV-309, a specific platelet-activating factor (PAF) antagonist suggested that PAF might play a role in the mechanism of increased MHC II expression. The purpose of the current study was two fold: 1) to investigate the mechanism of PAF-induced increased expression of MHC II in T lymphocytes, 2) to determine whether a specific PAF-antagonist, TCV-309, is capable of inhibiting the increased expression in an in vitro system. This study was subdivided, using four in vitro conditions: 1) purified resting T cells, 2) purified proliferating T cells, 3) PBL treated with PAF, and 4) PBL preincubated with TCV-309 and treated with PAF. The level of MHC II on T cells were measured by two color flow cytometry analysis (swine anti-CD3, MHC II-DR-β antibodies).Both MHC II intensity and the number of CD3+MHC+ T cells did not change in resting purified T cells once treated with PAF. Furthermore, MHC II intensity did not change in purified proliferating T cells treated with PAF. The number of CD3+MHC+ T cells, however, increased significantly (p<0.05) from day 1 to day 4 as compared with pre-treatment value (day 0) for purified proliferating T cells. Treatment of PBL with PAF (10−7 M) resulted in a significant (p<0.05) increase in MHC II expression from day 2 to day 4 post-treatment. The number of CD3+MHC+ T cells in PBL, however, did not change significantly upon treatment with PAF.The results of this study indicated that PAF did not have a direct effect on increased MHC II expression in resting or proliferating purified T lymphocytes. However, the mechanism of PAF-induced increased expression of MHC II in T cells may be via an indirect pathway involving accessory cells. TCV-309, a specific PAF receptor antagonist, is capable of inhibiting this PAF-induced increased expression of MHC II in T cells.

Changes in murine jejunal morphology evoked by the bacterial superantigen Staphylococcus aureus enterotoxin B are mediated by CD4+ T cells.

Bacterial superantigens (SAgs) are potent T-cell stimuli that have been implicated in the pathophysiology of autoimmune and inflammatory disease. We used Staphylococcus aureus enterotoxin B (SEB) as a model SAg to assess the effects of SAg exposure on gut form and cellularity. BALB/c, SCID (lacking T cells) and T-cell-reconstituted SCID mice were treated with SEB (5 or 100 microg intraperitoneally), and segments of the mid-jejunum were removed 4, 12, or 48 h later and processed for histochemical or immunocytochemical analysis of gut morphology and major histocompatibility complex class II (MHC II) expression and the enumeration of CD3+ T cells and goblet cells. Control mice received saline only. SEB treatment of BALB/c mice caused a time- and dose-dependent enteropathy that was characterized by reduced villus height, increased crypt depth, and a significant increase in MHC II expression. An increase in the number of CD3+ T cells was observed 48 h after exposure to 100 microg of SEB. Enteric structural alterations were not apparent in SEB-treated SCID mice compared to saline-treated SCID mice. In contrast, SEB challenge of SCID mice reconstituted with a mixed lymphocyte population or purified murine CD4+ T cells resulted in enteric histopathological changes reminiscent of those observed in SEB-treated BALB/c mice. These findings implicate CD4+ T cells in this SEB-induced enteropathy. Our results show that SAg immune activation causes significant changes in jejunal villus-crypt architecture and cellularity that are likely to impact on normal physiological processes. We speculate that the elevated MHC II expression and increased number of T cells could allow for enhanced immune responsiveness to other SAgs or environmental antigens.

128 ANTIGEN TRANSPORT AND PROCESSING ACROSS IFNγ-TREATED HT29-19A INTESTINAL CELLS.

The main function of Major Histocompatibility Complex class II (MHC II) molecules is to present antigenic peptides produced in antigen presenting cells to T lymphocytes. During intestinal inflammation, an overexpression of MHC II molecules in enterocytes is associated with an increased macromolecular transport. This study examines the relationship between MHC II expression and transepithelial transport and processing of an exogenous protein, horseradish peroxidase (HRP). Transwell-grown HT29-19A intestinal cells were treated on their basal or apical side with IFNγ (10 to 100 U/ml). After 48 hours, intracellular and surface expression of MHC II molecules as well as the lysosomal proteolytic activity (cathepsin B and D) were assessed. Transepithelial transport and processing of 3H-HRP were measured in HT29-19A cell monolayers mounted in Ussing chambers and ionic conductance (G) and 22Na and 14C-mannitol fluxes were used as markers of the paracellular pathway. HT29-19A cells did not express MHC II molecules in basal conditions. IFNγ induced an increase in MHC II molecules, but their transfer to the external membrane was small. The stimulation of MHC II molecules expression was not associated with a change in proteolytic cathepsin B or D activity. When IFNγ was placed on the basal compartment of intestinal monolayers, G, 22Na, 14C-mannitol and intact-HRP fluxes rose significantly (G: 7.2±0.2 vs 22.7±4.3 mS/cm2, 22Na: 63±2 vs 184±24 μg/h.cm2, 14C-Mannitol: 5.6±0.3 vs 25±5 μg/h.cm2, intact HRP: 31±4 vs 193±56 ng/h.cm2 in control vs IFNγ-treated cells) suggesting a paracellular leakage. Degraded-3H-HRP fluxes, reflecting a transcellular pathway, were also significantly increased in IFNγ-treated cells compared to controls. No such changes in permeability were observed when IFNγ was applied in the apical compartment. The chromatographic profiles of the tritiated peptides released in the basal compartment of Ussing chambers after transcytosis of 3H-HRP, were not strickingly different between control and IFNγ-treated intestinal cells. In the HT29-19A intestinal model stimulated by IFNγ, no direct relationship is found between MHC II molecules expression by enterocytes and the transepithelial transport and processing of exogenous antigens.

Interferon-gamma and tumor necrosis factor-alpha treatment of ex vivo human carcinoma cells potentiates their interaction with allogeneic lymphocytes.

Short-term exposure of ex vivo carcinoma and sarcoma cells to IFN-gamma and TNF-alpha induced or elevated to detectable levels the surface expression of MHC class I, class II, and ICAM-1 (CD54), but only rarely the B7 (CD80) molecules. The cytokine-treated tumor cells interacted more efficiently with allogeneic blood lymphocytes collected from healthy donors compared with untreated cells. This was demonstrated (1) by the induction of DNA synthesis and generation of cytotoxic activity in mixed cultures and (2) by the elevated susceptibility to the cytotoxic effectors. Although the cytokine-induced increase in MHC and ICAM-1 on the low-expressor tumors were probably important to the interaction with lymphocytes, it is likely that other properties were also induced that contributed to the phenomenon. This was indicated by the results obtained with several tumors that expressed indigenously high levels of these molecules but reacted with the allogeneic lymphocytes only or more efficiently after treatment with IFN-gamma and TNF-alpha. In these experiments B7 expression did not influence the efficiency of interactions between lymphocyte and tumor cells. The results also showed that, under the conditions used, the untreated tumor cells that did not activate allogeneic lymphocytes were sensitive to appropriately activated effectors. Thus the afferent and efferent arms of lymphocyte-tumor cell interactions appeared to have different requirements.

Immunogenicity and immunosensitivity of ex vivo human carcinomas: interferon gamma and tumour necrosis factor alpha treatment of tumour cells potentiates their interaction with autologous blood lymphocytes.

Human carcinoma cells vary appreciably in the expression of MHC class I, class II, ICAM-1 (CD54) and B7 (CD80) molecules. Short-term in vitro exposure of ex vivo carcinoma cells to interferon gamma and tumour necrosis factor alpha elevated/induced the surface expression of MHC class I, class II and ICAM-1, but only rarely of B7. We found that cytokine treatment elevated the cytotoxic susceptibility and the stimulatory potential of ex vivo tumour cells. This was demonstrated (a) by the increased frequency and elevated level of auto-tumour lysis and (b) by induction of DNA synthesis and generation of cytotoxic lymphocytes in autologous mixed lymphocyte/tumour cell culture (MLTC). The MHC class I and ICAM-1 molecules on the tumour cells were required for interaction with the lymphocytes as indicated by the inhibitory effect of specific mAb both in the stimulation and in the cytotoxic tests. While the cytokine-induced increases in MHC and ICAM-1 on the low-expression tumours were probably important for the modification of functional interaction with the autologous lymphocytes, it is likely that alterations in other properties of tumour cells were also induced which contributed to the phenomenon. This was indicated by the results obtained with several tumours, which expressed indigenously high levels of these molecules but activated the autologous lymphocytes only after cytokine treatment. In several experiments the untreated targets that did not activate the lymphocytes were sensitive to the cytotoxicity of the effectors activated in MLTC. The results show that the afferent and efferent arms of the immune response have different requirements for functional interactions between lymphocytes and tumour cells.

Resistance to NK Cell-Mediated Cytotoxicity (in K-562 Cells) does not Correlate with Class I MHC Antigen Levels

Natural Killer (NK) cells probably function as an early line of defense against virus-infected cells and tumor cells. In all cases, the killing by NK cell-mediated cytotoxicity (NK-CMC) is not MHC-restricted and the factors which determine the sensitivity to NK-CMC have not yet been identified. A positive correlation between resistance to NK-CMC and the level of class I MHC antigen (MHC I) expression on target cells has been reported in many studies, and in some cases a functional linkage between the two has been claimed. Several other studies have shown that there is no such correlation. By employing several experimental systems, we demonstrate here a lack of correlation between the level of MHC I and the sensitivity of K-562 cells to NK-CMC. Transfer of MHC I to MHC I-negative cells via vesicles had no effect on their resistance to NK-CMC. In addition, a decrease in resistance to NK-CMC and increase of MHC I levels was observed following target-cell membrane modulation by both application of cholesterol and hydrostatic pressure. Finally, no correlation between sensitivity to NK-CMC and MHC I expression was found in three sublines of K-562 cells. Since NK-CMC is a multistage process, it is concluded that components other than class I MHC antigens have a more prominent role in modulating the sensitivity of target cells to NK-CMC.

Phenotypic changes on hairy cells exposed in vitro to interferons: a quantitative FACS study

In vitro culture of hairy cells (HC) (five patients) with alpha IFN (100 U/ml) significantly enhanced MHC and CD22 antigen expression and reduced CD25, sIg and FMC7 positivity, together with consistent but not significant reductions in CD9, 19 and HC2. A sixth patient, who was refractory to the effects of alpha IFN in vivo, was also studied and none of these changes were seen in her HC when cultured with alpha IFN in vitro. gamma IFN (100 U/ml) produced a much less marked increase in MHC and reduced sIg. TPA (100 ng/ml) induced changes very similar to those observed with alpha IFN. alpha and gamma IFN had no effect on a range of other antigens in HCL (including CD23 and 38, and transferrin receptors) and also produced no significant antigenic changes in CLL cells. It is concluded that these findings are compatible with partial activation/maturation of HC by alpha IFN. It is suggested that the specificity of the phenotypic changes induced on HC by alpha IFN is a consequence of HC representing a stage of B-cell development at which alpha IFN has specific immunomodulatory effects.