Scandiatransplant acceptable mismatch program (STAMP) a bridge to transplanting highly immunized patients.

BACKGROUND: Highly immunized patients are a challenge for organ transplantation programs. One way of increasing the likelihood of transplantation in this group of patients is to expand the possible donations by defining acceptable HLA mismatches. In the Scandiatransplant Acceptable Mismatch Program (STAMP), a de-centralized approach has been implemented in 2009. AIMS: The program has been improved during the years from utilizing HLA-A, -B, -DR matching only to include typing of all deceased donors for HLA-A, -B, -C, -DRB1 and -DQB1. The calculation of a transplantability score (TS) has been introduced in order to take both HLA and AB0 into consideration resulting in a more realistic picture of the transplantability chance. MATERIALS AND METHODS: Patients were selected for eligibility and results of immunisation status were prepared in each of the 9 tissue typing laboratories, while access to the program is finally governed by a common steering group of immunologists and clinicians. RESULTS: In the period from March 2009 until February 2015, 96 patients were transplanted within this program. The mean recipient age was 49 years and 57% were females, 30% of the patients were first transplants and of these 93% were females. The majority of the patients had 2-5 HLA-A, -B. -DR mismatches. The allograft survival at 60 months was 79.1%. Applying the TS to the cohort confirmed that patients with a low TS score had longer waiting times. CONCLUSION: The program has matured during the years and now proves to be a valid approach for transplanting highly immunized patients.

Prevalence of venous thromboembolism at diagnosis of upper gastrointestinal cancer.

BACKGROUND: Venous thromboembolism (VTE) in patients with upper gastrointestinal (GI) cancer increases morbidity and mortality. This study aimed to determine the prevalence of VTE at diagnosis of upper GI cancer. METHODS: Patients admitted between February 2008 and February 2011 with upper GI cancer (pancreatic, extrahepatic biliary, lower oesophageal, gastro-oesophageal junction or gastric cancer) were investigated in a cross-sectional cohort study. At cancer diagnosis, all patients were examined for deep vein thrombosis (DVT) by means of bilateral compression ultrasonography. From February 2009 and onwards, computed tomographic pulmonary angiography (CTPA) was also performed for the diagnosis of pulmonary embolism (PE). RESULTS: Some 250 patients had ultrasonography; CTPA was performed in 143 patients on admission. DVT was detected in 13 (5·2 per cent) of the 250 patients, eight (3·2 per cent) of whom were asymptomatic. DVT was correlated with tumour location in the pancreaticobiliary tract (odds ratio (OR) 6·27, 95 per cent confidence interval 1·18 to 33·38; P = 0·031) and tumour stage IV (OR 19·34, 2·33 to 160·70; P = 0·006). PE was detected in 11 (7·7 per cent) of 143 patients, eight (5·6 per cent) of whom were asymptomatic. PE embolism was also significantly more common in patients with pancreaticobiliary tract cancer (OR 7·81, 1·28 to 47·62; P = 0·026) and in those with stage IV disease (OR 17·19, 1·83 to 161·50; P = 0·013). CONCLUSION: The prevalence of VTE at cancer diagnosis was significantly higher in patients with pancreaticobiliary tract cancer than in those with other forms of upper GI cancer, and in patients with advanced cancer stage.

Upregulated baseline plasma CCL19 and CCR7 cell-surface expression on monocytes in early rheumatoid arthritis normalized during treatment and CCL19 correlated with radiographic progression.

OBJECTIVES: The aim of this study was to measure, in early rheumatoid arthritis (RA) patients, the concentration of CC-chemokine ligand 19 (CCL19) in plasma and the cell-surface expression of CC-chemokine receptor 7 (CCR7) on circulating monocytes and CD4+ T lymphocytes and to analyse correlations with disease activity and 5-year radiographic progression. METHOD: In disease-modifying anti-rheumatic drug (DMARD)-naïve RA patients (disease duration < 6 months), we measured plasma CCL19 by enzyme-linked immunosorbent assay (ELISA) (n = 160) and CCR7 cell-surface expression on monocytes and CD4+ T lymphocytes by flow cytometry (n = 40) at baseline and after 1 year of treatment with methotrexate (MTX) or methotrexate+cyclosporin A (MTX/CyA). Radiographic progression was scored by the van der Heijde-modified Total Sharp Score (TSS) from 0 to 5 years. RESULTS: Increased baseline CCL19 (median 85 pg/mL, range 31-1008 pg/mL, p = 0.01) decreased after 1 year (median 31 pg/mL, range 31-1030 pg/mL, p < 0.001) and 5 years (median 31 pg/mL, range 31-247 pg/mL, p < 0.001) to a level below the controls (n = 45) (median 60 pg/mL, range 31-152 pg/mL). Baseline plasma CCL19 levels [p = 0.011, 95% confidence interval (CI) 0.0030-0.0176], anti-cyclic citrullinated peptide (anti-CCP) antibody status (p = 0.002, 95% CI 0.61-2.38), and TSS > 0 at baseline (p < 0.001, 95% CI 1.21-3.16) were independent predictors of 5-year radiographic progression evaluated by multiple logistic regression in contrast to never smoked, C-reactive protein (CRP), gender, age, number of tender (NTJ) and swollen joints (NSJ), and 28-joint Disease Activity Score (DAS28). Increased CCR7 expression on monocytes (p = 0.008) correlated to CRP (p = 0.006, r = 0.52) and normalized (n = 15) after 1 year (p = 0.02). CONCLUSIONS: In DMARD-naïve RA patients, CCL19 plasma level and CCR7 surface expression on monocytes were upregulated and normalized after 1 year of treatment. Increased baseline plasma CCL19 level, anti-CCP antibody status, and TSS > 0 at baseline correlated independently with 5-year radiographic progression.

Case report: binding of a clinically relevant human leukocyte antigen-DQα-specific antibody in a kidney graft recipient is inhibited by donor-type human leukocyte antigen-DQß chain.

In this case report, we have found what may be an immunization with donor-specific human leukocyte antigen (HLA)-DQα in combination with recipient-specific HLA-DQß. A renal allograft recipient who did not comply with immunosuppressive therapy during pregnancy had graft failure 23 months posttransplantation with biopsy-proven humoral and cellular rejection. Sera were tested in a Luminex-based single-antigen bead assay. We compared Luminex reactivity with the degree of eplet mismatching between the recipient's own HLA-DQ chains and the HLA-DQ chains bound to the Luminex beads. Eplet calculations were done with the HLAMatchmaker. HLA-DQ similarities were compared further by dissimilarity scoring in HistoCheck. We observed that Luminex beads with donor-type HLA-DQα and HLA-DQß bound less antibody than beads with donor-type HLA-DQα combined with recipient HLA-DQß. In HLAMatchmaker, we identified all eplet mismatches between donor and recipient HLA-DQ. Next, we counted how many of these eplets were represented on the various Luminex beads. We found that antibody binding to the bead increased with the number of such mismatches for HLA-DQα. Surprisingly, antibody binding decreased as the number of eplet mismatches for HLA-DQß increased, from a mean fluorescence intensity (MFI) value of 18,800 for no mismatched eplets to approximately 10,000 for 12 mismatched eplets. These findings were confirmed by comparing antibody binding with the structural dissimilarity score between the recipient HLA-DQ type and the HLA-DQ bound to the Luminex beads. In this patient, clinically relevant antibodies bound strongly to donor-like HLA-DQα chains when combined with recipient-like HLA-DQß. HLA-DQß chains more similar to those of the donor reduced the binding of donor- specific HLA-DQα antibody.

In active chronic rheumatoid arthritis, dipeptidyl peptidase IV density is increased on monocytes and CD4(+) T lymphocytes.

The effect of low-dose methotrexate (MTX) treatment on the CD26 density on circulating monocytes and CD4(+) T lymphocytes or levels of soluble CD26 (sCD26) has not yet been described in rheumatoid arthritis (RA). While CD26 in T lymphocytes is involved in the activation and proliferation of T lymphocytes, little is known of the role of CD26 in monocytes as it has only recently been localized to monocytes. We analysed the CD26 density by flow cytometry and levels of sCD26 in plasma before initiation of MTX treatment and 12 weeks later. This was done on 34 RA patients fulfilling the 1987 American College of Rheumatology (ACR) criteria followed for 16 weeks after starting MTX treatment. CD26 density on monocytes was increased in RA patients compared with healthy controls before MTX treatment (P < 0.01). After 12 weeks of MTX treatment, the CD26 density on monocytes decreased significantly in the ACR-50% group (P = 0.03), but not in the ACR-20% and the non-responder group (P = 0.15 and 0.87). The increased CD26 density on CD4(+) T lymphocytes (P < 0.01) was unaffected by the reduction in disease activity in relation to MTX treatment. The percentage of monocytes and CD4(+) T lymphocytes among peripheral blood circulating mononuclear cells did not change during MTX treatment. No effect of MTX treatment was observed on the plasma levels of sCD26. Active chronic RA is characterized by enhanced CD26 density on circulating monocytes and CD4(+) T lymphocytes. MTX treatment decreased CD26 density on monocytes in the ACR-50% responder group and was associated with decreased disease activity. The enhanced CD26 density on CD4(+) T lymphocytes was uninfluenced by MTX treatment.

Commonly used reference genes are actively regulated in in vitro stimulated lymphocytes.

Quantitative polymerase chain reaction (Q-PCR) studies of urine sediments have demonstrated an increased expression of cytotoxin genes during episodes of acute rejection of renal allografts. To compensate for differences in initial sample size and cDNA preparation, standard Q-PCR experiments involve normalization to a reference gene. Although stable expression of reference genes is a prerequisite for any Q-PCR analysis, commonly used reference genes have demonstrated a varying expression across tissues and various stimuli. In this study, cellular expression of several reference genes was investigated in a mixed lymphocyte reaction as a model of gene expression during alloreactive T-lymphocyte activation and acute rejection. Gene expression was quantified using Q-PCR, normalized to cell counts obtained by DNA quantification and corrected for cell polyploidy using flow cytometry. Examined reference genes were 18S rRNA, beta-actin (ACTB), hydroxymethylbilane synthase (HMBS), hypoxanthine phosphoribosyltransferase (HPRT1) and peptidylprolyle isomerase B (PPIB). This study also examined two novel T-lymphocyte-specific reference genes: CD3E and CD8B. HMBS and HPRT were 18.8- and 7.4-fold upregulated, respectively, ACTB was 5.3-fold upregulated, PPIB was 3.2-fold upregulated while 18S rRNA remained stably expressed. The T-lymphocyte-specific reference gene CD3E remained stable while CD8B was upregulated 2.3-fold. In conclusion, several commonly used reference genes were actively regulated during alloreactive T-lymphocyte activation. Additionally, we introduce two stable T-lymphocyte-specific reference genes that might be useful in a Q-PCR analysis of T-lymphocyte-specific cytotoxin genes in urine sediments, as they overcome the contribution of reference gene mRNA from cells irrelevant for diagnosis.

Differential effect of methotrexate on the increased CCR2 density on circulating CD4 T lymphocytes and monocytes in active chronic rheumatoid arthritis, with a down regulation only on monocytes in responders.

OBJECTIVES: To evaluate the effect of orally administered methotrexate (MTX) on the density of CC chemokine receptor 2 (CCR2) and CXC chemokine receptor 3 (CXCR3) on circulating monocytes, and the coexpression of CXCR3 and CCR2 on CD4 T lymphocytes in patients with active chronic rheumatoid arthritis. METHODS: All 34 patients with rheumatoid arthritis fulfilled the 1987 American Rheumatism Association criteria and were followed for 16 weeks after starting MTX. Peripheral blood mononuclear cells were analysed for CCR2 and CXCR3 density by three-colour flow cytometry before initiation of MTX and at week 12. RESULTS: 22 (65%) patients were non-responders, 12 (35%) patients responded to MTX by American College of Rheumatology (ACR)20% criteria, and 8 (24%) of these patients responded by ACR50%. In patients with active rheumatoid arthritis before starting MTX, CCR2 density on circulating monocytes, CD4(+) CXCR3(+) and CD4(+) CXCR3(-) T lymphocytes was increased compared with controls. During 12 weeks of MTX treatment, the CCR2 density on monocytes decreased significantly in the ACR50% group but not in the ACR20% and non-responder groups. The increased CCR2 density on CD4(+) CXCR3(+) and CD4(+) CXCR3(-) T lymphocytes was unaffected by the reduction in disease activity measured in relation to MTX treatment. The percentage of both monocytes and CD4(+) CXCR3(+) and CD4+ CXCR3(-) T lymphocytes among the peripheral circulating mononuclear cells did not change during MTX treatment. CONCLUSIONS: Active chronic rheumatoid arthritis is characterised by enhanced CCR2 density on circulating monocytes and CD4(+) CXCR3(+) and CD4(+) CXCR3(-) T lymphocytes. During MTX treatment, a decrease in CCR2 density on monocytes in the ACR50% responder group was associated with decreased disease activity. The increased CCR2 density on CD4(+) CXCR3(+) and CD4(+) CXCR3(-) T lymphocytes was uninfluenced by MTX and disease activity.

Serum concentration of the growth medium markedly affects monocyte-derived dendritic cells' phenotype, cytokine production profile and capacities to stimulate in MLR.

We have investigated how the maturation of monocyte-derived dendritic cells (Mo-DC) is affected by the serum concentration of the culture medium. Day 6 DC cultured in 1% human serum were a heterogeneous population of CD1a(-) and CD1a(+) DC that were separated by flow sorting. In contrast, Mo-DC generated in 10% human serum formed a homogenous population of CD1a(-) cells. Other phenotypically immature characteristics also varied, and three subsets were still distinguishable upon maturation in LPS. Furthermore, CD1a(-) DC and CD1a(+) DC from 1% culture conditions were excellent stimulators in MLR, while DC cultured in 10% serum were poor stimulators. Similarly, different cytokine profiles of the three subsets were identified. DC cultured in 1% serum had low expression of interleukin-12 (IL-12) p40 and IL-10 mRNA at day 6. Upon maturation, expression of IL-12 p40 mRNA was upregulated in CD1a(+) DC, whereas the level remained relatively low in CD1a(-) DC. In contrast, DC cultured in 10% had high levels of IL-10 mRNA at day 6 that was downregulated upon maturation. We conclude that the differentiation of monocytes into DC is significantly influenced by the serum concentration of the growth medium with effects on phenotype, cytokine profile and stimulatory activity.

Decreased urokinase receptor expression on granulocytes in HIV-infected patients.

Pericellular proteolysis initiated by receptor-bound urokinase-type plasminogen activator (uPA) is considered important for directed migration of granulocytes to inflammatory sites. Using flow cytometry and whole-cell binding of radiolabelled-uPA, we found a high level of uPA-receptor (uPAR) expression in granulocytes (3.9 x 104 +/- 0.9 x 104 sites/cell). Modulation of uPAR expression was assessed in the presence of chemoattractant gradients. Our findings demonstrate that interleukin (IL)-8, leukotriene B4(LTB4) and formyl-methionyl-leucyl-phenylalanine (f MLP) caused a dose-dependent upregulation of uPAR on granulocytes in healthy controls. Modulation of uPAR expression is known to regulate chemotactic response. As determined by flow cytometry, uPAR expression by granulocytes from human immunodeficiency virus (HIV)-infected patients was distinctly lower than that of healthy control cells (P < 0.001). However, upregulation of uPAR in response to chemoattractants was similar to that observed in healthy controls. In HIV-infected patients, the uPAR expression on granulocytes correlated (P < 0.001, n = 10) with the number of CD4+ blood cells. In contrast, the expression of IL-8 receptor, CD11b, CD18 and CD62 was not significantly altered in HIV-patients compared with healthy controls.

Effects of subcutaneous IL-2 therapy on telomere lengths in PBMC in HIV-infected patients.

In this study we investigated the effect of interleukin-2 (IL-2) on mean terminal restriction fragment (TRF) lengths in peripheral blood mononuclear cells (PBMC). Ten human immunodeficiency virus (HIV)-infected individuals were included and IL-2 was administered subcutaneously with 3 x 106 IU three times a week for 24 weeks. Mean TRF length was decreased on average by 267 bp at week 4 (P = 0.03) and 286 bp at week 8 (P = 0.09). Individual TRF changes at weeks 12, 16, 20 and 24 were highly variable. However, in the 12 weeks following therapy, TRF lengths generally increased reaching baseline levels by the end of the study. At baseline, mean TRF lengths were positively correlated to the ratio of naïve and memory phenotype within both CD4+ and CD8+ cells. This study shows that IL-2 treatment induces transient shortened mean TRF lengths in PBMC from HIV-infected individuals, indicating that IL-2 enhances the lymphocyte count by peripheral proliferation or recruitment of memory T cells into the blood.

In vitro migration of mononuclear cells towards synovial fluid and plasma from rheumatoid arthritis patients correlates to RANTES synovial fluid levels and to clinical pain parameters.

OBJECTIVE: To evaluate in vitro migration of mononuclear cells towards synovial fluid (SF) and plasma in relation to RANTES synovial fluid levels and clinical disease activity. METHODS: 31 RA patients with synovitis in one knee were included. Modified Boyden chamber technique was used to determine a migratory index defined as: 'In vitro migrating cells towards SF' divided by 'In vitro migrating cells towards plasma'. RANTES was quantified by ELISA. Disease activity was assessed by the swollen joint count, the Ritchie articular index (RAI), global assessment, pain on VAS, HAQ, ESR and CRP. RESULTS: A positive significant correlation was found between the migratory index and the RANTES levels in SF (r=0.48, p=0.006), the RAI (r=0.56, p=0.0001) and pain on VAS (r=0.43, p=0.04). The in vitro migration could be inhibited in 3 of 4 SF samples by neutralising antibodies towards RANTES (12-18%). CONCLUSION: The migratory index correlate to SF levels of RANTES and parameters for joint pain.

Migration of mononuclear cells in the modified Boyden chamber as evaluated by DNA quantification and flow cytometry.

In vitro migration of mononuclear cells in the modified Boyden chamber was evaluated using flow cytometry and DNA quantification (Hoechst 33258) of all adherent and nonadherent cells. The effects of different membrane pore sizes, cell concentrations and incubation times were studied. Pore sizes of 3 and 5 microm resulted in a reduction in the number of nonadherent cells compared with a pore size of 8 microm. Reducing the incubation time from 60 to 40 and 20 min resulted in too few migrating monocytes for analysis by flow cytometry. Flow cytometry showed that both monocytes and lymphocytes migrated and adhered to the membrane when using peripheral blood mononuclear cells (PBMC) to study monocyte migration. Migration of lymphocytes under these conditions is a novel observation. A substantial number of migrated cells could be identified by flow cytometry and quantified by DNA measurement as nonadherent below the membrane. Samples of synovial fluid (n = 49) and plasma (n = 133) as chemoattractants analysed in triplicate resulted in mean coefficient of variation (CV) values of 11 and 9%, respectively. Variation from assay to assay on the same day, using N-formyl-methionyl-leucyl-phenylanine (fMLP) 10(-7) M as chemoattractant resulted in a CV of 13%. Day-to-day variation, using fMLP 10(-7) M as chemoattractant and the same well on three different days, resulted in a CV of 21%. These results were obtained using a pore size of 5 microm, a PBMC concentration of 3 x 10(6)/ml and 60 min of incubation. The combination of DNA quantification and flow cytometry thus allowed characterization and quantification of subsets of migrating adherent as well as nonadherent mononuclear cells.

Subcutaneous interleukin-2 in combination with anti-retroviral therapy for treatment of HIV-1-infected subjects.

A total of 11 HIV-1 positive patients, with CD4+ cell counts between 200 and 500/microl, who were in stable anti-retroviral therapy, were treated with subcutaneous recombinant human IL-2 thrice weekly administered on an out-patient basis in a dose-escalating manner. Subcutaneous IL-2 was well tolerated and associated with only mild to moderate constitutional symptoms and local inflammation at the injection site. CD4+ cell count increased from 404 +/- 48/microl at baseline to 639 +/- 88/microl at week 6, with proportionate increases in naive cells and memory cells. Increased doses of IL-2 were then needed to sustain the number of CD4+ cells. After discontinuation of IL-2 treatment, CD4+ cell count returned to baseline levels. IL-2 induced a reduction in the percentage of CD8+ CD38+ and CD8+ HLA-DR+ cells, an increase in the fraction of CD8+ CD25+ and CD8+ CD122+, and an elevation in the number of NK-cells. IL-2 did not induce any clinically significant change in plasma HIV-RNA. In conclusion, IL-2 can safely be administered subcutaneously on an out-patient basis to HIV-infected individuals with CD4+ cell counts from 200/microl to 500/microl and with some improvement in immunological abnormalities. Continuous therapy, however, seems to result in the development of tachyphylaxia.

Effects of subcutaneous interleukin-2 therapy on phenotype and function of peripheral blood mononuclear cells in human immunodeficiency virus infected patients.

In the context of clinical therapy with recombinant human interleukin-2 (IL-2), we monitored immunological alteration in 10 human immunodeficiency virus type-I (HIV-1)-infected individuals, on stable antiretroviral therapy, who had a CD4+ cell count between 200 and 500 cells/mm3. Subcutaneous IL-2 was prescribed thrice weekly (at a dose of 3 x 10(6) IU) for 24 weeks and the patients were followed-up for 32 weeks. IL-2 treatment induced an increase in the CD4+ percentage (P<0.001) and CD4+ cell count (P<0.009). Furthermore. natural killer (NK) cell activity was increased (P<0.001) at week 8 of treatment, whereas lymphokine-activated killer (LAK) cell activity showed a transient, nonsignificant increase at week 8 and was reduced (P<0.001) at 32 weeks. However, the cytotoxic T-lymphocyte (CTL) activity decreased against HIV antigens, and the proliferative response to Candida, IL-2 and phytohaemagglutinin (PHA) declined during the first 8 weeks (P<0.05) and returned to baseline levels after 32 weeks. The HIV RNA level did not change during IL-2 therapy; however, after 8 weeks of follow-up a significant increase (P<0.001) in viral load was observed. In

Effects of methotrexate, sulphasalazine and aurothiomalate on polymorphonuclear leucocytes in rheumatoid arthritis.

The aim of the study was to evaluate the effects of treatment with methotrexate, sulphasalazine and aurothiomalate on polymorphonuclear leucocytes (PMN) in rheumatoid arthritis (RA). Circulating PMNs from 58 RA patients treated with either methotrexate (n = 27), sulphasalazine (n = 16) or aurothiomalate (n = 15) were assayed for their chemotactic capacity and generation of superoxide anions. The expression of CD18/CD11b was measured for 17 RA patients treated with methotrexate. Chemotaxis and generation of superoxide anions were not affected by any of the three drugs. We found no difference in the expression of CD18/CD11b in RA patients treated with methotrexate compared to healthy subjects. Further methotrexate and aurothiomalate did not in vitro alter chemotaxis, generation of superoxide anion or expression of CD18/CD11b on normal PMNs. In conclusion we found no evidence that the effect of methotrexate, aurothiomalate or sulphasalazine in RA can be explained by modulation of chemotactic ability or superoxide anion generation of peripheral circulating PMNs.

Internalization, lysosomal degradation and new synthesis of surface membrane CD4 in phorbol ester-activated T-lymphocytes and U-937 cells.

Protein kinase C activating phorbol esters downregulated membrane CD4 by endocytosis in U-937 and human T-cells. Half-time for internalization (approximately 15 min at 50 ng/ml PMA) was determined by FACS. CD4-bound 125I-labeled anti-CD4 mAb was rapidly degraded in PMA-activated cells, whereas degradation was low in resting cells. Endocytosis and/or degradation of anti-CD4 mAb was suppressed by H7, and by inhibitors of membrane traffic (Monensin) and lysosome function (methylamine, chloroquine). Immunocytochemistry localized CD4 to the surface of unstimulated T-cells. Upon PMA stimulation occasional labeling was seen in endosomes but whole cell CD4 decreased dramatically. However, methylamine-treated PMA blasts showed accumulation of CD4 in lysosomes and accordingly, pulse-chase experiments in biolabeled cell cultures suggested a manifest reduction of CD4 half-life in response to PMA. Despite their low surface CD4 density, PMA blasts exhibited uptake and accelerated degradation of anti-CD4 mAb. Also, inhibitors of protein synthesis enhanced the PMA-induced downregulation, and membrane CD4 reappeared on fully activated as well as unstimulated cells treated with trypsin. Ongoing CD4 synthesis in activated cells was further evidenced by metabolic labeling and Northern blot analysis demonstrating unaltered or slightly increased CD4 protein and mRNA levels resulting from PMA. Our findings demonstrate that phorbol esters downregulate the cellular CD4 pool by endocytosis and subsequent lysosomal degradation of membrane CD4. Transport of CD4 to the cell surface and CD4 synthesis is unaffected by activation.

Surface membrane CD4 turnover in phorbol ester stimulated T-lymphocytes. Evidence of degradation and increased synthesis.

Down-regulation of surface membrane CD4 (smCD4) in phorbol ester stimulated T-cells resulted from internalization. Internalization (T1/2 = 15 min at 50 ng PMA/ml) was followed by degradation of CD4-bound antibodies. Degradation in unstimulated T-cells was comparatively insignificant. Release of degradation products was PMA dose-dependent and could be inhibited by methylamine. Uptake and degradation continued after maximal down-regulation of surface membrane CD4, and methylamine did not inhibit reappearance of smCD4 antigens. Metabolic labelling of T-cells further showed that ongoing synthesis rather than recycling contributed to an accelerated smCD4 turnover in activated cells.

Cellular targets and receptors for interleukin-6. I. In vivo and in vitro uptake of IL-6 in liver and hepatocytes.

Interleukin-6 (IL-6) is a potent stimulator of the hepatic synthesis of acute-phase proteins. 125I-labelled IL-6 disappeared from the blood of rats with an overall half-time of about 1.5 min; 41% of the injected tracer dose was recovered in the liver by 15 min. The clearance was biphasic. The simultaneous injection of tracer and an excess of unlabelled IL-6 eliminated the initial rapid phase, and reduced the hepatic uptake to 14%. Light microscopic autoradiography showed 5% of the grains over non-hepatocytes, and 80% over hepatocytes, accumulating in areas around the bile canaliculi. Thereafter, degradation products accumulated in the bile. At 4 degrees C, isolated rat hepatocytes bound IL-6 with an apparent Kd of 39 pmol l-1 to a uniform class of 4500 receptors per cell with an apparent molar mass of 115-120 kg mol-1. The HepG2 human hepatocellular cell line bound IL-6 with an apparent Kd of 21 pmol l-1 to a uniform class of 1200 receptors per cell with an apparent molar mass of 155-160 kg mol-1. At 37 degrees C, both cell types endocytosed the bound ligand slowly, and degradation products appeared in the medium after a relatively long lag period (40 min in hepatocytes and 1 h in HepG2 cells).