Kinetics of leukocyte CD11b and CD64 expression in severe sepsis and non-infectious critical care patients.

BACKGROUND: Leukocyte surface molecules may improve sepsis diagnostics. Our aim was to study whether monocyte and neutrophil CD11b and CD64 expression differs between patients with severe sepsis (including septic shock) and intensive care unit (ICU) controls, and also to investigate the expression kinetics in patient groups. METHODS: Monocyte and neutrophil CD11b and CD64 expression was analyzed in 27 patients with severe sepsis, 7 off-pump coronary artery bypass (OPCAB) patients, and 8 ICU patients without systemic inflammation in the beginning of the treatment using quantitative flow cytometry. Blood samples were collected within 48 h of the beginning of severe sepsis, at admission to the ICU for non-systemic inflammatory response syndrome patients, and on the day of surgery before the skin incision for OPCAB patients, and on 2 consecutive days for all patients. Ten healthy individuals served as controls. RESULTS: Monocyte and neutrophil CD11b and neutrophil CD64 expression was higher in severe sepsis patients compared with the other groups (P < 0.05). In severe sepsis, the expression decreased over time (P < 0.05). In OPCAB patients, the monocyte and neutrophil CD64 expression increased after surgery (P < 0.05). Neutrophil CD64 expression had the highest and statistically significant area under curves (AUC) values for identification of severe sepsis during 3 consecutive days, the highest AUC being 0.990 on D0. CONCLUSION: Neutrophil CD64 as well as neutrophil and monocyte CD11b expressions were highest in severe sepsis compared with non-infectious conditions, and thus analyses of their expression may be promising approach for sepsis diagnosis in ICU population.

The effects of PMA and TFP and alterations in intracellular pH and calcium concentration on the membrane associations of phospholipid-binding proteins fodrin, protein kinase C and annexin II in cultured MDCK cells.

Annexin II, alpha-fodrin and protein kinase C (PKC) are associated with the cytoplasmic surface of the plasma membranes. When assayed with liposomes, they show affinity for acidic phospholipids and bind calcium ions. They also respond to or participate in cell signal transduction by altered membrane binding properties. In the present work we have studied the properties of these proteins in epithelial MDCK cells in response to elevated intracellular calcium ion concentration, lowered pH, treatment with tumor promoter phorbol myristoyl acetate (PMA) and calmodulin inhibitor trifluoperazine (TFP). In untreated polarized MDCK cells annexin II was seen both along the lateral walls and membranes of intracellular vesicles, fodrin was located along the lateral walls, whereas PKC was seen in the cytoplasm. There was no observable translocation of these proteins upon elevation of the intracellular calcium concentration using a calcium ionophore A23187. On the other hand, treatment with TFP led to a release of annexin II from the plasma membranes which was accompanied by a transient peak in the intracellular calcium. Treatment with PMA led to a loss of the cubic form of the cells, a slight elevation in the intracellular calcium concentration and a drop in the intracellular pH. Simultaneously fodrin was released from the lateral walls, but still remained insoluble in Triton X-100, PKC became associated with the intracellular membranes and fibers, whereas annexin II remained along the lateral walls. These changes could be prevented by clamping the intracellular pH neutral during PMA treatment. On the other hand, lowering of intracellular pH below 6.5 with the nigericin treatment led to a similar translocation of fodrin and PKC as PMA. This suggests that the protein redistribution is caused by cytoplasmic acidification and is due to an increased hydrophobicity and enhanced protonation of lipids and proteins. In contrast, no changes were seen in the annexin II distribution in response to altered pH. Hence, its release by TFP is presumably due to changes in the cationic properties of the inner phase of the plasma membrane. Thus, proteins which show similar binding properties with liposomes show different characteristics in their association with the intracellular membranes.

Effect of PMA on the integrity of the membrane skeleton and morphology of epithelial MDCK cells is dependent on the activity of amiloride-sensitive ion transporters and membrane potential.

The signaling pathways from an activation of protein kinase C (PKC) by phorbol myristate acetate (PMA) to the rearrangement of actin-based cytoskeleton and membrane skeleton of epithelial MDCK cells were studied by visualizing the cytoskeletal organization with immunofluorescence microscopy and by measuring intracellular pH, sodium ion concentration and membrane potential with the aid of fluorescent intracellular indicators. Upon PMA treatment the MDCK cells lost their cubic shape and acquired a spindle-like morphology. The stress fibers were depolymerized, and fodrin, the main component of the membrane skeleton, was released from the lateral walls to the cytosol. These changes were accompanied by depolarization of the cells, decrease in the intracellular pH and sodium ion concentration. In order to test the mutual correlation between the PMA-induced alterations we treated the cells with PMA in the presence of channel inhibitors or ionophores and in defined media. The effects of PMA on the membrane skeleton and morphology could be reversed in media lacking Na+ or K+ ions or by hyperpolarizing agents, dimethylamiloride and valinomycin, suggesting that the effects of PMA on the cytoskeleton were dependent on the ion gradients and membrane potential across the cell membrane. Moreover, the morphological changes and instabilization of the membrane skeleton of MDCK cells took place spontaneously without PMA in depolarizing conditions, in potassium gluconate buffer. We suggest that the membrane potential across the cell membrane of MDCK cells together with the activity of amiloride-sensitive cation transporters transmits signals in the protein kinase C (PKC) pathway leading from activation of PKC to fibroblast-like morphology and cytoplasmic localization of membrane skeleton components, features characteristic for cancer cells.

Glutamate-induced changes in the DNA-binding complexes of transcription factor YY1 in cultured hippocampal and cerebellar granule cells.

Transcription factor YY1 is a zinc finger protein which can interact and form complexes with several proteins. Depending on its binding partners, YY1 can either activate or repress the transcription of several genes. We have studied whether the regulation of YY1 is affected by glutamate-induced excitotoxic insult in cultured primary hippocampal and cerebellar granule cells. Using electrophoretic mobility shift (EMSA) technique, we observed that glutamate treatment induced a dramatic increase in the YY1-binding activity of the smallest of three complexes both in the hippocampal and cerebellar granule cells. Two larger complexes disappeared after glutamate treatment in cerebellar granule cells. Supershift assays with specific YY1 antibody showed that all three complexes contained YY1 protein. Western blot assays did not show any changes in the nuclear levels of YY1 protein. Our results show that excitotoxic treatment affects the regulation of YY1 transcription factor and suggest that YY1 is a significant nuclear target for stress-related signaling pathways in neuronal degeneration.

Distinct mode of apoptosis induced by genotoxic agent etoposide and serum withdrawal in neuroblastoma cells.

Here we compared the features of apoptosis induced by DNA-damaging agent, etoposide, and by withdrawal of the growth factors in NB 2a neuroblastoma cells. We showed that serum deprivation and etoposide induced a distinct pattern of regulation of c-Fos, c-Jun and p53 protein levels, as well as the differential changes in DNA-binding activity of AP-1 and NF-kappaB transcription factors. The late phase of apoptesis induced by serum withdrawal was associated with disintegration of nuclear DNA both into high molecular weight (HMW) and oligonucleosomal DNA fragments, whereas etoposide induced the formation of HMW-DNA fragments without internucleosomal DNA cleavage. Incubation of etoposide-treated cells without serum resulted in an additive effect on the pattern of DNA fragmentation. Differences in DNA fragmentation profiles induced by serum withdrawal and etoposide in NB 2a cells were reproducible in nonproliferating cerebellar granule cells and also in a cell free system assay after treatment of isolated normal nuclei with cytosolic extracts prepared from serum-deprived or etoposide-treated cells. Both HMW and oligonucleosomal DNA fragmentation in serum-deprived cells was inhibited by aurintricarboxylic acid and was completely abrogated by cycloheximide. In contrast, DNA fragmentation in etoposide-treated cells was insensitive to the inhibitory effect of aurintricarboxylic acid, and was not prevented by cycloheximide. Our results indicate that in NB 2a neuroblastoma cells etoposide and serum withdrawal induce a distinct mode of apoptosis which is associated with a distinct pattern of regulation of immediately early response genes in the early phase, and with recruitment of different mechanisms for DNA disintegration in the late phase of apoptosis.

Distinct mechanisms underlay DNA disintegration during apoptosis induced by genotoxic and nongenotoxic agents in neuroblastoma cells.

Exposure of mouse NB-2a neuroblastoma cells to genotoxic (etoposide or cytosine arabinoside) or nongenotoxic challenges (serum deprivation or okadaic acid) resulted in progressive cell death with biochemical and morphological characteristics typical of apoptosis. Apoptotic cell death induced by nongenotoxic agents was associated with the disintegration of nuclear DNA into high molecular weight (HMW) and oligonucleosomal-DNA fragments, while the formation of HMW-DNA fragments, but not oligonucleosomal-DNA ladder accompanied apoptosis induced by genotoxic agents. Combination of genotoxic and nongenotoxic insults, i.e. incubation of etoposide-treated cells in the serum-free medium, resulted in an additive effect on the profile of DNA disintegration, which involved both HMW fragmentation pattern as in etoposide alone treated cells and the oligonucleosomal-DNA ladder observed with serum-deprived cells. On the other hand, incubation of serum-deprived cells in the presence of Zn2+-ions led to the abrogation of internucleosomal DNA fragmentation but accumulation of HMW-DNA fragments. Differences in the pattern of DNA fragmentation were reproducible in a cell free apoptotic system after treatment of isolated normal nuclei with cytosolic extracts prepared from the cells treated with genotoxic or nogenotoxic apoptotic inducers. Cell free experiments also revealed that activities responsible for the formation of HMW- and oligonucleosomal-DNA fragments are separable in cytosolic extract prepared from the serum-deprived cells. Finally, DNA fragmentation induced by nongenotoxic apoptotic inducers was effectively prevented by cycloheximide and suramin, while both cycloheximide and suramin had only a slight inhibitory effect on DNA fragmentation induced by genotoxic agents. The results presented suggest that distinct pathways underlay disintegration of nuclear DNA during apoptosis induced by genotoxic and nongenotoxic inducers, and that the formation of HMW- and oligonucleosomal-DNA fragments proceeds via separate mechanisms in NB-2a neuroblastoma cells.

Comparative analysis of minimal residual disease detection by multiparameter flow cytometry and enhanced ASO RQ-PCR in multiple myeloma.

Multiparameter flow cytometry (MFC) and allele-specific oligonucleotide real-time quantitative PCR (ASO RQ-PCR) are the two most sensitive methods to detect minimal residual disease (MRD) in multiple myeloma (MM). We compared these methods in 129 paired post-therapy samples from 22 unselected, consecutive MM patients in complete/near complete remission. Appropriate immunophenotypic and ASO RQ-PCR-MRD targets could be detected and MRD analyses constructed for all patients. The high PCR coverage could be achieved by gradual widening of the primer sets used for clonality detection. In addition, for 13 (55%) of the patients, reverse orientation of the ASO primer and individual design of the TaqMan probe improved the sensitivity and specificity of ASO RQ-PCR analysis. A significant nonlinear correlation prevailed between MFC-MRD and PCR-MRD when both were positive. Discordance between the methods was found in 32 (35%) paired samples, which were negative by MFC-MRD, but positive by ASO RQ-PCR. The findings suggest that with the described technique, ASO RQ-PCR can be constructed for all patients with MM. ASO RQ-PCR is slightly more sensitive in MRD detection than 6-10-color flow cytometry. Owing to technical demands ASO RQ-PCR could be reserved for patients in immunophenotypic remission, especially in efficacy comparisons between different drugs and treatment modalities.

Alpha v beta 6 integrin down-regulates the MMP-13 expression in oral squamous cell carcinoma cells.

The integrin alphavbeta6, a receptor for fibronectin, vitronectin, tenascin and TGF-beta latency-associated peptide (LAP), is not detectable on normal oral epithelium but is neo-expressed in oral squamous cell carcinomas (OSCC) and epithelial dysplasia. Previously it has been shown that alphavbeta6 integrin can up-regulate MMP-3 and -9 expression in OSCC cells. Using beta6-transfected and control OSCC cells we demonstrate that alphavbeta6 integrin down-regulates MMP-13 expression at both mRNA and protein level. Although expressing less MMP-13, beta6-transfected cells were found to have similar collagenolytic activity as control cells and invade at similar levels through type I collagen. Growth of the tumour cells in organotypic culture and confocal microscopy confirmed low levels of MMP-13 in cells with high alphavbeta6 expression. Furthermore, human squamous cell carcinomas of the tongue with high expression of alphavbeta6 showed lower MMP-13 levels than carcinomas with low levels of alphavbeta6. Our results suggest that alphavbeta6 down-regulates MMP-13 expression in OSCC cells and that MMP-13 is not essential for the degradation of type I collagen by OSCC cells.

Different organizational states of fodrin in cultured MDCK cells are induced by treatment with low pH, calmodulin antagonist TFP, and tumor promoter PMA.

We have investigated the molecular mechanisms underlying dynamic organization of the fodrin network by treating the epithelial MDCK cells with various agents affecting intracellular pH, intracellular calcium ion concentration, intracellular calmodulin, and protein kinase C (PKC) activity. Elevation of intracellular calcium level by A23187 or treatment with trifluoperazine (TFP), a calmodulin inhibitor, did not have any drastic effect on the fodrin distribution as judged by immunofluorescence microscopy. A long-term incubation with phorbol-12-myristate-13-acetate (PMA), a protein kinase C activator, in contrast, released fodrin from the lateral walls of the MDCK cells, leading to a diffuse cytoplasmic distribution. TFP, along with PMA, accelerated destabilization of the fodrin skeleton. Treatment with TFP alone rapidly released the cells from the substratum, which, however, could be prevented by PMA. We have previously shown that lowering of intracellular pH (< 6.5) leads to a removal of fodrin from its basolateral residence (Eskelinen et al., 1992) and that this translocation is reversed upon returning normal pH. We now show that the rebuilding of the membrane skeleton can be prevented if TFP is added to the acidified cells. Moreover, in TFP-treated acidified cells, fodrin shows a clusterlike organization similar to that observed in resting lymphocytes. We also noticed that interconversions between these different organizational states of fodrin are independent of the intracellular calcium concentration. Thus manipulation of the intracellular pH and treatment with TFP and PMA reveals different organizational states of the fodrin skeleton. This suggests that fodrin may participate in PMA-, TFP- and pH-sensitive signal transduction pathways.

Regulation of the disassembly/assembly of the membrane skeleton in Madin-Darby canine kidney cells.

The effects of pH, temperature, block of energy production, calcium/calmodulin, protein phosphorylation, and cytoskeleton-disrupting agents (cytochalasin D, nocodazole) on the integrity of the membrane skeleton were studied in polarized MDCK cells. The intracellular distributions of alpha-fodrin, actin, and ankyrin were monitored by immunofluorescence microscopy. The membrane skeleton, once assembled, seemed to be quite stable; the only factors releasing alpha-fodrin from the lateral walls were the acidification of the cytoplasm and the depletion of extracellular calcium ions. Upon cellular acidification, some actin was also released from its normal location along the lateral walls and was seen in colocalization with alpha-fodrin in the cytoplasm, whereas ankyrin remained associated with the lateral walls. No accumulation of plasma membrane lipids was observed in the cytoplasm of acidified cells, as visualized by TMA-DPH. These results suggest that the linkages between the fodrin-actin complex and its membrane association sites are broken upon acidification. The pH-induced change in alpha-fodrin localization was reversible upon restoring the normal pH. Reassembly of the membrane skeleton, however, required temperatures above +20 degrees C, normal energy production, proper cell-cell contacts, and polymerized actin. Release of alpha-fodrin from the lateral walls to the cytoplasm was also observed upon depletion of extracellular calcium ions. This change was accompanied by the disruption of cell-cell contacts, supporting the role of proper cell-cell contacts in the maintenance of the membrane skeleton polarity. These results suggest that local alterations of the cytoplasmic pH and calcium ion concentration may be important in regulating the integrity of the membrane skeleton.

The polarity of the membrane skeleton in retinal pigment epithelial cells of developing chicken embryos and in primary culture.

We studied the morphogenesis and the membrane skeleton in the retinal pigment epithelium during chicken embryogenesis and in culture, by using immunofluorescence and electron microscopy. During embryogenesis two distinct membrane skeletal structures were formed, an apical and a basolateral one. The former was seen in the apical surface already in the 10-day-old embryos. It was comprised of ankyrin and alpha-fodrin and showed a codistribution with Na+,K(+)-ATPase and an as yet uncharacterized cadherin-like molecule. The basolateral membrane skeleton was seen in the lateral walls already in the 10-day-old embryos, and later, between the 13th and 17th embryonic days, it also appeared at the basal membrane, coincidentally with the formation of the basal infoldings. It consisted of ankyrin and alpha-fodrin, but did not codistribute with any of the integral membrane proteins studied (Na+,K(+)-ATPase and cadherins). In culture, the retinal pigment epithelial cells retained their polarized morphology. Compared with the situation in vivo, however, there was a distinct translocation of the membrane skeletal components fodrin and ankyrin from the apical surface to the lateral walls, accompanied by a similar redistribution of Na+,K(+)-ATPase and the cadherin-like molecule. The results suggest that (1) there is, in the retinal pigment epithelium, an apical Na+,K(+)-ATPase-membrane skeleton structure stabilized by contacts between the retinal pigment epithelium and the neural retina, possibly mediated by a cadherin-like molecule, and that (2) there is another fodrin/ankyrin-based membrane skeleton in the basolateral walls that is important for the maintenance of the extensive folding of these surface areas.

Low intracellular pH induces redistribution of fodrin and instabilization of lateral walls in MDCK cells.

We have studied the effect of intracellular pH on the establishment and maintenance of the cellular polarity in MDCK cells by utilizing nigericin which causes lowering of the cytoplasmic pH. At pH below 6.5, MDCK cells lost their polarized morphology and became roundish, with an increased apical area and shortened and unstable lateral walls. The lateral wall marker proteins uvomorulin and Na,K-ATPase remained segregated to the lateral walls in the acidified cells, as shown by immunofluorescence microscopy. Fodrin, on the other hand, was released from its normal basolateral residence and was found in the cytoplasm. Actin, which normally co-localizes with fodrin along the basolateral walls, showed a dotty distribution in the cytoplasm of acidified cells, while stress fibers remained intact. Microtubular network appeared flattened, but the Golgi complex retained its apical position. The pH change-induced alterations were readily reversible, as the normal basal-apical polarity (columnar shape, distinct apical and lateral domains with apposing and stiff lateral membranes) was reformed within 10 minutes after restoring the normal pH gradient across the cell membrane. This coincided with the translocation of fodrin from the cytoplasm to the lateral walls. The results show that lowering of intracellular pH leads to temporary segregation of fodrin from the other components of the membrane skeleton assembly, and that association of fodrin with the lateral walls seems to be a prerequisite for their close apposition and for the maintenance of normal basal-axial polarity.