Granulocyte Fraction Research Articles

Localization of serglycin in human neutrophil granulocytes and their precursors.

Serglycin is a major proteoglycan of hematopoietic cells. It is thought to play a role in the packaging of granule proteins in human neutrophil granulocytes. The presence of serglycin in myeloid cells has been demonstrated only at the transcriptional level. We generated a polyclonal antibody against recombinant human serglycin. Here, we show the localization of serglycin in humans during neutrophil differentiation. Immunocytochemistry revealed serglycin immunoreactivity in the Golgi area of promyelocytes (PM) and myelocytes (MC), as well as in a few band cells and mature neutrophil granulocytes. Granular staining was detected near the Golgi apparatus in some of the PM, and the major part of the cytoplasm was negative. Immunoelectron microscopy showed serglycin immunoreactivity located to the Golgi apparatus and a few immature granules of PM and MC. The decreasing level of serglycin protein during myeloid differentiation coincided with a decrease of mRNA expression, as evaluated by Northern blotting. Subcellular fractions of neutrophil granulocytes were obtained. Serglycin immunoreactivity was detected in the fraction containing Golgi apparatus, plasma membrane, and secretory vesicles by Western blotting and enzyme-linked immunosorbent assay. Serglycin was not detected in subcellular fractions containing primary, secondary, or tertiary granules. Together, these findings indicate that serglycin is located to the Golgi apparatus and a few immature granules during neutrophil differentiation. This is consistent with a function for serglycin in formation of granules in neutrophil granulocytes. Our findings contrast the view that native serglycin is present in mature granules and plays a role in packaging and regulating the activity of proteolytic enzymes there.

1006. Preliminary Results of a Phase I Trial Using Retroviral Gene Transfer of G156A MGMT To Protect Hematopoiesis during BG and BCNU Therapy of Advanced Malignancies

We have extensively studied the G156A MGMT mutation which confers O6alkylguanine DNA alkyltransferase (AGT) that is resistant to O6benzylguanine (BG). In a Phase I clinical trial at our institution, BG was shown to inhibit tumor AGT at 120mg/m2 and the maximum tolerated dose of BCNU was 40 mg/m2. In the laboratory, we have shown that the retrovirus MFG-G156A-MGMT confers BG and BCNU resistance ex vivo into human CD34+ cells. We have also reported that G156A-MGMT transduced murine hematopoietic progenitors repopulate the bone marrow and can be enriched up to 1000-fold after 2 cycles of BG and BCNU. These studies demonstrate the strong capacity of this model to confer BG and BCNU resistance and a selection advantage in vivo. Based on these data, we initiated a clinical trial using gene transfer into autologous CD34+ cells in patients with advanced malignancies. Patients undergo CD34+ mobilization with G-CSF and GM-CSF. CD34+ enriched cells (CliniMACS, Miltenyi) are transduced with MFG-G156A MGMT (produced in PG13 cells by the NGVL, K. Cornetta, Director) in the presence of the fibronectin fragment CH-296 (provided by Takara Bio Inc) and the cytokine combination SCF, Tpo, and Flt-3 ligand for 86 hours. The cell culture is harvested on day 4 and the entire culture is re-infused into the patient who received BG and BCNU 48 hours prior. Patients receive subsequent cycles of BG and BCNU every 6 weeks with analysis of marrow and blood cells for evidence of gene transfer prior to each cycle. In the first three patients, the frequency of gene transfer measured by proviral containing CFU in the post transduction culture was 20 ± 7% and 6.3 ± 3 % of the cells expressed G156A-AGT by flow cytometry. An average of 4.4 ± 2 x 107viable mononuclear cells with a CD34 purity of 93 ± 5% were infused. In one patient, 10% of bone marrow CFU showed presence of the provirus 5 weeks after infusion but prior to the second cycle of BG and BCNU. In this same patient, insertion site analysis by LAM-PCR showed a similar banding pattern in both the lymphocyte-monocyte and granulocyte fractions, indicating multilineage reconsitituion by a single transduced stem cell. Our preliminary results demonstrate efficient gene transfer into cytokine stimulated CD34+ cells using the MFG-G156A-MGMT vector and these cells can be infused without toxicity. Given the strong degree of selection observed in pre-clinical models, we anticipate enrichment for transduced cells following each cycle of chemotherapy.

Tissue factor in neutrophils: no

Tissue factor in neutrophils: no

Adoptive immunotherapy to increase the level of donor hematopoietic chimerism after nonmyeloablative marrow transplantation for severe canine hereditary hemolytic anemia

Severe hemolytic anemia in Basenji dogs secondary to pyruvate kinase deficiency can be corrected by allogeneic hematopoietic cell transplantation (HCT) from littermates with normal hematopoiesis after conventional myeloablative or nonmyeloablative conditioning regimens. If the levels of donor chimerism were low (<20%) after nonmyeloablative HCT, there was only partial correction of the hemolytic anemia. We next addressed whether allogeneic cell therapy after nonmyeloablative HCT would convert mixed to full hematopoietic chimerism, achieve sustained remission from hemolysis, and prevent progression of marrow fibrosis and liver cirrhosis. Three pyruvate kinase-deficient dogs were given HCT from their respective dog leukocyte antigen-identical littermates after nonmyeloablative conditioning with 200 cGy of total body irradiation. Postgrafting immunosuppression consisted of mycophenolate mofetil and cyclosporine. All 3 dogs engrafted and had mixed hematopoietic chimerism with donor levels ranging from 12% to 55% in bone marrow. In 2 of the 3 dogs, there were decreases in the levels of donor chimerism so that at 25 weeks after nonmyeloablative HCT, hemolysis recurred that was associated with increased reticulocyte counts. All 3 dogs then had 2 serial infusions of donor lymphocytes (DLI) from their respective donors at least 20 weeks apart to convert from mixed to full donor chimerism. Both dogs with recurrence of hemolytic anemia after nonmyeloablative HCT achieved higher levels of donor chimerism, with donor contributions ranging from 47% to 62% in the bone marrow and 50% to 69% and 16% to 25% in the granulocyte and mononuclear cell fractions of the peripheral blood, respectively, and with remission of the hemolytic anemia. One dog responded after the first DLI, and 5 weeks after the second DLI, the other dog converted to full donor chimerism. At last follow-up, all these dogs showed clinical improvement, as determined by increasing hematocrits and normal reticulocyte counts. Analysis of the marrow 3 years after HCT showed normal cellularity, a normal myeloid-erythroid ratio, and no or minimal marrow fibrosis. Liver biopsies demonstrated normal histologies with no or minimal fibrosis. We conclude that DLI after nonmyeloablative HCT can increase the levels of donor cells contributing to hematopoiesis in recipients, inducing remissions of the hemolytic process and preventing complications associated with iron overload.

Respiratory burst in neutrophilic granulocytes of carps (Cyprinus carpio): cytometric studies

Present studies aimed at flow cytometric analysis of respiratory burst in neutrophilic granulocytes in carps, at various stages of their ontogeny. Cytometric evaluation of the repiratory burst in PMN cells of carps demonstrated augmented values of the mean fluorescence channel of PMA-stimulated PMN cells, as compared to the value of the fluorescence channel noted for PMN cells which were not activated or did not respond to activation in response to the applied stimulator. Intensity of the fluorescence, compared to the mean fluorescence channel of the cells which were not stimulated by PMA, was most pronounced in 11- to 21-month old carps, average in the youngest carps aging 3 to 9 months, and the lowest in the eldest, 23- to 29-month old carps. In cases of analysis of the mean fluorescence channel in the selected fraction of PMA-stimulated PMN cells, as compared to the mean fluorescence channel of granulocytes which did not respond by the stimulation reaction, the most intense fluorescence was noted to develop in the eldest, i.e. 23- to 29-month old fish. In the remaining carps, on the other hand, the difference in the mean fluorescence channel was significantly lower. In parallel, the highest fraction of PMA-stimulated granulocytes was found in 11- to 21-month old carps, a lower fraction in the youngest carps aging 3 to 9 months and the lowest fraction of such granulocytes in the eldest carps, 23 to 29 months of age.

Are low-density granulocytes the major target cells of classical swine fever virus in the peripheral blood?

The target cells of classical swine fever (CSF) virus in the peripheral blood of pigs infected with recent field isolates from Germany were studied. Eight weaned pigs were inoculated oronasally with the CSF virus field isolate Visbek/Han 95 and three weaners were inoculated with the isolate Losten/Freese 98. All pigs showed severe clinical signs typical of CSF and died or had to be euthanized between 9 and 24 days post-infection (dpi). The first cells in the peripheral blood which became infected with CSF virus were mixed granulocytes (a combination of low- and high-density granulocytes). These cells yielded the highest infectivity for PK 15 cell cultures. On day 7 post-infection, the peripheral blood mononuclear cell (PBMC) fraction was virus positive, while the peripheral blood leucocyte (PBL), peripheral blood T lymphocyte (PBT) and high-density granulocyte fractions were either negative or their infectivity was lower than the infectivity of the PBMC fraction. These results indicate that PBMC contain more virus-positive cells than other fractions of leucocytes. These findings may also have diagnostic implications for the detection of CSF virus in blood samples. Because PBMC showed the highest infectivity in the early stages of CSF, it should be the sample of choice for CSF virus isolation.

Increased circulating levels of alpha1-antitrypsin and calprotectin are associated with reduced gas diffusion in the lungs.

The aim was to examine the relationship of serum inflammatory markers to the level of single-breath diffusing capacity of carbon monoxide (TL,CO). A stratified sample (n = 1,121) of a Norwegian general population aged 18-73 yrs was examined. The inflammatory markers measured were calprotectin, a prominent protein in the cytosol fraction of neutrophil granulocytes, and alpha1-antitrypsin (alpha1-AT), the major inhibitor of neutrophil elastase in the lower respiratory system. Both markers have increased circulating levels in the course of an acute inflammatory reaction. Subjects with a TL,CO < 80% of predicted value had a higher level of both alpha1-AT (p = 0.003) and calprotectin (p < 0.03) than those with a TL,CO > 100%. In multiple linear regression analyses, alpha1-AT was still significantly associated with TL,CO after adjusting for sex, age, smoking habits, haemoglobin, carboxyhaemoglobin, forced expiratory volume in one second and alveolar volume. In a similar analysis, no significant overall association was found between calprotectin and TL,CO, but in a stratified analysis, calprotectin was significantly related to TL,CO in females. However, no significant sex interaction in the relationship between the inflammatory markers and TL,COo was found. The findings suggest that increased levels of alpha1-antitrypsin and of calprotectin are risk factors for decreased diffusing capacity of carbon monoxide.

The effect of leukocyte-reduction method on the amount of human cytomegalovirus in blood products: a comparison of apheresis and filtration methods

This study examined the effectiveness of 3 leukocyte-reduction (LR) methods in depleting the residual level of cytomegalovirus (CMV) in blood products measured by quantitative polymerase chain reaction (QA-PCR). At 2 locations over 3 allergy seasons, apheresis platelets and whole blood were collected from 52 healthy CMV seropositive subjects having an elevated titer of CMV DNA (median = 2400 genome equivalents [GE]/mL) resulting in 32 evaluable LR apheresis platelets, 31 filtered platelets from whole blood, and 31 filtered red blood cells (RBCs) from whole blood. Leukoreduction by apheresis and filtration resulted in substantial reduction of detectable CMV DNA levels with 99.9% of the LR products expected to have less than 500 GE/mL of CMV DNA. No difference was found between methods (P =.52). CMV genomic leukocyte subset localization was determined by QA-PCR of fluorescence-activated cell sorter (FACS)-sorted peripheral blood from 20 seropositive subjects (n = 10 > 100 GE/mL, n = 10 QA-PCR negative). CMV was detected in monocyte (13 of 20) and granulocyte (3 of 20) fractions. Presence of competent virus in QA-PCR positive (> 100 GE/mL) peripheral blood samples was verified with 4 of 19 subjects positive in shell vial assay, and 8 of 18 positive for CMV gene products (messenger RNA). We observed a seasonal DNAemia variation in seropositive subjects. CMV seropositive subjects (n = 45) entered into longitudinal monitoring in March/April 1999 were QA-PCR negative at baseline. Subjects converted to a positive QA-PCR coincident with increased seasonal allergen levels (Norfolk 15 of 18 evaluable in 43.4 +/- 9.48 days; Denver, 16 of 23 evaluable in 96 +/- 26.3 days). These data demonstrate effective reduction of CMV load by LR during periods of DNAemia in CMV seropositive subjects. (Blood. 2001;97:3640-3647)

Methylation status of the p15INK4B gene in hematopoietic progenitors and peripheral blood cells in myelodysplastic syndromes.

We previously reported that the hypermethylation of the p15INK4B gene promoter was frequently observed in myelodysplastic syndromes (MDS), and that it may be associated with disease progression. An unanswered question is whether p15INK4B gene methylation is restricted to undifferentiated blastic cells, or whether differentiated cells such as granulocytes or erythrocytes of MDS origin also harbor this epigenetic alteration. In this study, we analyzed the methylation status of the p15INK4B gene in MDS by the methylation-specific PCR (MSP) method, which is more sensitive than Southern blotting. The bone marrow mononuclear cells (BM-MNCs) of 23 MDS patients were analyzed, and six of them showed p15INK4B methylation. Progenitor assay with methylcellulose medium was also performed in all patients. In two of the six patients with p15INK4B-methylated BM-MNCs, erythroid and/or non-erythroid colonies formed were subjected to molecular analysis. Colonies with and without p15INK4B methylation were detected in both patients. Furthermore, X-chromosome inactivation (XCI) pattern of each colony was simultaneously determined by MSP-based human androgen receptor gene analysis (HUMARA-MSP), and all p15INK4B-methylated colonies showed the same XCI pattern, which was dominant among the colonies, while p15INK4B-unmethylated colonies showed both patterns of XCI, in each of the two patients. We then examined the methylation status of the p15INK4B gene of granulocyte (PB-PMN) fractions from 10 patients with available peripheral blood cells. In all four patients with p15INK4B-methylated BM-MNCs, their PB-PMNs showed p15INK4B methylation. These results suggest that p15INK4B methylation in hematopoietic cells in MDS patients is restricted to the MDS clone but not necessarily to blast cells.

Quantitative assessment of retroviral transfer of the human multidrug resistance 1 gene to human mobilized peripheral blood progenitor cells engrafted in nonobese diabetic/severe combined immunodeficient mice

Mobilized peripheral blood progenitor cells (PBPC) are a potential target for the retrovirus-mediated transfer of cytostatic drug-resistance genes. We analyzed nonobese diabetic/severe combined immunodeficient (NOD/SCID) mouse-repopulating CD34+ PBPC from patients with cancer after retroviral transduction in various cytokine combinations with the hybrid vector SF-MDR, which is based on the Friend mink cell focus-forming/murine embryonic stem-cell virus and carries the human multidrug resistance 1 (MDR1) gene. Five to 13 weeks after transplantation of CD34+ PBPC into NOD/SCID mice (n = 84), a cell dose-dependent multilineage engraftment of human leukocytes up to an average of 33% was observed. The SF-MDR provirus was detected in the bone marrow (BM) and in its granulocyte fractions in 96% and 72%, respectively, of chimeric NOD/SCID mice. SF-MDR provirus integration assessed by quantitative real-time polymerase chain reaction (PCR) was optimal in the presence of Flt-3 ligand/thrombopoietin/stem-cell factor, resulting in a 6-fold (24% +/- 5% [mean +/- SE]) higher average proportion of gene-marked human cells in NOD/SCID mice than that achieved with IL-3 alone (P <.01). A population of clearly rhodamine-123(dull) human myeloid progeny cells could be isolated from BM samples from chimeric NOD/SCID mice. On the basis of PCR and rhodamine-123 efflux data, up to 18% +/- 4% of transduced cells were calculated to express the transgene. Our data suggest that the NOD/SCID model provides a valid assay for estimating the gene-transfer efficiency to repopulating human PBPC that may be achievable in clinical autologous transplantation. P-glycoprotein expression sufficient to prevent marrow aplasia in vivo may be obtained with this SF-MDR vector and an optimized transduction protocol. (Blood. 2000;95:1237-1248)

Separation of European flat oyster, Ostrea edulis, haemocytes by density gradient centrifugation and SDS-PAGE characterisation of separated haemocyte sub-populations

A two-step gradient centrifugation with Percoll and Ficoll successively as density medium was developed to separate European flat oyster, Ostrea edulis, haemocytes into three sub-populations representing granulocytes, large hyalinocytes and small hyalinocytes, respectively. After a Percoll gradient centrifugation, granulocytes and agranulocytes were separated and a pure fraction of granulocytes was obtained. The agranulocytes were further separated by centrifugation through a Ficoll gradient, and two haemocyte sub-populations representing large hyalinocytes and small hyalinocytes were obtained. No significant impact on the haemocyte viability was detected after separation with this two-step density gradient centrifugation. The three haemocyte sub-populations showed different protein patterns in SDS-PAGE.

Different kinetics of the respiratory burst response in granulocytes, induced by serum from blood coagulated in contact with polymer materials

Tubes of different polymer materials were filled with blood collected by venous puncture. The blood was allowed to clot for 10 min, and the serum was collected. Complement activation was demonstrated through assessment of the C3-level by radial immunodiffusion. Phospholipid fingerprints were made after lipid extraction of serum and separation by thin-layer chromatography. The granulocyte fraction of venous blood was separated on a Percoll gradient and the cells were either loaded with a calcium probe, or incubated with luminol. These cells were used as a biological test for inflammatory mediators. Serum from blood coagulated in contact with different materials was added to the test cells. The intracellular calcium level was recorded by Calcium Green-1 ® fluorescence and the respiratory burst of the test cells was recorded by luminol-amplified chemiluminescence. Serum from blood coagulated in contact with glass tubes, methylised glass tubes and teflon (PTFE) tubes induced a transient increase of the cellular calcium level, indicating a G protein-coupled activation of the test cells. Serum from blood coagulated in contact with glass tubes, methylised glass tubes, and PTFE tubes primed the test cells for a subsequent f-MLP response. Serum from blood coagulated in contact with polyurethane and polypropylene induced a direct biphasic respiratory burst response in the test cells and serum from blood coagulated in contact with methylised glass induced a direct monophasic respiratory burst response in the test cells. Complement activation was demonstrated after blood contact with hydrophobic glass and PTFE. Different fingerprints of phospholipid content were found in sera after blood contact with different materials. The data show that different inflammatory mediators are released during blood coagulation in contact with different materials. The method may be valuable as a screening test for blood compatibility of materials.

Immunomagnetic separation for enrichment and sensitive detection of disseminated tumour cells in patients with head and neck SCC

Screening for malignant cells in the blood and bone marrow was introduced as a strategy for the improved detection of tumour spread and may predict the development of distant metastases. The sensitivity of these approaches depends on several factors, including the choice of antibody for immunocytochemistry (ICC) and the number of cells examined. In this study criteria have been defined for scoring cells reactive with a pan-cytokeratin antibody as tumour, by comparing immunostained cells in clinical samples obtained from head and neck cancer patients and a control group without epithelial malignancy. When leucocyte subfractions are prepared by density gradient separation (DGS) from central venous blood obtained from patients with advanced head and neck squamous cell carcinoma (SCC) and screened by ICC, epithelial tumour cells sediment preferentially with the mononuclear cells but may also be detected in the granulocyte (GC) fraction. Some cases were found to have more tumour cells in the GC fraction. Similar results were seen in model experiments. To increase the sensitivity of the ICC approach, the efficiency of positive immunomagnetic selection (IMS) using Dynabeads coated with an antibody recognizing the Ber-EP4 epitope has been compared with negative IMS using anti-CD45 Dynabeads. Tumour cells were recovered from bone marrow aspirates for 2/17 cases using the positive enrichment technique and for 11/17 patients following negative IMS. These findings justify prospective studies incorporating negative IMS to establish the prognostic significance of these disseminated tumour cells for this group of patients. Copyright © 1999 John Wiley & Sons, Ltd.

Immunomagnetic separation for enrichment and sensitive detection of disseminated tumour cells in patients with head and neck SCC.

Screening for malignant cells in the blood and bone marrow was introduced as a strategy for the improved detection of tumour spread and may predict the development of distant metastases. The sensitivity of these approaches depends on several factors, including the choice of antibody for immunocytochemistry (ICC) and the number of cells examined. In this study criteria have been defined for scoring cells reactive with a pan-cytokeratin antibody as tumour, by comparing immunostained cells in clinical samples obtained from head and neck cancer patients and a control group without epithelial malignancy. When leucocyte subfractions are prepared by density gradient separation (DGS) from central venous blood obtained from patients with advanced head and neck squamous cell carcinoma (SCC) and screened by ICC, epithelial tumour cells sediment preferentially with the mononuclear cells but may also be detected in the granulocyte (GC) fraction. Some cases were found to have more tumour cells in the GC fraction. Similar results were seen in model experiments. To increase the sensitivity of the ICC approach, the efficiency of positive immunomagnetic selection (IMS) using Dynabeads coated with an antibody recognizing the Ber-EP4 epitope has been compared with negative IMS using anti-CD45 Dynabeads. Tumour cells were recovered from bone marrow aspirates for 2/17 cases using the positive enrichment technique and for 11/17 patients following negative IMS. These findings justify prospective studies incorporating negative IMS to establish the prognostic significance of these disseminated tumour cells for this group of patients.

Detection of micrometastasis by cytokeratin 20 RT-PCR is limited due to stable background transcription in granulocytes.

The reverse transcription polymerase chain reaction (RT-PCR) amplification of cytokeratin 20 (CK20) mRNA is considered a promising candidate method for the detection of circulating tumour cells in bone marrow and peripheral blood of cancer patients. In this study we have investigated the diagnostic specificity of the CK20 mRNA detection in samples from healthy donors (HD; n = 33), intensive care units patients (ICU; n = 20) and bone marrow obtained from patients suffering from chronic inflammatory diseases (CID; n = 14). RNAs purified from stabilized lysates showed positive results in 24% of the HD group (8/33), 35% of the ICU group (8/20) and in 40% of the CID group (5/14). The use of Ficoll gradients to separate nucleated cells completely restored the specificity of this CK20 RT-PCR assay. The CK20-expressing cells are positively identified to belong to the granulocyte fraction of leucocytes, which appear to express the gene on a background level. Our results demonstrate for the first time that CK20 mRNA expression is not limited to epithelium. Its occurrence in normal granulocytes has to be considered in tests designed to detect circulating cancer cells or micrometastases. © 1999 Cancer Research Campaign

Pathogenesis of borna disease virus: granulocyte fractions of psychiatric patients harbor infectious virus in the absence of antiviral antibodies.

Borna disease virus (BDV) causes acute and persistent infections in various vertebrates. During recent years, BDV-specific serum antibodies, BDV antigen, and BDV-specific nucleic acid were found in humans suffering from psychiatric disorders. Furthermore, viral antigen was detected in human autopsy brain tissue by immunohistochemical staining. Whether BDV infection can be associated with psychiatric disorders is still a matter of debate; no direct evidence has ever been presented. In the present study we report on (i) the detection of BDV-specific nucleic acid in human granulocyte cell fraction from three different psychiatric patients and (ii) the isolation of infectious BDV from these cells obtained from a patient with multiple psychiatric disorders. In leukocyte preparations other than granulocytes, either no BDV RNA was detected or positive PCR results were obtained only if there was at least 20% contamination with granulocytes. Parts of the antigenome of the isolated virus were sequenced, demonstrating the close relationship to the prototype BDV strains (He/80 and strain V) as well as to other human virus sequences. Our data provide strong evidence that cells in the granulocyte fraction represent the major if not the sole cell type harboring BDV-specific nucleic acid in human blood and contain infectious virus. In contrast to most other reports of putative human isolates, where sequences are virtually identical to those of the established laboratory strains, this isolate shows divergence in the region previously defined as variable in BDV from naturally infected animals.

Human T-cell leukemia virus type 1 can infect a wide variety of cells in mice.

Analysis of human T‐cell leukemia virus type 1 (HTLV‐1)‐infected cell types and the interplay of these infected cells in vivo should provide valuable information to elucidate the pathogenesis of HTLV‐1‐associated diseases in humans and in animal models. In this study, HTLV‐1‐infected cell types were identified in HTLV‐1‐infected C3H/HeJ mice. Pan T, CD+4, CD8+8, granulocyte and pan B cell fractions in the splenocytes of MT‐2 cell‐inoculated mice were sorted by use of their cell surface high‐density expression of CD3e, CD4, CD8, Gr‐1 and B220 antigens, respectively, with a fluorescence‐activated cell sorter. the pX sequence of HTLV‐1 provirus in the lysate of each fraction was amplified by polymerase chain reaction and detected by Southern hybridization. Interestingly, in addition to the CD+4 cell fraction, the pX sequence was also found in CD8+ cell, B cell and granulocyte fractions. The broad cell spectrum of HTLV‐1 infection in mice is consistent with the situation in humans. Our findings indicate that HTLV‐1 receptor or coreceptor is widely distributed among different cell types in mice.

Molecular cloning and characterization of leukotriene B4 receptor.

Leukotriene B4 (LTB4) is a potent proinflammatory and chemotactic factor biosynthesized in various tissues, which is involved in inflammation, immune responses and host defense against infection.1 LTB4 activates inflammatory cells by binding to its cell-surface receptor (BLT). Specific binding for [3H]LTB4 was reported in the membrane fractions of granulocytes, T lymphocytes, alveolar macrophages, spleen, brain, THP-1 cells, and differentiated HL-60 cells.2–5 By addition of GTPγS, low affinity sites of [3H]LTB4 binding appeared in spleen membranes.5 LTB4 stimulated a dose-dependent increase in GTP hydrolysis in HL-60 cells.6 LTB4 induced increase in intracellular calcium, D-myo-inositol-l,4,5-triphosphate (InsP3) accumulation, O2-production, and activation of GTPase in a pertussis toxin (PTX)-sensitive manner.7,8 On the other hand, LTB4-induced hyperadhesiveness of vascular endothelial cells was reported to be PTX-insensitive.9 These data indicated the presence of BLT coupled with G-protein(s). However, BLT had not been purified to homogeneity or cDNA-cloned.

Major injury induces increased production of interleukin-10 in human granulocyte fractions.

Patients with severe trauma or polytrauma frequently acquire alterations in immune functions which are correlated to dysbalanced cytokine synthesis. In these settings the role of polymorphonuclear neutrophil granulocytes (PMN) as cytokine-producing cells is less well characterized. The immunosuppressive role of interleukin (IL)-10 is well known, and increased systemic IL-10 levels are related to the severity of injury and to posttraumatic complications. We determined concentrations of IL-10 in culture supernatants of 30 individual PMN fractions isolated from 18 severely traumatized patients (15 polytraumata, Injury Severity Score: 18-41, 3 severely burned patients) admitted to intensive care units. IL-10 was analyzed by ELISA (R&D Systems, Wiesbaden, Germany). PMN were isolated from EDTA-anticoagulated peripheral blood employing a one-step procedure based on a discontinuous double Ficoll gradient. The cells [1 x 10(6)/ml RPMI 1640 supplemented with 10% fetal calf serum and 25 mM N-(2-hydroxyethyl)-piperazine-N'-(2-ethanesulfonic acid] were stimulated with 0.05% heat-killed Staphylococcus aureus (Pansorbin, Calbiochem-Novabiochem, Bad Soden, Germany) for 24 h using cell culture conditions. Our results show that PMN fractions of traumatized patients produce significantly (P<0.008) higher amounts of IL-10 (354+/-95 pg/ml, n = 30) than normal healthy donor cells (125+/-95 pg/ml, n = 7). IL-10 release from PMN fractions exceeded the release from isolated patients' peripheral blood mononuclear cells induced by similar stimulation or by stimulation with toxic shock syndrome toxin-1 (10 ng) and concanavalin A (2 microg). Our results provide evidence that PMN fractions play an active role in the development of posttraumatic immunosuppression by autocrine or paracrine mechanisms, for example, by suppressing one's own antimicrobial activities or determining the development of T-cell responses via their ability to release IL-10.

Granulocytes in the subarachnoid space of humans and rabbits with bacterial meningitis undergo apoptosis and are eliminated by macrophages.

The contribution of leukocyte apoptosis to the resolution of meningeal inflammation in bacterial meningitis was studied in the cerebrospinal fluid (CSF) and in meningeal infiltrates of humans and rabbits by in situ tailing, flow cytometry, agarose gel electrophoresis, and electron microscopy. In humans, the rate of apoptotic granulocytes was 21.0+/-20.8% (n=11) in cytocentrifuge preparations and 3.3+/-3.4 (n=14) in putrid infiltrates of autopsy cases (P=0.02). In rabbits, CSF pleocytosis peaked 8 h after the initiation of antibiotic treatment (5311+/-3122/microl). At this time, the rate of apoptotic granulocytes was 15.2+/-7.3% in CSF and 1.8+/-1.4% in the meningeal infiltrates (each group n=6, P=0.007). Thereafter, the rate of apoptotic granulocytes in CSF declined below 10%. In humans and rabbits, bands representing internucleosomal fragments of approximately 180 base pairs and multiples thereof were documented on agarose gels. Phagocytosis of apoptotic granulocytes by macrophages was visualized by light and electron microscopy. In conclusion, during resolution of subarachnoid space inflammation in bacterial meningitis, a substantial fraction of granulocytes undergoes apoptosis. These granulocytes are removed by phagocytosis by macrophages. Apoptosis is more frequent in granulocytes floating in the CSF than in adherent cells.