Studies on control of granulopoiesis in man II. Influence of circulating neutrophil count on release of labelled bone marrow cells

Leukocyte kinetic studies were performed by labelling neutrophilic leukocytes in vivo with radioactive diisopropylfluorophosphate and measuring the time taken for the label to traverse the myelocyte compartment, marrow granulocyte reservoir and blood (myelocyte-to-tissue transit time). Leukocyte specific activity (LSA) reached background levels within 17 to 22 days in normal subjects and more rapidly than normal in patients with neutropenia due to hypersplenism or marrow aplasia. When neutropenia was partially or completely corrected by splenectomy the label disappeared more slowly. In patients with stable neutrophilia the label disappeared slowly and a terminal plateau in the LSA curve was encountered. Analysis of LSA curves suggests that disappearance of labelled neutrophil cohorts is influenced by circulating neutrophil levels, not only by an effect on release of cells from the marrow granulocyte reservoir but also, directly or indirectly, by an effect on myelocyte proliferation.     

The Mechanism of Action of Splenic Irradiation in Chronic Myelogenous Leukemia

The mechanism of action of splenic irradiation in the induction of a remission in chronic myelogenous leukemia was investigated in six patients using a leukocyte kinetic approach. The leukocytes were labelled in vitro with radioactive diisopropylfluorophosphate-32 and returned to the circulation. The effect of treatment on the rate of change of leukocyte specific activity was determined. The results suggest (1) that irradiation of the spleen damages granulopoietic cells as they cycle back and forth between the spleen, blood and other extravascular compartments; (2) that damage to exchangeable granulopoietic cells in transit through the irradiated spleen may explain the long remission often encountered after this form of therapy.     

Increased endothelial cell-leukocyte interaction in murine schistosomiasis: possible priming of endothelial cells by the disease

Background and Aims

Schistosomiasis is an intravascular parasitic disease associated with inflammation. Endothelial cells control leukocyte transmigration and vascular permeability being modulated by pro-inflammatory mediators. Recent data have shown that endothelial cells primed in vivo in the course of a disease keep the information in culture. Herein, we evaluated the impact of schistosomiasis on endothelial cell-regulated events in vivo and in vitro.

Methodology and Principal Findings

The experimental groups consisted of Schistosoma mansoni-infected and age-matched control mice. In vivo infection caused a marked influx of leukocytes and an increased protein leakage in the peritoneal cavity, characterizing an inflamed vascular and cellular profile. In vitro leukocyte-mesenteric endothelial cell adhesion was higher in cultured cells from infected mice as compared to controls, either in the basal condition or after treatment with the pro-inflammatory cytokine tumor necrosis factor (TNF). Nitric oxide (NO) donation reduced leukocyte adhesion to endothelial cells from control and infected groups; however, in the later group the effect was more pronounced, probably due to a reduced NO production. Inhibition of control endothelial NO synthase (eNOS) increased leukocyte adhesion to a level similar to the one observed in the infected group. Besides, the adhesion of control leukocytes to endothelial cells from infected animals is similar to the result of infected animals, confirming that schistosomiasis alters endothelial cells function. Furthermore, NO production as well as the expression of eNOS were reduced in cultured endothelial cells from infected animals. On the other hand, the expression of its repressor protein, namely caveolin-1, was similar in both control and infected groups.

Conclusion/Significance

Schistosomiasis increases vascular permeability and endothelial cell-leukocyte interaction in vivo and in vitro. These effects are partially explained by a reduced eNOS expression. In addition, our data show that the disease primes endothelial cells in vivo, which keep the acquired phenotype in culture.     

Leukocyte- and platelet-derived microvesicle interactions following in vitro and in vivo activation of toll-like receptor 4 by lipopolysaccharide

Background

Pro-coagulant membrane microvesicles (MV) derived from platelets and leukocytes are shed into the circulation following receptor-mediated activation, cell-cell interaction, and apoptosis. Platelets are sentinel markers of toll-like receptor 4 (TLR4) activation. Experiments were designed to evaluate the time course and mechanism of direct interactions between platelets and leukocytes following acute activation of TLR4 by bacterial lipopolysaccharide (LPS).

Methodology/Principal Findings

Blood from age-matched male and female wild type (WT) and TLR4 gene deleted (dTLR4) mice was incubated with ultra-pure E. coli LPS (500 ng/ml) for up to one hour. At designated periods, leukocyte antigen positive platelets, platelet antigen positive leukocytes and cell-derived MV were quantified by flow cytometry. Numbers of platelet- or leukocyte-derived MV did not increase within one hour following in vitro exposure of blood to LPS. However, with LPS stimulation numbers of platelets staining positive for both platelet- and leukocyte-specific antigens increased in blood derived from WT but not dTLR4 mice. This effect was blocked by inhibition of TLR4 signaling mediated by My88 and TRIF. Seven days after a single intravenous injection of LPS (500 ng/mouse or 20 ng/gm body wt) to WT mice, none of the platelets stained for leukocyte antigen. However, granulocytes, monocytes and apoptotic bodies stained positive for platelet antigens.

Conclusions/Significance

Within one hour of exposure to LPS, leukocytes exchange surface antigens with platelets through TLR4 activation. In vivo, leukocyte expression of platelet antigen is retained after a single exposure to LPS following turn over of the platelet pool. Acute expression of leukocyte antigen on platelets within one hour of exposure to LPS and the sustained expression of platelet antigen on leukocytes following a single acute exposure to LPS in vivo explains, in part, associations of platelets and leukocytes in response to bacterial infection and changes in thrombotic propensity of the blood.     

Production of macrophage migration inhibitory factor and lymphotoxin by leukocytes from normal and Wiskott-Aldrich syndrome patients

The production of macrophage migration inhibitory factor (MIF) and lymphotoxin (LT) by cultured leukocytes from patients with Wiskott-Aldrich syndrome (WAS) and normal controls was studied. The presence of these lymphokines in leukocyte culture supernatants usually correlated directly with the dose of stimulant used. Doses of nonspecific mitogens and specific antigens, which produced maximal in vitro lymphocyte transformation, stimulated maximal production of these mediators. When the incorporation of tritiated thymidine by stimulated leukocyte cultures from patients with Wiskott-Aldrich syndrome (WAS) was deficient, they usually produced less MIF and lymphotoxin than normal. However, when their in vitro lymphoproliferative responses were normal, the lymphotoxin activity in supernatants of WAS leukocyte cultures was normal.     

Computational generation and screening of RNA motifs in large nucleotide sequence pools

Although identification of active motifs in large random sequence pools is central to RNA in vitro selection, no systematic computational equivalent of this process has yet been developed. We develop a computational approach that combines target pool generation, motif scanning and motif screening using secondary structure analysis for applications to 10¹²–10¹⁴-sequence pools; large pool sizes are made possible using program redesign and supercomputing resources. We use the new protocol to search for aptamer and ribozyme motifs in pools up to experimental pool size (10¹⁴ sequences). We show that motif scanning, structure matching and flanking sequence analysis, respectively, reduce the initial sequence pool by 6–8, 1–2 and 1 orders of magnitude, consistent with the rare occurrence of active motifs in random pools. The final yields match the theoretical yields from probability theory for simple motifs and overestimate experimental yields, which constitute lower bounds, for aptamers because screening analyses beyond secondary structure information are not considered systematically. We also show that designed pools using our nucleotide transition probability matrices can produce higher yields for RNA ligase motifs than random pools. Our methods for generating, analyzing and designing large pools can help improve RNA design via simulation of aspects of in vitro selection.     

Tight junction proteins expression and modulation in immune cells and multiple sclerosis

The tight junction proteins (TJPs) are major determinants of endothelial cells comprising physiological vascular barriers such as the blood–brain barrier, but little is known about their expression and role in immune cells. In this study we assessed TJP expression in human leukocyte subsets, their induction by immune activation and modulation associated with autoimmune disease states and therapies. A consistent expression of TJP complexes was detected in peripheral blood leukocytes (PBLs), predominantly in B and T lymphocytes and monocytes, whereas the in vitro application of various immune cell activators led to an increase of claudin 1 levels, yet not of claudin 5. Claudins 1 and 5 levels were elevated in PBLs of multiple sclerosis (MS) patients in relapse, relative to patients in remission, healthy controls and patients with other neurological disorders. Interestingly, claudin 1 protein levels were elevated also in PBLs of patients with type 1 diabetes (T1D). Following glucocorticoid treatment of MS patients in relapse, RNA levels of JAM3 and CLDN5 and claudin 5 protein levels in PBLs decreased. Furthermore, a correlation between CLDN5 pre‐treatment levels and clinical response phenotype to interferon‐β therapy was detected. Our findings indicate that higher levels of leukocyte claudins are associated with immune activation and specifically, increased levels of claudin 5 are associated with MS disease activity. This study highlights a potential role of leukocyte TJPs in physiological states, and autoimmunity and suggests they should be further evaluated as biomarkers for aberrant immune activity and response to therapy in immune‐mediated diseases such as MS.     

Evidence for the identification of lymphocyte activating factor as the adherent cell-derived mediator responsible for enhanced antibody synthesis by nude mouse spleen cells

Human adherent peripheral blood leukocytes spontaneously elaborate both a thymocyte proliferative factor and a factor which augments the in vitro anti-sheep erythrocyte (SRC) plaque-forming cell (PFC) response of nu/nu mouse spleen cells. Nonadherent leukocytes do not spontaneously elaborate either factor. The adherent cell-derived factors appear to have an identical molecular weight (approximately 14,500 Daltons) as determined by Sephadex gel filtration. The data support the hypothesis that the molecule(s) mediating both enhancing activities is identical to the previously described adherent leukocyte product, LAF.     

Binding of L-selectin to its vascular and extravascular ligands is differentially regulated by pH

Ligands for L-selectin, a leukocyte adhesion molecule, are expressed in high endothelial venules (HEVs) in lymph nodes and extravascular tissues, such as renal tubules. Here, we report that the binding of L-selectin to its vascular and extravascular ligands is differentially regulated by pH. The optimal L-selectin-dependent binding of leukocytes to HEVs was observed at pH 7.4, a physiological pH in the blood. In contrast, the optimal binding of leukocytes to the renal tubules was observed at pH 5.6. Consistently, optimal binding of soluble recombinant L-selectin to a major vascular ligand, 6-sulfo sialyl Lewis X, was observed at pH 7.4. Binding to extravascular ligands, such as chondroitin sulfate (CS) B, CS E and heparan sulfate, occurred at pH 5.6. Under physiological shear stress ranging from 1 to 2 dynes/cm², maximal leukocyte rolling on vascular ligands was observed at pH 6.8 to 7.4, and no rolling was detected at pH conditions below 5.6. These findings suggest that the pH environment is one important factor that determines leukocyte trafficking under physiological and pathological conditions.     

Antigen-independent leukocyte random locomotion inhibitory activity in human ultrafiltrated leukocyte extracts

Human ultrafiltrated leukocyte extracts (MW < 5000) were fractionated by Sephadex G-10 column chromatography and the effects of these fractions on leukocyte random locomotion were investigated in vitro. Fr-4, one of these fractions, had significant leukocyte random locomotion inhibitory activity, independent of the presence of mononuclear leukocytes. This inhibitory activity was not due to cytotoxic effects on leukocytes. As seen by scanning electron microscopy, the number of cell surface pseudopods on leukocytes incubated with Fr-4 was reduced. Fr-4A, one of three fractions separated from Fr-4 by Sephadex G-25 column chromatography, significantly inhibited leukocyte random locomotion. Fr-4A contained numerous components, one of which was identified as 2-deoxyribose, on the basis of thin-layer chromatography. Biologically 2-deoxyribose showed an inhibitory effect on leukocyte locomotion and a reduction of the extrusion of pseudopods on the surface of leukocytes, at the range of assayed concentrations. This inhibitory activity is probably derived from 2-deoxyribose.     

Leukocyte migration inhibition studies with Epstein-Barr virus (EBV) determined nuclear antigen (EBNA) in relation to the EBV-carrier status of the donor

Cell extracts from EBV-genome-carrying cell lines inhibit the migration of leukocytes from EBV-positive but not seronegative healthy donors. In the present study extracts from EBV-negative lines and their own in vitro EBV-converted sublines were used to induce migration inhibition with leukocytes from seronegative and seropositive individuals. A clear difference was found between the extracts from EBV-negative and positive cell lines. Significant migration inhibition could be obtained with antigen(s) associated with the virus nonproducer state. Since EBNA is known to be expressed by all nonproducer EBV-genome-carrying cells, we have compared the effect of partially purified EBNA and correspondingly prepared mock-EBNA on the leukocyte migration. Purified EBNA inhibited the leukocyte migration of EBV seropositives, whereas mock-EBNA had no such effect.     

Computer-assisted morphometry of the intracapillary leukocyte pool in the rabbit lung

Computer-assisted morphometry was performed to evaluate the number and cell characteristics of capillary and alveolar leukocytes in rabbit lungs. An image-processing system and a programmable spreadsheet program were used, which allowed morphometric analysis of a large reference area. Neutrophils represented the largest intracapillary leukocyte population (2.2×10⁷/ml parenchyma, which corresponds to an approximately 104-fold microvascular enrichment of this cell type related to cell counts calculated for the capillary blood volume). In addition, large numbers of intracapillary lymphocytes (1.7×10⁷/ml parenchyma; 47-fold enrichment) and monocytes (0.3×10⁷/ml parenchyma; 86-fold enrichment) were detected. The total count of pulmonary leukocytes thus approximated the total number of pulmonary endothelial cells; and the total circulating pools of the different leukocytes were surpassed by the corresponding lung capillary pools, 3.2-fold for neutrophils, 1.2-fold for lymphocytes and 4.8-fold for monocytes. In contrast, alveolar cell numbers ranged from 1–2% of the capillary counts for all types of leukocytes. We conclude that the rabbit lung microvasculature harbours large pools of immunocompetent cells, which may contribute to host-defense mechanisms at the gas-exchange area.     

Protein synthesis rates of human PBMC and PMN can be determined simultaneously in vivo by using small blood samples

Immune cell functions can be evaluated in vivo by measuring their specific protein fractional synthesis rates (FSR). Using stable isotope dilution techniques, we describe a new method allowing simultaneous in vivo assessment of FSR in two leukocyte populations in healthy human subjects, using small blood samples. Peripheral blood mononuclear cell (PBMC) and polymorphonuclear neutrophil (PMN) FSR were measured during primed continuous intravenous infusion of L-[1-¹³C]leucine. Immune cells from 6 ml of whole blood were isolated by density gradient centrifugation. In a first study, we calculated the FSR using plasma [¹³C]leucine or -[¹³C]ketoisocaproate (KIC) enrichments as precursor pools. In a second study, we compared protein FSR in leukocytes, using enrichments of either intracellular or plasma free [¹³C]leucine as immediate precursor pools. The present approach showed a steady-state enrichment of plasma and circulating immune cell free [¹³C]leucine precursor pools. The linearity of labeled amino acid incorporation rate within mixed PBMC and PMN proteins also was verified. Postabsorptive protein FSR was 4.09 ± 0.39%/day in PBMC and 1.44 ± 0.08%/day in PMN when plasma [¹³C]KIC was the precursor pool. The difference between PBMC and PMN FSR was statistically significant, whatever the precursor pool used, suggesting large differences in their synthetic activities and functions. Use of the plasma [¹³C]KIC pool led to an underestimation of leukocyte FSR compared with the intracellular pool (PBMC: 6.04 ± 0.94%/day; PMN: 2.98 ± 0.30%/day). Hence, the intracellular free amino acid pool must be used as precursor to obtain reliable results. In conclusion, it is possible to assess immune cell metabolism in vivo in humans by using small blood samples to directly indicate their metabolic activity in various clinical situations and in response to regulating factors. peripheral blood mononuclear cells; polymorphonuclear neutrophils; protein metabolism; stable isotopes; leucine     

Real-Time Imaging Reveals the Dynamics of Leukocyte Behaviour during Experimental Cerebral Malaria Pathogenesis

During experimental cerebral malaria (ECM) mice develop a lethal neuropathological syndrome associated with microcirculatory dysfunction and intravascular leukocyte sequestration. The precise spatio-temporal context in which the intravascular immune response unfolds is incompletely understood. We developed a 2-photon intravital microscopy (2P-IVM)-based brain-imaging model to monitor the real-time behaviour of leukocytes directly within the brain vasculature during ECM. Ly6C^hi monocytes, but not neutrophils, started to accumulate in the blood vessels of Plasmodium berghei ANKA (PbA)-infected MacGreen mice, in which myeloid cells express GFP, one to two days prior to the onset of the neurological signs (NS). A decrease in the rolling speed of monocytes, a measure of endothelial cell activation, was associated with progressive worsening of clinical symptoms. Adoptive transfer experiments with defined immune cell subsets in recombinase activating gene (RAG)-1-deficient mice showed that these changes were mediated by Plasmodium-specific CD8⁺ T lymphocytes. A critical number of CD8⁺ T effectors was required to induce disease and monocyte adherence to the vasculature. Depletion of monocytes at the onset of disease symptoms resulted in decreased lymphocyte accumulation, suggesting reciprocal effects of monocytes and T cells on their recruitment within the brain. Together, our studies define the real-time kinetics of leukocyte behaviour in the central nervous system during ECM, and reveal a significant role for Plasmodium-specific CD8⁺ T lymphocytes in regulating vascular pathology in this disease.     

Letter: Limited licensure.

We examined the relationship of leukocyte colony-stimulating activity (CSA) in vitro to neutrophil count in vivo. Using a standard two-layer system, cultures of 10⁶ leukocytes were assayed for their ability to stimulate colony formation by human bone marrow colony-forming cells. The total leukocyte CSA per ml (TLCSA) of blood varied directly with the blood neutrophil count in a group of patients with a wide range in blood neutrophil count, and in two patients recovering from neutropenia in whom serial observations were made. In the latter two patients the rise in TLCSA did not antedate the rise in blood neutrophil count, suggesting that blood leukocyte colony-stimulating factor (CSF) per se probably has little biologic significance. However, release into the circulation of cells which generate CSF could be an important way of controlling the amount of CSF acting within the marrow. In one patient the CSA of dialyzed serum increased after the rise in TLCSA, while undialyzed serum contained no CSA.     

Inhibition of an vitro cell-mediated response: further experiments on the cell lineage and target of suppressor cells

The contribution of lymphotoxin to guinea pig leukocyte natural cytotoxicity was evaluated with [³H]TdR release and colony-inhibition assays of 104C1 benzo(a)pyrene in vitro-transformed and tumorigenic, tumor-specific transplantation antigen-negative, syngeneic strain 2/ N fibroblasts. Cytolethal ³H-release activities of mitogen (PHA)¹-stimulated nonimmune and ovalbumin (OA) immune as well as OA-stimulated OA immune unfractionated, adherent (macrophage-enriched) and nonadherent peritoneal leukocytes are qualitatively similar. ³H release is maximal by 48 hr, increases with antigen or mitogen concentration, is greatest with unfractionated leukocytes, and is least with adherent macrophages. Lymphotoxin produced by peritoneal leukocytes, alone or in combination with the leukocytes does not or only minimally induces ³H release even after 6 days of incubation with guinea pig target cells although guinea pig lymphotoxin possesses cytolytic activity as indicated by ³H release from αL929 mouse tumor cells. In contrast to the absent or very weak cytolytic activity of guinea pig lymphotoxin for the guinea pig target cells nonimmune macrophages, nonadherent leukocytes, and lymphotoxin all exhibit readily detectable colony-inhibitory (CI) activity for the syngeneic tumor cells. Macrophage and lymphotoxin CI, moreover, are additive, whereas nonadherent leukocyte and lymphotoxin CI are synergistic. The latter may be due to additional lymphotoxin induced by target cell antigens or other mechanisms of target cell stimulation of effector lymphoid cells and result from very high local levels of lymphotoxin released by the effector cells. Lymphotoxin CI, furthermore, can be cytostatic or cytolethal as indicated by resumption of 104C1 but not αL929 colony growth following removal of lymphotoxin, indicating that natural cell-mediated cytotoxicity consists of lymphotoxin-dependent and -independent cytostatic and cytolethal effector mechanisms.     

HLA-B8 phenotype associated with an increased mixed leukocyte reaction

Peripheral blood leukocytes from normal volunteers and from patients with untreated Hodgkin's diseases have been assayed for the magnitude of their responses to pools of selected stimulating cells in a quantitative one-way mixed leukocyte reaction (MLR). Responses by leukocytes from individuals withHLA-B8 segregated above the median response regardless of whether the donors were healthy or were patients with Hodgkin's disease. These results suggest the presence of a gene (or genes) in the responding cells that has an effect on the magnitude of the MLR. This gene is in nonrandom association with the gene coding forB8 at theB region of theHLA complex. The observed linkage disequilibrium ofB8 with a gene controlling the proliferative response to alloantigens may be the basis for the increased frequency ofB8 in long-term survivors in carcinoma of the breast and Hodgkin's disease, and for the association ofB8 with several autoaggressive diseases.     

Isolation of Leukocytes from the Human Maternal-fetal Interface

Pregnancy is characterized by the infiltration of leukocytes in the reproductive tissues and at the maternal-fetal interface (decidua basalis and decidua parietalis). This interface is the anatomical site of contact between maternal and fetal tissues; therefore, it is an immunological site of action during pregnancy. Infiltrating leukocytes at the maternal-fetal interface play a central role in implantation, pregnancy maintenance, and timing of delivery. Therefore, phenotypic and functional characterizations of these leukocytes will provide insight into the mechanisms that lead to pregnancy disorders. Several protocols have been described in order to isolate infiltrating leukocytes from the decidua basalis and decidua parietalis; however, the lack of consistency in the reagents, enzymes, and times of incubation makes it difficult to compare these results. Described herein is a novel approach that combines the use of gentle mechanical and enzymatic dissociation techniques to preserve the viability and integrity of extracellular and intracellular markers in leukocytes isolated from the human tissues at the maternal-fetal interface. Aside from immunophenotyping, cell culture, and cell sorting, the future applications of this protocol are numerous and varied. Following this protocol, the isolated leukocytes can be used to determine DNA methylation, expression of target genes, in vitro leukocyte functionality (i.e., phagocytosis, cytotoxicity, T-cell proliferation, and plasticity, etc.), and the production of reactive oxygen species at the maternal-fetal interface. Additionally, using the described protocol, this laboratory has been able to describe new and rare leukocytes at the maternal-fetal interface.     

Enteric loss of plasma-proteins in Strongyloides-infection of pigs

Three out of six piglets at the age of 8 weeks were subcutaneously infected with 10, 000 third-stage larvae of Strongyloides ransomi per kg body weight (Group A). The other three animals served as controls (Group B). Ten days post-infection each piglet received 5 μCi ⁵¹Cr-albumin intravenously. Daily measurements of ⁵¹Cr in plasma, faeces and urine showed that the infected animals excreted 6·5 times more ⁵¹Cr in the faeces than the controls during the 10-day experimental period. The total plasma volume was higher in the infected group than in the controls. The plasma-albumin was significantly reduced by the infection, however the total plasma protein concentration decreased without statistical significance.A ⁵¹Cr-plasma concentration curve calculated on the basis of daily faecal plus urinary ⁵¹Cr-excretion was different from the concentration curve obtained by daily ⁵¹Cr-measurements in plasma. From the difference between both curves the amount of ⁵¹Cr lost to an extravascular pool of the body was calculated. In both groups this extravascular ⁵¹Cr-pool reached its maximum within one day. Due to higher faecal ⁵¹Cr-excretion in the infected group the extravascular ⁵¹Cr decreased more rapidly with time than in the control group (2·44% of dose/day: 0·86% of dose/day). It is concluded that the extravascular pool of ⁵¹Cr is built up by ⁵¹Cr-albumin and free trivalent ⁵¹Cr.