PMN Subpopulations Research Articles

Resting and activated bovine neutrophils and eosinophils differ in their responses to adrenergic agonists

Polymorphonuclear cells (PMN) provide a rapid response to infection and tissue damage and stress can modify these critical innate immune defences. The study of adrenergic receptor (AR) expression and function in bovine PMNs is limited but both neutrophils and eosinophils express numerous AR genes but differ significantly in their expression of individual AR genes. A flow cytometric technique was developed to differentiate between bovine neutrophils and eosinophils so both neutrophil and eosinophil responses to adrenergic agonists could be analysed. Neutrophils and eosinophils displayed significantly different changes in CD11b, L-selectin, and CD44 expression when activated by bovine serum opsonized zymosan and recombinant bovine interferon gamma. The responses of activated and resting neutrophils and eosinophils were then compared following stimulation with endogenous adrenergic agonists, epinephrine (E) norepinephrine (NE), and synthetic agonists targeting α1-, α2-, or β-ARs. Both resting and activated neutrophils and eosinophils displayed differences in iROS, CD44, and L-selectin expression following stimulation with E and NE. Resting neutrophils displayed pro-inflammatory responses to both E and NE, while resting eosinophils displayed a pro-inflammatory response to only NE. No single synthetic adrenergic agonist fully recapitulated responses observed with either E or NE and responses to adrenergic agonists were dose-dependent. In conclusion, bovine eosinophils and neutrophils responded to multiple adrenergic agonists by altering expression of proteins involved in immune surveillance and pro-inflammatory responses. Significant differences in neutrophil and eosinophil responses to adrenergic agonists are consistent with their differences in AR gene expression. This highlights the importance of analysing separately these two PMN subpopulations when investigating the effects of either endogenous or synthetic AR agonists.

KPT-330, a potent and selective CRM1 inhibitor, exhibits anti-inflammation effects and protection against sepsis

Sepsis, a systemic inflammatory response caused by infection or injury, is still one of the most important causes of death in clinical patients. The ongoing search for the pathogenesis of sepsis and novel therapeutic methods are highly urgent. In this study, we hypothesized that KPT330, a potent and specific small molecule inhibitor of CRM1, could reduce inflammation and attenuate the severity of sepsis. In LPS-induced sepsis model in vivo, administration of KPT330 increased survival rate and ameliorated LPS-induced lung injury, with suppressed levels of TNF-α, IL-6 and HMGB1 in the circulation and decreased macrophage and PMN subpopulations in peritoneal cavity. In vitro investigations showed that KPT330 dose-dependently inhibited LPS-triggered proinflammatory cytokines production including TNF-α, IL-6 and HMGB1 in macrophages. Furthermore, KPT330 treatment significantly suppressed TNF-α and IL-6 mRNA expression and inhibited HMGB1 necleocytoplasmic translocation by inhibiting CRM1 distribution. Moreover, the mechanism analysis demonstrated that KPT330 exerted anti-inflammation effects by inhibiting the production of pro-inflammatory cytokines through suppressing activation of NF-κB and p38 signaling. Thus, pharmacologic stimulation of KPT330 may present a promising therapeutic strategy for sepsis.

Class I odorant receptors, TAS1R and TAS2R taste receptors, are markers for subpopulations of circulating leukocytes.

Our cellular immune system has to cope constantly with foodborne substances that enter the bloodstream postprandially. Here, they may activate leukocytes via specific but yet mostly unknown receptors. Ectopic RNA expression out of gene families of chemosensory receptors, i.e., the ∼400 ORs, ∼25 TAS2R bitter-taste receptors, and the TAS1R umami- and sweet-taste receptor dimers by which we typically detect foodborne substances, has been reported in a variety of peripheral tissues unrelated to olfaction or taste. In the present study, we have now discovered, by gene-specific RT-PCR experiments, the mRNA expression of most of the Class I ORs (TAS1R) and TAS2R in 5 different types of blood leukocytes. Surprisingly, we did not detect Class II OR mRNA. By RT-qPCR, we show the mRNA expression of human chemosensory receptors and their cow orthologs in PMN, thus suggesting an evolutionary concept. By immunocytochemistry, we demonstrate that some olfactory and taste receptors are expressed, on average, in 40-60% of PMN and T or B cells and largely coexpress in the same subpopulation of PMN. The mRNA expression and the size of subpopulations expressing certain chemosensory receptors varied largely among individual blood samples, suggesting a regulated expression of olfactory and taste receptors in these cells. Moreover, we show mRNA expression of their downstream signaling molecules and demonstrate that PTX abolishes saccharin- or 2-PEA-induced PMN chemotactic migration, indicating a role for Gi-type proteins. In summary, our data suggest "chemosensory"-type subpopulations of circulating leukocytes.

The effect of supportive E. coli mastitis treatment on PMN chemiluminescence and subpopulations of T lymphocytes

The aim of this field study was to assess the impact of a single i.m. injection of lysozyme dimer and flunixin meglumine in combination with intramammary and systemic antibiotic on chemiluminescence of PMN (polymorphonuclear leucocytes) and subpopulations of lymphocyte T in blood of cows with E. coli mastitis. Examinations were performed on 30 dairy cows affected with naturally occurring acute form of E. coli mastitis. Cows were randomly divided into three groups according to the method of treatment. The first group was treated with approved intramammary antibiotic product, the same antibiotic in i.m. injection and one injection of flunixin meglumine on the first day of therapy. Next group was treated with the same antibiotic and additionally one injection of lysozyme dimer on the first day of therapy. The third one was treated only with an antibiotic and served as a control group. Blood samples were taken before treatment and on days 3 and 7. In samples haematology indices were determined, spontaneous and opsonised zymosan stimulated CL and PMA measurements were performed and the subpopulations of T lymphocyte (CD2(+), CD4(+), CD8(+)) were assayed in whole blood. There was no effect of the applied supportive treatment on the value of morphological blood indices. A significant influence of the time of sample collection on the level of CL and dynamics of lymphocytes T subpopulation was demonstrated. A single injection of flunixin meglumine or lysozyme dimer on the day of the beginning of treatment of E. coli mastitis, does not affect the level of neutrophil chemiluminescence and the percentage of T lymphocytes in the blood of mastitic cows in the analysed period of time.

Biogenic amines activate blood leukocytes via trace amine-associated receptors TAAR1 and TAAR2

Certain biogenic amines, such as 2-PEA, TYR, or T1AM, modulate blood pressure, cardiac function, brain monoaminergic systems, and olfaction-guided behavior by specifically interacting with members of a group of rhodopsin-like receptors, TAAR. A receptor that is absent from olfactory epithelia but had long been identified in the brain and a variety of peripheral tissues, TAAR1 has been found recently in blood B cells, suggesting a functional role of TAAR1 in these cells. With the present study, we have set out to clarify the expression and functional roles of TAAR in different isolated human blood leukocyte types. Here, we report the functional expression of TAAR1 and its closest relative TAAR2 in blood PMN and T and B cells. Both receptors are coexpressed in a subpopulation of PMN, where they are necessary for the chemosensory migration toward the TAAR1 ligands 2-PEA, TYR, and T1AM, with EC50 values of 0.43 ± 0.05 nM, 0.52 ± 0.05 nM, and 0.25 ± 0.04 nM, respectively. The same amines, with similar potencies, triggered cytokine or Ig secretion, in purified blood T or B cells, respectively. Notably, 2-PEA regulated mRNA expression of 28 T cell function-related genes, above all of the CCL5. In siRNA-guided experiments, TAAR1 and TAAR2 proved to be necessary for amine-induced blood leukocyte functions. In summary, our results demonstrate that biogenic amines potently regulate blood cell functions via TAAR1 and TAAR2 and open the perspective of their specific pharmacological modulation.

Neonatal Neutrophils with Prolonged Survival Secrete Mediators Associated with Chronic Inflammation

Background: The resolution of inflammation involves the efficient removal of apoptotic neutrophils (PMN). However, a subpopulation of PMN that are resistant to apoptosis may contribute to PMN persistence in tissues, an early hallmark of chronic inflammation. We previously made observations that neonatal PMN with prolonged survival had augmented expression of CD18/CD11b, an adhesion molecule critical to inflammation. Objectives: The objectives of this study were to test the hypothesis that surviving neonatal PMN retain the capacity to secrete key mediators associated with chronic inflammation. Methods: We profiled cytokine and chemokine secretion patterns of lipopolysaccharide (LPS)-stimulated neonatal and adult PMN using multicytokine array and ELISA. Results: We observed that surviving 24-hour neonatal PMN stimulated with LPS had enhanced secretion of interleukin (IL)-8, a chemokine involved in PMN activation and recruitment. In addition, 24-hour neonatal PMN secreted levels of monocyte inhibitory protein (MIP)-1β that were higher than those secreted by 0-hour PMN, but amounts of IL-1 receptor antagonist (IL-1Ra) were lower. Conclusions: The results of the present study extend previous observations of augmented function in surviving neonatal neutrophils, and further suggest their potential contribution to the pathogenesis of inflammatory disorders in neonates.

Priming study of human phagocytes oxidative burst by using flow cytometry.

In response to a variety of stimuli, e.g. pathogens, phagocytes release reactive oxygen species which are essential for bacterial killing and also potentiate inflammatory reactions. We have used flow cytometry measurements to study the priming process of phagocyte oxidative burst in whole blood, in order to avoid introducing artefacts due to the purification process and to stimulate the in vivo situation more closely. In these conditions, we examined the in vitro effects of proinflammatory cytokines (TNF-alpha, IL-1 alpha, IL-1 beta, IL-6, IL-8 and GM-CSF) on the PMN oxidative burst. We found that none of the cytokine tested directly activated the PMN oxidative burst. In contrast, TNF, GM-CSF and IL-8 strongly primed a subpopulation of PMN which produced large amounts of H2O2 in response to fMLP, suggesting that these cytokines may play a critical role in bactericidal killing in vivo. Furthermore, we reported a decreased H2O2 production by TNF or IL-8 primed PMN in HIV-infected patients. This impairment, which correlated with the clinical stage of the disease, could contribute to the increased susceptibility to bacterial infections in HIV-infected patients. In addition, we reported the case of a child with severe recurrent infections due to intracellular microorganisms which could be related to an impairment of the phagocyte priming process of the oxidative burst [corrected].

The role of cellular maturation in neutrophil heterogeneity

Previous studies have shown that many neutrophil (PMN) characteristics are heterogeneous but the origin of PMN heterogeneity is unknown. It is unclear if PMN functional heterogeneity is secondary to maturational differences or due to distinct subpopulations of cells that possess different functional capacities. The PMN 31D8 antigen is a useful probe for evaluation of PMN subpopulations. The majority of PMNs (approximately 85%) exhibit a high intensity fluorescence after 31D8 monoclonal antibody (MoAb) labeling (31D8 enriched or “brightxyc PMNs) as determined by flow cytometric analysis. These cells are more functional than cells with low intensity fluorescence (31D8 diminished or “dull” PMNs). Various immunologic, clonogenic and functional techniques were used to study the expression of the 31D8 antigen in HL-60 cells and myeloid cells in order to evaluate antigenic and functional heterogeneity during morphologic maturation. The results of this study indicate that the percentage of 31D8 antigen positive (31D8 antigen enriched and diminished) bone marrow cells increases from 20 +/- 11% in myeloblast cells to 68 +/- 10% in promyelocytes, 93 +/- 2% in myelocytes and 99 +/- 1% in bands and PMNs. 31D8 antigen enriched cells first appear at the myelocyte stage (32 +/- 10%) and increase in bands (52 +/- 13%), marrow PMNs (62 +/- 13%) and peripheral blood PMNs (88 +/- 4%). These data indicate that the heterogeneous expression of 31D8 antigen in PMNs is due, at least in part, to maturational differences within the PMN population and raise the possibility that other heterogeneously expressed PMN characteristics are also maturationally derived. They also suggest that 31D8 antigenic expression may be a more precise indicator of myeloid functional maturation than maturation as identified by cellular morphology.

The Role of Cellular Maturation in Neutrophil Heterogeneity

Previous studies have shown that many neutrophil (PMN) characteristics are heterogeneous but the origin of PMN heterogeneity is unknown. It is unclear if PMN functional heterogeneity is secondary to maturational differences or due to distinct subpopulations of cells that possess different functional capacities. The PMN 31D8 antigen is a useful probe for evaluation of PMN subpopulations. The majority of PMNs (approximately 85%) exhibit a high intensity fluorescence after 31D8 monoclonal antibody (MoAb) labeling (31D8 enriched or "bright" PMNs) as determined by flow cytometric analysis. These cells are more functional than cells with low intensity fluorescence (31D8 diminished or "dull" PMNs). Various immunologic, clonogenic and functional techniques were used to study the expression of the 31D8 antigen in HL-60 cells and myeloid cells in order to evaluate antigenic and functional heterogeneity during morphologic maturation. The results of this study indicate that the percentage of 31D8 antigen positive (31D8 antigen enriched and diminished) bone marrow cells increases from 20 +/- 11% in myeloblast cells to 68 +/- 10% in promyelocytes, 93 +/- 2% in myelocytes and 99 +/- 1% in bands and PMNs. 31D8 antigen enriched cells first appear at the myelocyte stage (32 +/- 10%) and increase in bands (52 +/- 13%), marrow PMNs (62 +/- 13%) and peripheral blood PMNs (88 +/- 4%). These data indicate that the heterogeneous expression of 31D8 antigen in PMNs is due, at least in part, to maturational differences within the PMN population and raise the possibility that other heterogeneously expressed PMN characteristics are also maturationally derived. They also suggest that 31D8 antigenic expression may be a more precise indicator of myeloid functional maturation than maturation as identified by cellular morphology.

Deficiency of glycosyl‐phosphatidylinositol anchored proteins on paroxysmal nocturnal haemoglobinuria (PNH) neutrophils and monocytes: heterogeneous deficiency of decay‐accelerating factor (DAF) and CD16 on PNH neutrophils

Glycosyl-phosphatidylinositol (GPI) anchored membrane proteins have been reported to be deficient on affected paroxysmal nocturnal haemoglobinuria (PNH) blood cells. In the present study we investigated the deficiency of several GPI anchored membrane proteins on PNH neutrophils (PMN) and monocytes from 10 patients with PNH. Decay-accelerating factor (DAF) and Fc gamma R-III (CD16) on PMN, DAF and CD14 on monocytes, were investigated by two-colour immunofluorocytometry. Neutrophil alkaline phosphatase activity was also assayed on PNH neutrophils. Normal human PMN were always shown phenotypically to be DAF+/CD16+. A DAF-/CD16- subpopulation of PMN was demonstrated in all the patients studied. In six out of the 10 patients, deficiencies of DAF and CD16 were found simultaneously on affected PNH PMN. The percentage of DAF- PMN showed a positive correlation with the neutrophil alkaline phosphatase (NAP) score. However, it should be noted that, in four out of the 10 patients with PNH, a DAF+/CD16- subpopulation of PMN was also clearly found. This may indicate that the deficiencies of DAF and CD16 on PNH PMN are heterogeneous. Normal human monocytes were demonstrated to be DAF+/CD14+, whereas PNH monocytes consisted of subpopulations of DAF+/CD14+ and DAF-/CD14-. In the same patients with PNH, the deficiencies of DAF on PMN and monocytes correlated well with each other. These results suggest that, at least in some patients with PNH, the mechanisms which induce the membrane defects of PNH blood cells are heterogeneous.

HETEROGENEITY OF NEUTROPHIL (PMN) FUNCTION AND MONOCLONAL ANTIBODY (MAb) BINDING IN NEONATES AND ADULTS

Studies have shown that PMN from adults and neonates exhibit heterogeneity of function and 31D8 MAb binding. The heterogenity of 31D8 binding correlates with PMN motile heterogeneity. MOI MAb binds to PMN glycoproteins which allow the PMN to adhere and move normally. We further studied PMN heterogeneity in 10 neonates and 10 adults by placing PMN in the upper compartment of chemotactic chambers and allowing them to transverse 10 μm polycarbonate filters in response to C5 frag. PMN were analyzed for viability, band count and MAb binding with 31D8 and MO1. The % of non-motile (NM) neonatal and adult PMN recovered from the upper compartment after 90 min. were 7+1% and 4 ± 1%, respectively (P<0.01). The % highly motile (HM) neonatal and adult PMN from the lower compartment were 5 ± 2% and 11 ± 2%, respectively (P<0.01). The viability and % bands in NM PMN were not significantly different from control PMN. The NM PMN had significantly less 31D8 and MO1 MAb binding than the HM PMN sub-populations. Neonatal NM and HM PMN had significantly less 31D8 and MO1 MAb binding than adult NM and HM PMN. These data suggest that variable 31D8 and MO1 antigen expression may contribute to differences in motile subpopulations of PMN in neonates and adults and are at least in part responsible for differences between neonatal and adult PMN motility. Decreased PMN chemotaxis in neonates may also result from a larger subpopulation of NM PMN and a smaller subpopulation of HM PMN.

Functional analysis of the marginating pool of human polymorphonuclear leukocytes.

The intravascular pool of human polymorphonuclear leukocytes (PMN) is composed of one compartment which is circulating and another that is marginated to the vascular endothelium. Administration of B-adrenergic agonists leads to a rapid demargination with an increase in the circulating PMN pool. The marginating PMN has previously been stated to represent an older PMN based on a higher cytochemical alkaline phosphatase activity. With the understanding that circulating PMN are heterogeneous with respect to function and size we undertook the present study to evaluate the contribution of the marginating PMN to functional and volume-dependent heterogeneity. We found that PMN isolated 7 min after epinephrine administration, presumably enriched by marginating PMN, were not different in volume, biochemically measured alkaline phosphatase activity, stimulated superoxide anion release, degranulation, or phagocytosis. These data suggest that the circulating and marginating pools of PMN are interchangeable and that the marginating PMN are not enriched by a particular subpopulation of PMN.

Interaction of Ia antigen-bearing polymorphonuclear leukocytes and murine splenocytes.

Polymorphonuclear leukocytes (PMN) were induced in the peritoneum of a Balb/c mouse by ip injection of Fusobacterium nucleatum (FN) (greater than 95% PMN). A subpopulation of PMN harvested bore Ia surface antigens and stimulated a mixed lymphocyte reaction (MLR) when cultured with C57B1/6J splenocytes. The reaction was blocked by a short prior incubation of PMN with anti-Ia antibody or PMN cell depletion by the same antibody plus complement. The Ia antigen-bearing PMN were capable of antigenic modulation since incubation of PMN for 24 h rendered the cells incapable of stimulating an MLR. The Ia antigen-bearing PMN produced a soluble material that enhanced the phytohemagglutinin (PHA) response of murine splenocytes and the active material was a product of live cells since the supernatants contained no detectable lactate dehydrogenase activity. The data suggest that murine PMN subpopulations, defined by surface Ia antigen, can modulate mitogenic responses by production of an enhancing factor(s).

Modifications of granulocyte subpopulations and of their adherence property during filtration leukapheresis in man

Summary. Polymorphonuclear leucocytes (PMN) isolated from the peripheral blood of 23 blood donors undergoing filtration leukapheresis were examined with regard to their number, expression of membrane receptors for rabbit IgG or human C3b and their ability to adhere to nylon fibres. All the donors showed an early granulocytopenia followed by a gradual return of PMN into the circulation, but differed in the number of PMN appearing in the rebound phase. Evaluation of rosette forming cells (RFC) and cells adhering to nylon fibres in this phase led to the recognition of two main groups of donors. In one of these groups the number of PMN, RFC and adhering cells remained unchanged compared to the pre‐apheresis values. The other group showed marked leucocytosis accompanied by a relative increase in the number of non‐RFC and non‐adhering cells. Possible mechanisms involved in the changes of PMN subpopulations are discussed.

Modifications of granulocyte subpopulations and of their adherence property during filtration leukapheresis in man.

Polymorphonuclear leucocytes (PMN) isolated from the peripheral blood of 23 blood donors undergoing filtration leukapheresis were examined with regard to their number, expression of membrane receptors for rabbit IgG or human C3b and their ability to adhere to nylon fibres. All the donors showed an early granulocytopenia followed by a gradual return of PMN into the circulation, but differed in the number of PMN appearing in the rebound phase. Evaluation of rosette forming cells (RFC) and cells adhering to nylon fibres in this phase led to the recognition of two main groups of donors. In one of these groups the number of PMN, RFC and adhering cells remained unchanged compared to the pre-apheresis values. The other group showed marked leucocytosis accompanied by a relative increase in the number of non-RFC and non-adhering cells. Possible mechanisms involved in the changes of PMN subpopulations are discussed.