Leukocyte migration: dismantling inhibition

A new study demonstrates that the Src-family kinases Fgr and Hck inhibit chemokine signaling in polymorphonuclear leukocytes and dendritic cells by phosphorylation of PIR-B, an inhibitory receptor expressed on leukocytes. In resting cells, PIR-B phosphorylation is constitutive but is decreased transiently by addition of chemokines. In Fgr/Hck-deficient cells, constitutive PIR-B phosphorylation is markedly decreased. These Src-family kinases have a novel role in tonic inhibition of cell activation that must be overcome to allow the phenotypic effects of chemokine signaling through G-protein-coupled receptor ligands.     

Fibroblastic reticular cells guide T lymphocyte entry into and migration within the splenic T cell zone

Although a great deal is known about T cell entry into lymph nodes, much less is understood about how T lymphocytes access the splenic white pulp (WP). We show in this study that, as recently described for lymph nodes, fibroblastic reticular cells (FRCs) form a network in the T cell zone (periarteriolar lymphoid sheath, PALS) of the WP on which T lymphocytes migrate. This network connects the PALS to the marginal zone (MZ), which is the initial site of lymphocyte entry from the blood. T cells do not enter the WP at random locations but instead traffic to that site using the FRC-rich MZ bridging channels (MZBCs). These data reveal that FRCs form a substrate for T cells in the spleen, guiding these lymphocytes from their site of entry in the MZ into the PALS, within which they continue to move on the same network.     

Leukocyte migration in experimental inflammatory bowel disease

Emigration of leukocytes from the circulation into tissue by transendothelial migration, is mediated subsequently by adhesion molecules such as selectins, chemokines and integrins. This multistep paradigm, with multiple molecular choices at each step, provides a diversity in signals. The influx of neutrophils, monocytes and lymphocytes into inflamed tissue is important in the pathogenesis of chronic inflammatory bowel disease. The importance of each of these groups of adhesion molecules in chronic inflammatory bowel disease, either in human disease or in animal models, will be discussed below. Furthermore, the possibilities of blocking these different steps in the process of leukocyte extravasation in an attempt to prevent further tissue damage, will be taken into account.     

Leukocyte migration inhibition reaction in trophoblastic tumor patients]

Inhibition of leukocyte migration was studied in 40 patients with trophoblastic tumours under the effect of protein extract from chorionepithelioma. A marked inhibition of leukocyte migration was revealed in patients with signs of active tumour process before and during the treatment. Four patients in whom no inhibition was revealed either before or during the treatment were an exception. In the majority of the patients the inhibition effect was absent against the background of clinical well-being achieved as a result of surgical and chemotherapeutic, or chemotherapeutic treatment alone. Leukocytes of healthy donors failed to respond to the tumour extract in all 24 cases.     

Leukocyte polarization in cell migration and immune interactions.

Cell migration plays a key role in a wide variety of biological phenomena. This process is particularly important for leukocyte function and the inflammatory response. Prior to migration leukocytes undergo polarization, with the formation of a lamellipodium at the leading edge and a uropod at the trailing edge. This cell shape allows them to convert cytoskeletal forces into net cell-body displacement. Leukocyte chemoattractants, including chemokines, provide directional cues for leukocyte motility, and concomitantly induce polarization. Chemoattractant receptors, integrins and other adhesion molecules, cytoskeletal proteins and intracellular regulatory molecules change their cellular localization during cell polarization. A complex system of signal transduction molecules, including tyrosine kinases, lipid kinases, second messengers and members of the Rho family of small GTPases is thought to regulate the cytoskeletal rearrangements underlying leukocyte polarization and migration. The elucidation of the mechanisms and signals that control this complex reorganization will lead to a better understanding of critical questions in cell biology of leukocyte migration and polarity.     

Leukocyte transepithelial migration in lung induced by DMSA functionalized magnetic nanoparticles

Magnetic nanoparticles surface-covered with meso-2,3-dimercaptosuccinic acid (MNPs-DMSA) constitute a promising approach for tissue- and cell-targeted delivery of therapeutic drugs in the lung. However, they can also induce a transient transendothelial migration of leukocytes in the organ as a side effect after endovenous administration of MNPs-DMSA. We demonstrated that monocytes/macrophages constitute the main subpopulation of leukocytes involved in this process. Our recent research found that MNPs-DMSA upregulated the mRNA expression of E-, L- and P-selectin and macrophage-1 antigen and increased concentration of tumor necrosis factor α (TNFα) in lung, in a time dependent manner. The critical relevance of the β₂ integrin-dependent pathway in leukocyte transmigration elicited by MNPs-DMSA was demonstrated by use of knockout mice. Our work characterizes mechanisms of the pro-inflammatory effects of MNPs-DMSA in the lung and identifies β₂ integrin-targeted interventions as promising strategies to reduce pulmonary side effects of MNPs-DMSA during biomedical applications. In addition, MNPs-DMSA could be used as modulators of lung immune response.Key words: magnetic nanoparticles, DMSA, nanobiotechnology, transepithelial migration, cell adhesion molecules, integrins, monocytes, lungNanotechnology deals with structures of 100 nm or smaller in at least one dimension and has the potential to create many new materials and devices with a vast range of applications. Materials can be produced that are nanoscale in one dimension (for example, very thin surface coatings), in two dimensions (for example, nanowires and nanotubes) or in all three dimensions (for example, nanoparticles).Magnetic nanoparticles (MNPs) are a class of nanoparticles that can be manipulated using a magnetic field. MNPs are traditionally ferrite-based materials with the general formula MFe₂O₄, where M is a doubly charged metal-ion, such as iron, nickel or cobalt. Magnetic fluids (MFs) are colloidal mixtures composed of MNPs suspended in a carrier fluid, usually an organic or inorganic solvent. There is an increasing interest in developing biocompatible MFs for biomedical applications¹ for instance, for detection of circulating tumor cells,² contrast agents for magnetic resonance imaging³ and in an experimental cancer treatment called magnetic hyperthermia in which the fact that nanoparticles heat when they are placed in an alternative magnetic field is used.⁴ Another potential use includes attaching magnetic nanoparticles to drug/gene for targeting purposes.⁵ In order to be used for medical applications, magnetic nanoparticles are coated with a surfactant to prevent their agglomeration (due to van der Waals and magnetic forces) and allow the association of MNPs surface with different molecules.^6,7In previous studies, we have shown that MNPs surface-coated with meso-2,3-dimercaptosuccinic acid (MNPs-DMSA) (Fig. 1), with average diameter of about 9 nm, presented preferential distribution in the lung tissue, after intravenous administration in mice.^8–10 This target specificity of MNPs-DMSA offers a unique property that may be successfully exploited for the treatment of lung diseases.¹¹ In addition, we reported that the presence of MNPs-DMSA in the lung led to trafficking of leukocytes from blood vessels into pulmonary parenchyma and airspace and that interleukin-1 (IL-1) and interleukin-6 (IL-6) were overexpressed.¹² IL-1 acts as a trigger that activates a cascade of cytokine production and induces the production of a wide range of immunomodulatory cytokines.¹³ IL-6 is among the mediators regulated by IL-1 and is often increased in inflammatory processes in the lung.¹³ These differential expressions were particularly associated with blood vessels and cells of airway ducts suggesting that they could have some role during the recruitment process of inflammatory cells, as observed in histological analyses. In fact, these cytokines are commonly associated with the activation of cells concerning the expression of adhesion surface proteins.¹³ This is in agreement with several studies that described the requirement of IL-1α production in rat airways for full polymorphonuclear cell migration in models for immune-complex deposition or inhalation of cement dust, coal dust or diesel exhaust particles.^14–16Open in a separate windowFigure 1Schematic representation of DMSA-functionalized maghemite nanoparticles.Cell migration plays a key role in a wide variety of biological phenomena. This process is particularly important for leukocyte function and the inflammatory response. A mechanistic understanding of cellular interactions with synthetic surfaces, particularly in the context of inflammatory and healing responses, has been a major goal of biomaterial science.Leukocyte trafficking in the lung involves transendothelial migration, migration in tissue interstitium and transepithelial migration. In addition, leukocyte emigration involves regulatory mechanisms including complement activation, cytokine regulation, chemokine production, activation of adhesion molecules and their respective counter receptors. The process is presumably initiated and modulated by the production of early response cytokines such as IL-1 and tumor necrosis factor (TNF) from lung cells, especially from alveolar macrophages, setting the stage for leukocyte migration through endothelium.¹⁷ On the other hand, ensuing production of interleukin-10 (IL-10) brings into play powerful anti-inflammatory factors that strongly regulate inflammatory responses, functioning as intrinsic regulators of the lung inflammatory response.^18,19Tissue infiltration by circulating leukocytes is a three-step process involving rolling on the endothelium, attachment to the endothelium and transmigration across the endothelial cells lining blood vessel walls (Fig. 2). Leukocyte migration out of the blood is initiated by leukocyte rolling on the luminal side of the endothelium, as mediated by the low-affinity receptors selectins (E-, L- and P-selectin).^20–22 Binding of selectins on leukocytes stimulates “outside-in” signals in these cells, increasing the affinity of the integrin family of receptors (cell surface receptors consisting of an α- and a β-subunit, which are grouped in distinct subfamilies based on β-subunit utilization), which then bind to endothelial cell adhesion molecules such as intercellular adhesion molecule-1 [(ICAM-1)/CD54] and vascular cellular adhesion molecule-1 (VCAM-1). Function-blocking studies have identified the β₁ (CD29) and β₂ (CD18) integrins as the major players involved in leukocyte adhesion and migration.²³ Leukocyte integrin affinity is also rapidly increased by “inside-out” signals from leukocyte chemokine receptors triggered by chemokines displayed on the surface of endothelial cells.²⁴ With an increase in leukocyte integrin receptor affinity, leukocyte rolling is arrested.²⁴Open in a separate windowFigure 2Schematic representation of leukocyte endothelial migration into lung parenchyma.Using immunohistochemistry, we demonstrated that following injection of MF-DMSA, the distribution pattern of E-selectin and members of the β₂ integrin subfamily (macrophage-1 antigen, Mac-1; leukocyte function associated antigen-1, LFA-1) was changed in the lung vessels, but not of β₁ integrin.¹⁰ For L and P selectins no differences were observed between treated and control animals. However, for E-selectin, labeling was found in the endothelium of veins and venules 12 h after MF-DMSA administration, but not in the lung''s vascular compartments of the control and 4 h treatment groups.¹² Concerning integrins, in the control group, leukocytes labeled with Mac-1 and LFA-1 were found only in post-capillary sites. Four hours after MF-DMSA administration, leukocytes expressing these β₂ integrins were also found in capillaries.¹⁰ Our findings expand on other studies showing that the capillary network constitutes an important migration site in the lung.²⁵ Thus, the modulation of Mac-1 and LFA-1 expression in leukocytes located inside capillaries supports the importance of these integrins and capillaries for migratory activity in the lung, in this case after MF-DMSA administration. However, we cannot discard the participation of larger vessels in the migration induced by MNPs-DMSA. In fact, some images from our laboratory have showed that this is also a route used by the leukocytes after injection of these nanoparticles (Fig. 3).Open in a separate windowFigure 3Light microscopy image of leukocytes containing MNPs-DMSA inside a vein. Note that the cells (yellow arrows) are close or attached to the endothelium.It is worth noting that 12 h after MF-DMSA administration, leukocytes labeled with LFA-1 were observed only in post-capillary sites, similar to the control. We speculated that the absence of LFA-1 labeling in capillaries in the period of 12 h after MF-DMSA administration is due to the accentuated decrease of LFA-1 expression levels in the leukocyte over the course of time. In fact, as will be discussed below, we obtained a decrease in the LFA-1 mRNA 12 h after MNPs-DMSA administration. This point of view is in agreement with other studies that demonstrated the distinct contribution of LFA-1 and Mac-1 to transendothelial migration in the lung.²⁶ While both Mac-1 and LFA-1 participate in transendothelial migration at the beginning of the inflammatory process, over time Mac-1 becomes the predominant member of the β₂ integrin subfamily mediating migration of leukocytes.²⁶These results raised several questions related to MNPs-DMSA administration, such as: what is the time profile of leukocyte migration into the airspace? Which is the principal leukocyte subpopulation involved in this process? Is it a fact that the mechanism by which the presence of MNPs-DMSA induces transendothelial migration of leukocytes into the lung is based on their ability to somehow change the expression of cell adhesion molecules on leukocytes and lung vascular endothelial cells? Is β₂ or β₁ integrin, or both, the main receptor involved in MNPs-DMSA leukocyte-induced migration?Recently, we uncovered some of these answers including the main adhesion molecules that are involved in this migration. We first determined that the number of leukocytes in the bronchoalveolar lavage fluid reached its peak 12 h after MNPs-DMSA administration, decreasing to normal values in 48–72 h. Cytologic and FACS analysis demonstrated that the main subpopulation of leukocytes involved in this process was monocyte/macrophage.²⁷It is well known that the reticuloendothelial system, in particular macrophage cells, actively neutralizes and eliminates foreign matter from the body, including nonbiological particles. These and other particulated materials in the lung may lead to lung damage. In fact, transmission electron microscopy analysis clearly demonstrated an uptake of MNPs-DMSA by monocyte/macrophage cells,²⁷ indicating that this may be a mechanism of nanoparticle clearance used by the lung in order to avoid further damage. It is worth noting that an increase in the relative percentage of lymphocytes after MNPs-DMSA administration was also observed. The importance of this finding was not addressed in the paper, but we speculate that it could be important for the control of the inflammatory process initiated by the MNPs-DMSA injection. Failure in control of the inflammatory processes could potentially lead to chronic inflammatory diseases and pulmonary fibrosis. In spite of the fact that we did not determine which was the main source of the production of two different cytokines, one considered pro-inflammatory (TNFα) and the other anti-inflammatory (IL-10), we found an increase in the ratio of IL-10/TNFα cytokine release 12 h after MNPs-DMSA administration. This is clearly a signal that the inflammatory process was being controlled, in agreement with previous reports showing that IL-10 is able to limit the induction of cell adhesion molecules in the lung.²⁸ We presume that lymphocytes are taking part in this process. Further studies are necessary to clarify this point.The nature of the cells present in the pulmonary tissue parenchyma was not determined in this study. However, these cells were not able to cause tissue damage in the lung. We observed no histological or ultrastructural damage in the lung of animals treated with MNPs-DMSA, indicating that the nanoparticle-induced inflammation is not enough to cause chronic disease, such as pulmonary fibrosis.We then determined the effect of MNPs-DMSA on mRNA expression of selectins, integrin β₁ and integrin β₂.²⁷ We found that MNPs-DMSA upregulated the mRNA expression of E-, L- and P-selectin, as well as Mac-1. Further, using knockout mice (deficient in the β₂-subunit common to all β₂ integrins), we observed that, compared to wild-type mice, the recruitment of leukocytes to the airspace following administration of MNPs-DMSA was completely blocked in the former.²⁷ The fact that transmigration of β₂ integrin-deficient monocytes was affected when compared with wild-type monocytes strongly argues in favor of a major contribution by β₂ integrins to monocyte trans-epithelial migration in our system, which is additionally supported by the increase of mRNA of β₂ integrins, as cited above.We should remember, however, that the absence of change in LFA-1 and very late antigen-4 (VLA-4) mRNA does not exclude a role for them in leukocyte migration induced by MNPs-DMSA. Integrins are cell adhesion molecules constitutively expressed on the cell surface and also stored within intracellular vesicles.^29,30 In addition, transendothelial migration of leukocytes depends not only on the number of integrins on the cell surface but also on the change in conformation of these molecules reflecting their activation.³² Therefore, our results did not exclude the possibility that MNPs-DMSA induce the activation of LFA-1 and VLA-4 constitutively located on the surface of leukocytes or the translocation of these integrins from intracellular vesicles to the plasma membrane. On the other hand, the absence of a significant change in the mRNA expression of VCAM-1, which is the major endothelial cell ligand for VLA-4, can be regarded as an indirect indicator that VLA-4 is not involved in this process.The fact that an increase in the mRNA of Mac-1 occurred and there is no change in the mRNA levels of VLA-4 (and LFA-1) corroborates the hypothesis that migration of leukocytes induced by MNPs-DMSA is mainly dependent on β₂ integrins and not β₁ integrins pathway. In addition, we can presume that MAC-1 is the main β₂ integrin molecule involved in the process of leukocyte trafficking.The increased use of nanoparticles in medicine has raised concerns on their ability to gain access to privileged sites in the body. In fact, a study has shown that, in some cases, they can potentially cause damage to tissues located behind cellular barriers. Therefore, it is fundamental to understand the mechanisms underlying interactions between nanoparticles and the body, for their safe and effective use. In the case of MNPs-DMSA, we can use this knowledge for treatment of lung diseases when associated with drugs, as well as for downregulation or upregulation of the local immune system.One important question still unanswered about the use of magnetic nanoparticles in lung disease treatments is what could be expected if more than one dose is necessary in a short period of time. Recent research of Mejias et al.³¹ was close to answer this question. In their study the authors injected repeated doses (nine in total) of magnetic nanoparticles stabilized with DMSA, but unfortunately, they did not analyze the lungs, assuming that the particles would be stocked in the liver, spleen and kidney. For these organs, however, the authors did not refer to any observed damage. We believe that the answer to this question is related with several factors such as physical-chemical features of the nanoparticles (size, hydrodynamic radius, etc.) interval between the injections, amount of iron injected, among others. These features are also important for a second open question: what happens if the organ has a preexistent disease? Further studies are necessary to clarify this point. It is important to minimize, in all cases, the amount of injected iron, increasing, when possible, the amount of drug attached to the nanoparticles. The use of magnetic nanoparticles is already a reality as a contrast agent. It is possible that in the future they also can be used as drug delivery carriers.In resume our work characterizes mechanisms of the pro-inflammatory effects of MNPs-DMSA in the lung and identifies β₂ integrin-targeted interventions as promising strategies to reduce pulmonary side effects of MNPs-DMSA during biomedical applications. In addition, MNPs-DMSA could be used as modulators of lung immune response.     

Nanotopography-guided migration of T cells

T cells navigate a wide variety of tissues and organs for immune surveillance and effector functions. Although nanoscale topographical structures of extracellular matrices and stromal/endothelial cell surfaces in local tissues may guide the migration of T cells, there has been little opportunity to study how nanoscale topographical features affect T cell migration. In this study, we systematically investigated mechanisms of nanotopography-guided migration of T cells using nanoscale ridge/groove surfaces. The velocity and directionality of T cells on these nanostructured surfaces were quantitatively assessed with and without confinement, which is a key property of three-dimensional interstitial tissue spaces for leukocyte motility. Depending on the confinement, T cells exhibited different mechanisms for nanotopography-guided migration. Without confinement, actin polymerization-driven leading edge protrusion was guided toward the direction of nanogrooves via integrin-mediated adhesion. In contrast, T cells under confinement appeared to migrate along the direction of nanogrooves purely by mechanical effects, and integrin-mediated adhesion was dispensable. Therefore, surface nanotopography may play a prominent role in generating migratory patterns for T cells. Because the majority of cells in periphery migrate along the topography of extracellular matrices with much lower motility than T cells, nanotopography-guided migration of T cells would be an important strategy to efficiently perform cell-mediated immune responses by increasing chances of encountering other cells within a given amount of time.     

Leukocyte migration inhibition reaction--an index of the antibacterial effectiveness of stimulants of the body's nonspecific resistance

The presence of correlation between the protective action of prodigiosane and yeast RNA and the character of reaction in the direct capillary variant of the leukocyte migration inhibition (LMI) test, made with the blood of mice immunized with tularemia vaccine, has been revealed. The possibility and expediency of using the LMI test for evaluating the effectiveness of the stimulators of the nonspecific anti-infective resistance of the body has been shown.     

Leukocyte migration in guinea pigs. II. Partial characterization of a leukocyte migration inhibitory factor distinct from macrophage migration inhibitory factor.

In this study we present data on the partial biological and biochemical characterization of guinea pig leukocyte migration inhibition factor (LIF) and migration inhibition factor (MIF). The results indicate that guinea pig LIF and MIF are distinct mediators of cellular immunity, in terms of indicator cells affected and molecular weight. This is in agreement with previous reports showing distinctions between human LIF and MIF. Partial characterization of guinea pig LIF suggested that it is a heat-stable protein of molecular weight 68,000–158,000 and does not contain terminal sialic acid groups.     

Leukocyte migration inhibition test (LMIT) in systemic lupus erythematosus.

A leukocyte migration inhibition test (LMIT) utilizing the agarose gel technique was performed with native DNA as an antigen in ten patients with systemic lupus erythematosus (SLE) and five normal subjects. Irrespective of disease activity, supernatants obtained at different time intervals during lymphocyte culture in eight patients with SLE showed significant alteration of migration, either enhancement or inhibition, of normal leukocytes. However, supernatants in the control experiments produced no significant alteration of migration. Polyacrylamide gel electrophoresis of supernatants obtained from the SLE group revealed that the inhibitory activity was present in the albumin region, whereas the enhancement activity was found in the beta-globulin region. These results indicate that the hitherto employed estimation of the leukocyte migration inhibition test based on the total activity of these two factors is insufficient for accurate evaluation of chemical mediators from sensitized lymphocytes and that the separation of these two factors may be important for a greater understanding of cellular immunity.     

The adult scent glands and scent of nine bugs of the superfamily Coreoidea

In most of the six coreoid genera examined there are differences in the scent gland complex. This comprises a median, ventral, metathoracic reservoir with either one, two, or three pairs of accessory glands.

The nine adult bugs examined all produced a colourless, single-phase scent, the components of which were similar although the relative proportions varied. Either n-hexanal or n-hexyl acetate, or both, were usually the major constituents (about 90 per cent of the total) and n-hexanol and acetic acid were also present in amounts varying from traces to about 10 per cent. In one species n-butanal was detected and in two species n-butyl butyrate and (probably) butyric acid were present.

The characteristic, ester odour of these coreoid bugs is quite unlike that of pentatomid bugs examined so far and the two groups do not possess a single component in common; nevertheless there are interesting analogies between the scent components of both groups.     

Altered migration, recruitment, and somatic hypermutation in the early response of marginal zone B cells to T cell-dependent antigen

The early responses of follicular (Fo) and marginal zone (MZ) B cells to T cell-dependent Ag were compared using anti-hen egg lysozyme (HEL+) B cells capable of class switch recombination and somatic hypermutation (SHM). Purified CD21/(35int)CD23high Fo and CD21/35(high)CD23low MZ splenic B cells from SW(HEL) Ig-transgenic mice were transferred into wild-type recipients and challenged with HEL-sheep RBC. Responding HEL+ B cells from both populations switched efficiently to IgG1, generated syndecan-1+ Ab-secreting cells, and exhibited equivalent rates of proliferation. However, the expansion of HEL+ MZ B cells lagged significantly behind that of HEL+ Fo B cells due to less efficient homing to the outer periarteriolar lymphatic sheath and reduced recruitment into the proliferative response. Despite the equivalent rates of class switch recombination, the onset of SHM was delayed in the MZ subset, indicating that these two activation-induced cytidine deaminase-dependent events are uncoupled in the early response of MZ B cells. Migration of HEL+ B cells into germinal centers coincided with the onset of SHM, occurring more rapidly with Fo vs MZ responders. These results are consistent with the concept that Fo and MZ B cells have evolved to specialize in T cell-dependent and T-independent responses respectively.     

Insect odorant receptors: channeling scent

Odorant detection in insects involves heterodimers between an odorant receptor (OR) and a conserved seven-transmembrane protein called Or83b, but the exact mechanism of OR signal transduction is unclear. Two recent studies in Nature (Sato et al., 2008; Wicher et al., 2008) now reveal that these OR-Or83b heterodimers form odorant-gated ion channels, revealing a surprising new mode of olfactory transduction.     

Courtship learning: scent of a woman

Learning to predict an outcome based on previous experience is of considerable selective advantage. Getting it wrong can be costly. In a complex environment, however, using the appropriate predictor is not necessarily a trivial task.     

Trends in floral scent chemistry in pollination syndromes: floral scent composition in hummingbird-pollinated taxa

We studied an assemblage of 17 species of bird-pollinated Ecuadorian plants (from 14 angiosperm families), including taxa pollinated by short-billed (trochiline) and sickle-billed (hermit) hummingbirds. Hummingbirds are widely supposed to ignore fragrance while visiting flowers. We collected floral headspace odours in order to test the general prediction that specialist hummingbird-pollinated flowers are scentless. In nine out of 17 of these species we failed to detect any odours using gas chromatography-mass spectrometry (GC-MS), whereas the remaining eight species produced trace levels of volatile compounds. Most of these odour compounds were of terpenoid or lipoxygenase derivation and are commonly emitted by vegetative as well as floral plant tissues. Further studies will be required to determine whether these weak odours attract alternative pollinators, repel enemies or represent vestiges of a scented ancestry.  © 2004 The Linnean Society of London, Botanical Journal of the Linnean Society , 2004, 146 , 191–199.