Barbara Plytycz

Role of mast cells in zymosan-induced peritoneal inflammation in Balb/c and mast cell-deficient WBB6F1 mice

Zymosan‐induced peritonitis was investigated in mast cell‐deficient WBB6F1 mice and in Balb/c mice pretreated with mast cell stabilizer (cromolyn) or antagonists of histamine receptors (mepyramine, triprolidine, cimetidine, or ranitidine). The inherited mast cell deficiency in W/Wv knockouts of WBB6F1 mice impaired significantly the level of histamine and plasma exudation (measured 30 min after stimulation) as well as the influx of exudatory leukocytes, accumulation of plasma and exudate chemoattractants, and the release of proinflammatory cytokines (TNF‐α, IL‐1β, and IL‐6) measured at 6 h of inflammation. All of those factors were fully restored after selective intraperitoneal reconstitution of W/Wv mice with bone marrow‐derived mast cells from their control +/+ counterparts. Cromolyn pretreatment of Balb/c mice reduced exclusively the early plasma exudation and histamine influx. Blocking of histamine receptors inhibited not only the early plasma exudation but also temporarily diminished primary leukocyte influx and levels of MCP‐1 and IL‐1β. In conclusion, mast cells play an important role in the initiation of zymosan‐induced peritonitis and modulate its further course.

Riboflavin as a Source of Autofluorescence in Eisenia fetida Coelomocytes

Abstract Immunocompetent cells of earthworms (coelomocytes) contain adherent amoebocytes and large eleocytes (chloragocytes); the latter are filled with numerous granules. We have previously shown that eleocytes of several (but not all) earthworm species exhibit strong autofluorescence detectable by fluorescent microscopy and flow cytometry. In the present article, the molecular origin of eleocytes autofluorescence was elucidated in coelomocytes expelled via dorsal pores in the integument of Eisenia fetida subjected to electric shock (1 min at 4.5 V). Spectrofluorometry (excitation and emission spectra and fluorescence lifetime), together with HPLC analysis of coelomocyte suspensions and supernatants, indicated that riboflavin but not FMN (flavin mononucleotide) or FAD (flavin-adenine dinucleotide) is the main fluorophore responsible for eleocyte fluorescence in this species. Additionally, lipofuscins are suspected to participate in this phenomenon.

Effect of heavy metals on coelomocytes of the earthworm Allolobophora chlorotica

Earthworms are sensitive bioindicators of soil pollution. The aim of present investigations was to study the effects of heavy metals on earthworms and on their coelomocytes involved in the defence reactions. Adult individuals of Allolobophora chlorotica collected in Krakow (K) soil were kept in the laboratory either in the K soil, or were transferred to unpolluted soil from the rural area Sierbowice (S) or to the heavily polluted (Zn>Pb>Cd>Cu) soil from the industrial area, Bukowno (B). They were kept there at 22 °C for up to 8 weeks. Cocoons and juveniles appeared in S and K soil samples. The number and activity of the coelomocytes of worms maintained in S and K soils were unaffected despite some accumulation of heavy metals in the earthworm tissues. In contrast, in the B soil samples, bioaccumulation of metals was strongest, high mortality of adults was recorded, body mass was reduced, and reproduction completely inhibited. Coelomocytes retrieved from the B soil survivors exhibited significant impairment of pinocytosis and plastic adherence. Perhaps impairment of immune functions contributed to the poor survival under conditions of heavily polluted B soil samples.

Morphine modulation of peritoneal inflammation in Atlantic salmon and CB6 mice.

Peritoneal inflammation is a convenient model for comparisons of modulatory effects of morphine in phylogenetically distant vertebrates. Both in salmon and mice morphine injected intraperitoneally together with an irritant (thioglycollate) significantly inhibits inflammation as estimated by the number of peritoneal leukocytes. The low number of exudate cells in morphine‐treated animals seems to be compensated by their high activity, as evidenced by the enhanced phorbol myristate acetate‐induced respiratory burst. The morphine‐inhibited influx of leukocytes into the irritated peritoneal cavity correlates with the morphine‐lowered level of plasma chemotactic factors both in fish and mice. It implies that morphine impairs the level of plasma chemotactic factor either directly (affecting their release from the resident peritoneal cells) or indirectly (decreasing the number of inflammatory leukocytes by inhibition of their migration from hemopoietic sites). The inhibitory effects of morphine on both the cell number and chemoattractant level are completely reversed by the naltrexone pretreatment, which implicates the involvement of opioid receptors. J. Leukoc. Biol. 65: 590–596; 1999.

Inflammatory macrophages, and not only neutrophils, die by apoptosis during acute peritonitis.

The central paradigm says that during inflammation, after completing their function, granulocytes die apoptotically in periphery to avoid destruction of self-tissues. Here we aimed to investigate the kinetic aspect of inflammatory leukocyte apoptosis and verify whether apart from neutrophils also other inflammatory leukocytes numerously undergo apoptosis. We observed that in physiological conditions, less than 7% of either resident peritoneal macrophages or lymphocytes die apoptotically. The studies on a model of acute zymosan-induced peritoneal inflammation revealed that there are two waves of inflammatory leukocyte apoptosis. The first wave corresponds to the time of maximal neutrophil accumulation in peritoneum (6h) and the apoptotic death indeed concerns mostly neutrophils (over 30% of those cells), but also more macrophages die at this time (>10%). The second wave (at 3 days) concerns mostly macrophages (20% versus 3-6% for other populations) and coincides with the resolution of inflammation and the dominant presence of macrophages. In contrast, numbers of apoptotic T (1-3%) and B (approximately 5%) cells do not significantly change during the whole peritonitis. The two waves of apoptosis concur with an increase of caspase-8, -9 and -3 at the transcript and activity levels. The apoptosis inducer TNF-alpha is produced only during first hours while nitric oxide throughout all inflammation. Moreover, during the whole course of peritonitis the expression of pro-apoptotic Bax dominates over anti-apoptotic Bcl-2. In conclusion, we characterized kinetics of apoptotic death of inflammatory leukocytes during acute peritoneal inflammation and revealed that both phagocyte populations (neutrophils and macrophages) die numerously in peritoneum.

Riboflavin content of coelomocytes in earthworm (Dendrodrilus rubidus) field populations as a molecular biomarker of soil metal pollution.

The effect of Pb + Zn on coelomocyte riboflavin content in the epigeic earthworm Dendrodrilus rubidus inhabiting three metalliferous soils and one reference soil was measured by flow cytometry and spectrofluorimetry. A reciprocal polluted<-->unpolluted worm transfer experiment (4-week exposure) was also performed. High proportions of autofluorescent eleocytes were counted in worms from all localities, but intense riboflavin-derived autofluorescence was detectable only in reference worm eleocytes. Other findings were: (i) fluorophore(s) other than riboflavin is/are responsible for eleocyte autofluorescence in residents of metalliferous soils; (ii) riboflavin content was reduced in the eleocytes of worms transferred from unpolluted to metal-polluted soil; (iii) the riboflavin content of D. rubidus eleocytes is a promising biomarker of exposure; (iv) COII mitochondrial genotyping revealed that the reference population is genetically distinct from the three mine populations; (v) metal exposure rather than genotype is probably the main determinant of inter-population differences in eleocyte riboflavin status.

Effects of temperature and soil pollution on the presence of bacteria, coelomocytes and brown bodies in coelomic fluid of Dendrobaena veneta

Adult Dendrobaena veneta (Annelida; Oligochaete; Lumbricidae), were kept for 2-4 weeks at either 22 °C or 10 °C in control (C) heavy metal-free commercial soil, or in relatively unpolluted Krakow (K) urban soil, or in heavily polluted (Zn>Pb>Cd) Bukowno (B) industrial soil. At the end of exposures, the numbers of coelomocytes, brown bodies, and bacterial content was measured in coelomic fluid, while heavy metal accumulation was recorded in the animal tissues. The most drastic changes, with high mortality of animals (30 %), were recorded in D. veneta kept for 4 weeks at 22 °C in heavily polluted B soil samples; the number of free coelomocytes in the surviving worms in this treatment group was decreased, whilst the numbers of brown bodies and of bacteria (both free in coelomic fluid and entrapped in the brown bodies) were significantly increased. These changes were absent in earthworms exposed to B soil at 10 °C, as well as at both temperatures in relatively uncontaminated K soil.

Enhanced early vascular permeability in gelatinase B (MMP-9)-deficient mice: putative contribution of COX-1-derived PGE2 of macrophage origin.

Increased vascular permeability leading to vascular leakage is a central feature of all inflammatory reactions and is critical for the formation of an inflammatory exudate. The leakage occurs because of gap formation between endothelial cells and breakdown of the basement membrane barriers. The present study aimed to investigate the role of gelatinase B [matrix metalloproteinase 9 (MMP‐9)], known to be involved in neutrophil exudation, in changes of vascular permeability at the early stages of acute zymosan peritonitis. We show that although MMP‐9 is being released already within the first minutes of peritonitis, its lack, induced pharmacologically or genetically, does not decrease but rather increases vasopermeability. In mice treated with an inhibitor of gelatinases (A and B), a tendency to increased vasopermeability existed, and in MMP‐9−/− mice [knockout (KO)], the difference was statistically significant in comparison with their controls. Moreover, in intact KO mice, significantly augmented production of prostaglandin E2 (PGE2) of cyclooxygenase 1 (COX‐1) origin was detected, and depletion of peritoneal macrophages, but not mast cells, decreased vasopermeability in KO mice. Thus, the increase of vasopermeability observed on KO mice is a result of the increased production of COX‐1‐derived PGE2 by peritoneal macrophages. We conclude that genetic deficiency in gelatinase B might lead to the development of a compensatory mechanism involving the COX pathway.

Neutrophil elastase activity compensates for a genetic lack of matrix metalloproteinase‐9 (MMP‐9) in leukocyte infiltration in a model of experimental peritonitis

Extracellular proteolysis of basement membranes and matrix is required for leukocyte diapedesis and migration to the inflammatory focus. Neutrophil elastase (NE) and matrix metalloproteinases (MMPs) are among the enzymes involved in these processes, as shown in mice genetically deprived of such enzymes. However, studies with MMP‐9−/− mice revealed that albeit neutrophil influx is impaired initially in these animals versus controls, neutrophilia is subsequently augmented during later stages of zymosan peritonitis. MMP‐9 as a MMP and NE as a serine protease belong to different enzyme classes. As MMP‐9 and NE are produced by neutrophils and have similar biological effects on matrix remodeling, it was evaluated whether enhanced NE activity might compensate for the lack of MMP‐9. In genetically uncompromised mice, two waves of NE expression and activity during zymosan peritonitis were observed in inflammatory neutrophils and macrophages at the time of influx of the respective cell populations into the peritoneum. Additionally, NE expression was associated with the activity of resident peritoneal mast cells and macrophages, as their depletion reduced NE activity. Most importantly, the NE mRNA and protein expression and activity were enhanced significantly in MMP‐9−/− mice during late stages of zymosan peritonitis. In addition, the application of a selective NE inhibitor restrained enhanced neutrophil accumulation significantly. In conclusion, during acute peritoneal inflammation, NE expression and activity increase gradually, facilitating leukocyte influx. Moreover, increased NE activity might compensate for a genetic lack of MMP‐9 (as detected in MMP‐9−/− mice), resulting in delayed accumulation of neutrophils during late zymosan peritonitis.

Studies on the growth and longevity of the yellow-bellied toad, Bombina variegata, in natural environments

Yellow-bellied toads were studied in their natural environment in a mountain locality in southeastern Poland. 608 specimens were captured, marked by yellow skin autografts placed in different parts of their dorsal surface according to body length, and released. Some of them were recaptured and measured from one to nine years later to estimate their growth and longevity. Yellow-bellied toads grew rapidly in early life; thereafter their growth was very limited. Body size was not an accurate age indicator of an individual of this species. The body length 51-55 mm was maximal in this locality. Yellow-bellied toads were long-lived in nature, some individuals surviving for much more than ten years, and perhaps even more than 20 years. The skeletochronological technique (counting the growth lines in phalangeal cross-sections of the clipped toes of some marked individuals) underestimated the actual age of these animals.