M. Quillec

Natural killer activity of kurloff cells: A direct demonstration on purified kurloff cell suspensions

In order to study their natural killer effect, guinea pig splenic Kurloff cells were fractionated by Percoll discontinuous density gradient centrifugation. Kurloff cells were collected and tested for cytotoxicity in a 24-hr chromium-release test. Comparison of different splenic cellular samples (of males or estrogenized females) with increasing percentage of Kurloff cells, revealed a highly significant positive correlation (r = 0.93, alpha less than 0.01) with the cellular cytotoxicity developed against the K 562 target cells.

New data on the cytolytic effects of natural killer cells (Kurloff cells) on a leukemic cell line (guinea pig L2C)

L2C leukemia is a leukemia that occurs in strain two guinea pigs. The L2C cells are natural killer-sensitive. The Kurloff cell (KC), a guinea pig NK cell, develops a 3-fold increase in lysosomal enzyme activity and the number of KC cells increases during leukemogenesis, leading to KC cell-mediated L2C cytolysis. This paper shows that conjugates are produced by incubating KC and L2C for 4 h, with 34% of L2C showing chromatin compaction and shrinkage of the cytoplasm. There was also a reorientation of the KC cytoplasmic organelles to face the target cell and an elongation of the KC to produce arms that engulfed the L2C. The L2C had either necrotic or apoptotic characteristics. L2C DNA fragmentation was demonstrated in situ with the comet and the TUNEL assays. 22.2% of the viable L2C lost their membrane asymmetry during KC-L2C conjugation as shown by incubation with Annexin V-FITC. These results provide new evidence that the death of L2C is due, at least partly, to apoptosis. The cytolytic effect of the NKKC might be a model of the cytological changes that occur in NK cell-leukemic cell conjugates.

Collagen-associated sulphated proteoglycans. Ultrastructure after formaldehyde-cetylpyridinium chloride fixation

SummaryIn the course of an ultrastructural cytochemical study of intracellular sulphated proteoglycans involving the addition of cetylpyridinium chloride in the primary aldehyde fixative, a remarkable ultrastructural preservation of the collagenassociated sulphated proteoglycans was observed. Together with the preservation of their localization among the collagen fibrils (with, for some of them, a 50nm periodic association with d-bands) and of their native elongated shape, previously observed under similar technical conditions, these stick-shaped and chondroitinase ABC-sensitive proteoglycans exhibited a typical pattern with several dense longitudinal parallel tracks (periodicity: 3–4nm) not described as yet. Readily observable without high iron diamine-staining, the morphology of these cetylpyridinium chloride-precipitated and collagen-associated polyanions was particularly enhanced after incubation in the diamine solution which ascertained their sulphate content.Such a common ultrastructural organization with parallel tracks for both intracellular (i.e., in eosinophilic polymorphonuclear cells and Kurloff cells) and extracellular CPC-precipitated sulphated proteoglycans could correspond to intrinsic properties of the complexed molecules and could be related to ‘double track’ proteoglycans observed under other technical conditions in basement membranes.

The proteoglycan skeleton of the Kurloff body as evidenced by cuprolinic blue staining.

SummaryThis study deals with the ultrastructure of the chondroitin sulphate proteoglycans of the Kurloff body, a large lysosome organelle, metachromatic towards Toluidine Blue, of a blood cell unique to the guinea pig and called the Kurloff cell. Splenic Kurloff cell from oestrogen-treated guinea pig cells were examined after staining with Cuprolinic Blue, a cationic phthalocyanine-like dye, in the presence of MgCl2 in a critical electrolyte concentration method. Better results were obtained when the fixation-staining by the glutaraldehyde Cuprinolinic Blue MgCl2 mixture was preceded by a glutaraldehyde pre-fixation. On light microscopy, Kurloff bodies generally exhibited an overall pink and glassy metachromasia, sometimes with additional darker metachromatic small dots at their peripheries. At the ultrastructural level, the metachromatic central matrix of the Kurloff body usually exhibited, as a major feature, a typical network pattern of ribbon-like or stellate electron-dense precipitates suggesting the presence of a skeleton of Cuprolinic Blue-reactive filamentous structures. Taking into account their high anionicity (as shown by the stability of the dye binding in the presence of 0.3 m MgCl2) and their susceptibility to chondroitinase ABC, these anionic structures were assumed to be related to the proteochondroitin-4-sulphate previously characterized as the only major sulphated glycoconjugate of the Kurloff cell.

Uneven Distribution and Size of Rabbit Lens Capsule Proteoglycans

To document the ultrastructural distribution of lens capsule proteoglycans, rabbit lens capsules were fixed and stained overnight in 50 mM sodium acetate, pH 5.6, containing 2.5% glutaraldehyde, 0.2% Cuprolinic Blue and 0.2 M MgCl2. They were rinsed, stained with 1% aqueous sodium tungstate, embedded in Epon, sectioned (60 nm), and examined with an electron microscope at 60 kV.Proteoglycan–Cuprolinic Blue complexes mainly appeared as networks of small electron-dense filaments throughout the posterior and anterior capsules. The posterior capsule was a single layer with a network of small proteoglycan filaments gradually decreasing in size from the humoral side (90×10 nm) to the lenticular side (30×8 nm). The humoral side of the anterior capsule had a thin lamina (400 nm) containing large (180×40 nm), very electron-dense proteoglycan–Cuprolinic Blue complexes plus small proteoglycans. Below this lamina, the complexes were only seen as filaments slightly smaller than those in the corresponding area of the posterior capsule.Cuprolinic Blue binding of the anterior and posterior lens capsules revealed differences in the size and distribution of their sulphated proteoglycans which do not correspond to the patterns of their immunoreactivity with anti-heparan sulphate proteoglycan. The humoral lamina in the anterior capsules, with large proteoglycan structures, might be a distinct structural and functional compartment.

Kurloff cell ultrastructure after combined formaldehyde-cetylpyridinium chloride fixation and high-iron diamine staining.

SummaryThis study deals with the ultrastructure of the chondroitin sulphate proteoglycans of the Kurloff body, a large lysosomal organelle that stains metachromatically with Toluidine Blue and which is present in Kurloff cells (a blood cell unique to the guinea pig). Splenic tissues were fixed with 1% cetylpyridinium chloride (CPC) added to 4% paraformaldehyde and examined either after Spicers high-iron diamine staining for sulphated anionic sites followed by post-fixation with ferrocyanide-osmium tetroxide or after a simple post-fixation with ferrocyanide-osmium tetroxide. CPC-precipitated sulphated sites were preferentially located at the periphery of the Kurloff body but, unexpectedly, were absent in the central matrix. Although their electron opacity was lower, these anionic sites were readily observable in the absence of HID-staining after sole post-fixation by ferrocyanide-reduced osmium. CPC-precipitated sulphated anionic sites were either associated with the myelin figures or constituted unexpected structures. They contained (i) tightly-stacked lamellae, with a very regular 4 nm periodicity, and (ii) groups of 2, 3, 4 short dense lines with a 3–5 nm periodicity. By taking into account the susceptibility of these HID-reactive structures towards chondroitinase ABC, these different sulphated components were assumed to be related to the proteochondroitin-4-sulphate previously characterized as the only major sulphated glycoconjugate of the Kurloff cell. Their colocalization with phospholipidic structures was suggested following, observation of sections treated by a chloroform-methanol mixture.

Ultrastructure of proteoglycans in the specific granules of guinea-pig basophilic leukocytes as demonstrated by cuprolinic blue staining

The ultrastructure of sulphate proteoglycans in basophil granules was examined using cytochemical procedures designed to stabilize and visualize these highly anionic macromolecules in situ. Unfixed or glutaraldehyde-prefixed guinea-pig spleen cells were submitted to fixation/staining in 2.5% glutaraldehyde, 0.2% cuprolinic blue (CB; a cationic phthalocyanin dye) and 0.2 or 0.3M MgCl2 with or without glycosidase treatments. Abundant electron-dense precipitates were present throughout the granule matrix. The stained structures were often arranged in a quasi-crystalline typical banded pattern. Negative control basophils had no electrondense precipitates. Digestion with chondroitinase ABC destroyed the CB-positive electron-dense banded or filamentous patterns while sialidase treatment did not, but led to larger CB-positive filaments in the cytoplasm near the granules. Taking into account their high anionicity, as shown by the stability of dye binding in the presence of 0.3M MgCl2, and their susceptibility to chondroitinase ABC, the CB-precipitates are assumed to be related to the sulphated proteoglycans previously characterized in basophil granules. The CB-positive crystalline or filamentous network of the granule matrix is also assumed to reflect the in situ location and organization of these intracellular proteoglycans and may be involved in maintaining the shape of the granule.

GLYCOCONJUGATES WITH Neu5Ac(α2,6)Gal(β1,4)GlcNAc SEQUENCE. A SELECTIVE AFFINOHISTOCHEMICAL PROPERTY OF KURLOFF CELLS IN GUINEA PIG THYMUS

.COHPARATIVE FIHE STRUCTURE OF TIlE FIN DERHOSKELETON DURING REGENERATION IN TIlE GUPPY (Poecil io reCicu[ata) AHD ZESRAFISH (~rachydanio r e r io ) . Jacquel ine GERAUDIE(I), HarieJos~phe HONNOT (I) and Marcus SINGER (2). ( 1 ) Laboao~o.i~e de 8Lo~ouie du P~ve.~oppe.me~t de~ g e . ~ b ~ l n ~ e u ~ , figLue~Lt~ de PARIS-SUP XI, 8~t.441, 91405 O~ag ¢~dex (F~a~ee), mtd [2] P e p o ~ e ~ o~ ~ o m g , Sehoo~ of MedLe~ne, Ca~e ~ e ~ t e ~ ~e~e~ve U~veA~itg, C~eveeand, OH. 44106 (L~A). The dermoskeleton of a t e l eos t f in comprises the collagenous a c t i n o t r i e h i a inser ted at the d i s t a l extremity and between the tgo pa ra l l e l demi-rays ghich form the bony rays or l ep ido t r i ch ia sus ta in ing the appendage. Amputation of the pectora l f ins in Guppy or Zebrafish is followed by a rapid r e s to ra t ion of the missing d i s t a l part . The study of the process of regrowth has been followed by u l t r a s t ruc tu ra l means. Results are s imilar in both species and emphasize the ubiquitous mechanisms of dermoskeleton regenerat ion in t e l eos t . Regeneration of the acCinotr ichia occurs at the t ip of regenerate , adjacent to the wound epider mis ~hich is not separated from the mesenchymal compartment by any organized basal lamina. This locat ion favors an epidermal o r ig in of the components although molecules present in the mesodermal compartment my be instrumental in the complex organizat ion of the f ina l a c t i n o t r i c h i a ( I ) . Subsequent gro~Ch in length and ~idth occurs according to a d i s t a l Co proximal d i r ec t i on in the growing f in regenerate,were two sub-populations merge according co t he i r spat ia l o r i en t a t i on . Regeneration of the l ep ldocr ich la occurs in the opposite d i r ec t ion , from the level of amputation towards the Clp of the groging regenerate. Regenerating l e p i d o t r i c h i a l bone is formed in the dermoepidermal boundary of the f in regenerate ~hi le at the level of Cite stump, Cite bone is located a~ay from the epidermis. This spa t i a l d i spos i t ion is reminiscent of bone oncogenesis and this pa t tern of bone d i f f e r e n t i a t i o n is under study. Hineral isacion is reminiscent o£ ghat occurs during bone ontogenesis (2) but the biophysical study of the supposed hydroxypaCite c rys ta l s observed is under way.

Scanning electron microscopy of pure Kurloff cell suspensions

SummaryScanning electron microscopy has been used to examine suspensions of Kurloff cells, which are present only in guinea pig. The cells are round with a surface characterized by uniformly distributed short stubby microvilli and some ridges. Some cells show one or more pores surrounded by a smooth margin, others a smooth globular expansion about 10 μm in diameter which may correspond to a Kurloff body undergoing exocytosis. The Kurloff body is released into the medium where it can exist freely. The surface of the free body and of the expansion display the same smooth appearance. Although the nature of the Kurloff cell remains unknown, this study provides further information concerning its morphology and describes an extrusion process not observed previously.

The Kurloff cell: Distribution, seasonal variations and effects of artificial illumination cycles

Abstract In 372 full grown non‐gravid female guinea pigs, the average number of Kurloff cells in blood was 402/mm3, s.d.: 366.5. The distribution was positively skewed. This distribution is subject to seasonal variations since the figures are significantly higher from the beginning of May to the end of October than from the beginning of November to the end of April. In other samples of guinea pigs, both total darkness and continuous illumination significantly lowered the number of Kurloff cells in blood. The average number of Kurloff cells is stabilized during an LD 12:12 on both sides of vernal equinox. The Kurloff cell is a new example of a photoperiodically sensitive blood cell. Variations in its production do not appear to be related to the estrogen peak of the estrus cycle. These variations may perhaps be a function of the derivative of the duration of illumination.