Circulating leukocytes are thought to extravasate from venules through open interendothelial junctions. To test this paradigm, we injected N-formyl-methionyl-leucyl-phenylalanine (FMLP) intradermally in guinea pigs, harvesting tissue at 5–60 min. At FMLP-injected sites, venular endothelium developed increased surface wrinkling and variation in thickness. Marginating neutrophils formed contacts with endothelial cells and with other neutrophils, sometimes forming chains of linked leukocytes. Adherent neutrophils projected cytoplasmic processes into the underlying endothelium, especially at points of endothelial thinning. To determine the pathway by which neutrophils transmigrated endothelium, we prepared 27 sets of serial electron microscopic sections. Eleven of these encompassed in their entirety openings through which individual neutrophils traversed venular endothelium; in 10 of the 11 sets, neutrophils followed an entirely transendothelial cell course unrelated to interendothelial junctions, findings that were confirmed by computer-assisted three-dimensional reconstructions. Having crossed endothelium, neutrophils often paused before crossing the basal lamina and underlying pericytes that they also commonly traversed by a transcellular pathway. Thus, in response to FMLP, neutrophils emigrated from cutaneous venules by a transcellular route through both endothelial cells and pericytes. It remains to be determined whether these results can be extended to other inflammatory cells or stimuli or to other vascular beds.

Cloned mouse mast cells resemble, by ultrastructure, immature mast cells observed in vivo. These mast cell clones can be grown in the absence of any other cells, facilitating direct investigations of their biochemistry and function. We find that cloned mast cells express plasma membrane receptors (Fc~R) that bind mouse IgE with an equilibrium constant (KA) similar to that of normal mouse peritoneal mast cells. In addition, cloned mast cells do not display detectable la antigens and cannot enhance Ig secretion when added to lymphocyte cultures or mediate natural killer lysis. In the presence of I mM sodium butyrate, cloned mast cells stop dividing and acquire abundant electron-dense cytoplasmic granules similar to those of mature mast cells. Their histamine content increases concomitant with cytoplasmic granule maturation and may exceed that of untreated mast cells by 50-fold. Unlike peritoneal mast cells, cloned mast cells incorporate 35S04 into chondroitin sulfates rather than heparin. 1-hese findings demonstrate that, unlike fully differentiated mouse peritoneal mast cells, cloned immature mouse mast cells contain no heparin and low levels of histamine. In addition, they establish that high-affinity Fc, R are expressed early in mast cell maturation, well before completion of cytoplasmic granule synthesis and mediator storage.We have previously reported methods to clone mast ceils with normal karyotypes from mouse hematopoietic tissue in vitro (30). Our cloned mast cells contain less histamine than normal mouse peritoneal mast cells and resemble immature mast cells by morphology. Others have described similar findings with uncloned cells (22,32,38,42,43,51,53). Although some mast cells synthesize heparin when fully differentiated (23, 54), certain mast cell tumors are devoid of heparin and incorporate 3BSO4 into chondroitin sulfate exclusively (18). Histochemical evidence suggests that normal immature connective tissue mast cells (6) and the mast cells in the intestinal lamina propria of rodents (25, 50) also may synthesize glycosaminoglycans other than heparin, but this notion has not been confirmed directly.In this report, we show that cloned mast cells closely resemble, by ultrastructure, immature mast cells found in vivo.Cloned mast cells incorporate 35804 preferentially into chondroitin sulfates, confirming histochemical evidence that immature mast cells contain little heparin. In addition, cloned mast cell proliferation, cytoplasmic granule synthesis, and mediator storage can be modulated in vitro, permitting direct analysis of mast cell maturation. MATERIALS AND METHODS AntiseraLyt-1.2 and Lyt-2.2 antisera, prepared as described (45), were kindly donated by F. W. Shen~; monoclonal antibody against Thy-l.2 (mc-a-Thy-l.2) was donated by Ed Clark2; and mc-a-Lyt-1 and mc-a-Lyt-2 were gifts from J. Ledbetter and L Herzenberg a. Mast cells were examined for la antigens using ATH-a-ATL alloantisera (16) or mc-a-Ia (10-3.6, reference 35) generously provided by John Freed and J. M. Kupinski 4. Mast Cells...

Vascular permeability factor/vascular endothelial growth factor (VPF/VEGF) interacts with two high-affinity tyrosine kinase receptors, VEGFR-1 and VEGFR-2, to increase microvascular permeability and induce angiogenesis. Both receptors are selectively expressed by vascular endothelial cells and are strikingly increased in tumor vessels. We used a specific antibody to localize VEGFR-2 (FLK-1, KDR) in microvascular endothelium of normal mouse kidneys and in the microvessels induced by the TA3/St mammary tumor or by infection with an adenoviral vector engineered to express VPF/VEGF. A pre-embedding method was employed at the light and electron microscopic levels using either nanogold or peroxidase as reporters. Equivalent staining was observed on both the luminal and abluminal surfaces of tumor- and adenovirus-induced vascular endothelium, but plasma membranes at interendothelial junctions were spared except at sites connected to vesiculovacuolar organelles (VVOs). VEGFR-2 was also localized to the membranes and stomatal diaphragms of some VVOs. This staining distribution is consistent with a model in which VPF/VEGF increases microvascular permeability by opening VVOs to allow the transendothelial cell passage of plasma and plasma proteins.