Purified monocyte-derived angiogenic substance (angiotropin) induces controlled angiogenesis associated with regulated tissue proliferation in rabbit skin.

Angiotropin is a differentiation factor for microvascular endothelial cells isolated from serum-free cultures of lectin-activated, porcine monocytes. We used an ear lobe model in rabbits, single intradermal injection of angiotropin to induce phenotypical changes of the endothelial cells in capillaries and postcapillary venules, vascular engorgement, and subsequent angiogenesis in dose-dependent manner. The vascular changes are associated with epidermal and stromal cell proliferation. Angiogenesis and tissue proliferation occur in the absence of tissue necrosis and do not lead to scar formation. Angiotropin-induced angiogenesis is not inhibited by local dexamethasone although it involves a defined turnover of inflammatory cells. Proliferation is transient and regressive events follow. The overall tissue reaction resembles changes found in the undamaged skin margin of a primary healing wound during the inflammatory/proliferative phase. From these observations we conclude that angiotropin is an important secretory product of activated peripheral macrophages that triggers inflammatory and proliferative reactions in wound healing by activating microvascular endothelial cells.

The filter technique for measuring leucocyte locomotion in vitro. Comparison of three modifications.

Three forms of filter technique for measuring random and directional locomotion of leucocytes have been compared: (1) the conventional one filter technique of Boyden (lower surface count method); (2) the two filter system with a lower cell-impermeable filter designed to count the cells at the underside of the upper filter as well as those on the lower filter (two filter count method); and (3) two filter systems counting only cells associated with the lower filter (lower filter count method). In some instances all three methods produce qualitatively similar results. In others totally different results are reproducibly obtained with identical cell preparations, media and attractants. Compared to the two filter count method, the lower surface count method and the lower filter count method are not sufficiently reliable. The discrepancies are partly due to errors in measuring the response. They are caused by variable cell adhesion to the filters resulting in a varying distribution of cells between the upper and lower filter and/or detachment of neutrophils from the upper filter. Some of the discrepancies are not due to errors in assessing the response, but to differences in gradient formation and drift of chemokinetic and chemotactic materials from one compartment to the other.

Biotechnological production and functional characterization of leukocytic cytokine(s) with cytotoxic effect on resting but not activated thymocytes.

In the present report we describe the analysis of suppressive and cytotoxic factor(s) derived from Con A-induced porcine leukocyte culture supernatant solution(s) (PCAS). The supernatant solution(s) had been produced on a biotechnological scale using porcine peripheral blood leukocytes isolated from 200 to 1000 1 of porcine blood. The supernatant solution(s) were tested for their effect during murine T cell activation. We found that crude supernatant solution(s) contain suppressive and cytotoxic factor(s) (SF) which have a similar selectivity for non-activated thymocytes as suppressive factor(s) previously detected in the supernatant solution(s) of allo-antigen-stimulated murine spleen cell cultures. SF was obtained from crude serum-free culture supernatants by fractionated ammonium-sulphate precipitation between 35 and 45% saturation. SF has the following characteristics: its release into the culture supernatant is dependent on mitogenic stimulation, it acts during early stages of T lymphocytic activation, the activity towards naive thymocytes is most likely caused by an irreversible cytolytic mechanism, and phenotypically and functionally immature thymocytes are preferentially affected through exposure to SF.

Neomorphogenesis of blood vessels in rabbit skin induced by a highly purified monocyte-derived polypeptide (monocyto-angiotropin) and associated tissue reactions.

Chemotropic morphogenesis of new blood vessel patterns with active haemodynamics in the skin of rabbits was induced by intradermal injection of highly purified monocyto-angiotropin (MAT), one of the low-molecular-weight (4,500) polypeptide monokines exuded by activated mammalian (porcine) monocytes (macrophage type) on their culture in serum-free, fully synthetic media. Evidence is presented that neohypervascularization of the skin is associated with enhanced tissue function as represented by hair growth. Exclusively in the area of MAT injection, numerous capillary-like vessel formations arose from preexisting vessels, and the quiescent hair follicles (telogen) were activated to growing ones (anagen) within 1-2 weeks.

Chemotaxis is not a special case of haptotaxis.

Serum peptides containing classical anaphylatoxin (CAT) produce marked chemotatic orientation of human neutrophil granulocytes without modifying cell attachment to the substratum. Furthermore gradients of adhesion produced with gammaglobulins fail to induce morphological orientation of neutrophils. The results suggest that chemotaxis is not a special case of haptotaxis.

A transient rise in cyclic AMP levels following chemotactic stimulation of neutrophil granulocytes.

A short transient rise of cyclic AMP is observed within 1 minute after primary stimulation of neutrophils with chemotactic serum peptides containing classical anaphylatoxin (CAT). A second administration of these peptides after two minutes failed to produce a second peak of cAMP. Human serum albumin (HSA) which has chemokinetic but no chemotactic activities did not change cAMP levels. There was no significant change in cGMP levels within 1 minute following stimulation of rabbit neutrophils with chemotactic peptides or HSA.

Diverging effects of chemotactic serum peptides and synthetic f-Met-Leu-Phe on neutrophil locomotion and adhesion.

The chemotactic serum peptide preparations CAT 1.6.1. and C5adesArg induced marked directional locomotion over a wide concentration range without significant effects on random locomotion and adhesion of human neutrophils in Gey's solution containing 2% HSA. In contrast, f-Met-Leu-Phe produced marked negative chemokinetic effects and its capacity to induce directional locomotion was more limited with respect to magnitude and concentration range. The negative chemokinetic effect of f-Met-Leu-Phe correlated closely with increased spreading and cell adhesion.

[Diagnostic-therapeutic trial in infantile genetic agranulocytosis with leucorecruitin]. / Infantile genetische Agranulozytose (IGA). Leukorecrutin im diagnostisch-therapeutischen Versuch.

Subject of this report is the usefulness of a biologically specific mediator of the leucocytosis reaction (leucorecruitin) in the diagnostic evaluation of granulopenic diseases. According to our trial the application of leucorecruitin in healthy humans resulted in a significant increase of granulocytes in circulating blood. The therapeutic effect depends on the neutrophil storage pool. In a diagnostic-therapeutic trial in infantile genetic agranulocytosis with depleted storage pools, leucorecruitin failed to lift the low number of circulating granulocytes to a normal level. No side effects could be observed so far.

Purified monocyte-derived angiogenic substance (angiotropin) stimulates migration, phenotypic changes, and "tube formation" but not proliferation of capillary endothelial cells in vitro.

Activated monocytes (macrophages, histiocytes) induce the formation of new blood vessels by secretion product(s). From conditioned serum-free media of porcine peripheral monocytes treated with concanavalin A, a substance with very strong angiogenic activity in vivo, designated as angiotropin, has been isolated and purified to homogeneity. We investigated the biological action of the monocyte-derived angiogenic substance on cultured capillary and large vessel (aorta) endothelial cells and on 3T3 fibroblasts, mimicking steps of the angiogenic pathway in vitro. We found that angiotropin does not stimulate the proliferation of capillary endothelial and 3T3 cells; however, in concentrations less than 1 ng/ml, it enhances random migration of capillary endothelial cells but not of 3T3 cells. On confluent monolayers of capillary and aortic endothelial cells angiotropin leads to defined changes of cell morphology that are dose dependent and reversible. In the presence of angiotropin, capillary endothelial cells rapidly form tubelike structures on gelatinized plates. This organizational state is not reached with aortic endothelial cells. The results indicate that the biological action of monocytic angiotropin is different from that of the angiogenic growth factors that stimulate the proliferation of endothelial cells and nonlymphoid mesenchymal cells and keep endothelial cells in the contact-inhibited epitheloid cell phenotype. We propose that angiotropin is directly involved in monocyte-induced angiogenesis.

Distinct chemokinetic and chemotactic responses in neutrophil granulocytes.

Evidence has been presented to show that chemokinesis and chemotaxis of human neutrophil granulocytes can be controlled by distinct cellular mechanisms and by different chemicals. Materials such as human serum albumin or fibrinogen had chemokinetic but no chemotactic properties. Highly purified preparations of serum-derived peptides containing classical anaphylatoxin had detectable chemotactic activity only. Chemokinetic as well as chemotactic substances were required for the expression of chemotaxis in the form of efficient directional locomotion. The roles of chemokinesis and chemotaxis in the regulation of directional locomotion have been analyzed. Further experiments showed that the chemotactic response conforms to the law of Weber-Fechner. The number of cells which had accumulated in response to a chemotactic simulus was proportional to the logarithm of the concentration of the chemical substance. Readaptation from high to low chemotactic stimulus occurred only to a limited extent.

A fast-acting cytotoxin derived from Con A-activated porcine leukocytes. I. Biochemical characteristics and target cell specificity.

We here describe a new cytotoxin which was detected in serum-free culture supernatants of mitogen stimulated porcine leukocytes. The factor was precipitated at 35-45% (NH4)2 SO4 concentration, sensitive to heat, low pH, and trypsin, thus indicating its protein nature. Column chromatography on hydrophilic or hydrophobic matrices revealed amphiphilic properties. The smallest unit which was sufficient to induce complete target cell lysis had a molecular weight of 33K. This porcine cytotoxin (PCT) could be distinguished functionally from lymphotoxin (LT) and tumor necrosis factor (TNF). It is a fast acting mediator which exerts strong cytostatic and cytotoxic anti-tumor activity using a panel of T lymphoma target cells.

Relation between stimulus intensity and neutrophil chemotactic response.

The effect of chemotactic peptides which lack chemokinetic activity has been investigated. The neutrophil response is proportional to the logarithm of the stimulus intensity, or alternatively a power function with an exponent of 0.3. Equal responses are obtained for equal ratios between the peptide concentration in the lower compartment and the threshold concentration. The significance of Weber-Fechner's law in leucocyte chemotaxis is discussed.

Enzymatic cleavage of the epsilon-peptide bond in alpha- and epsilon-substituted glycyl- and phenylalanyl-lysine peptides.

Lysine peptides, X-Lys-OH (Formula: see text) were synthesized, following classic or non-classic routes. Some bacterial and mammalian enzymes, endo- and exo-peptide hydrolases of the enzyme nomenclature type EC 3.4., were tested for their ability to split the epsilon-peptide bond in the above substrates. Kinetic constants (Km,kcat) were evaluated with leucine aminopeptidase from hog kidney and eye lens with aminopeptidase I from yeast. Aminopeptidase M (hog pancreas) and hog intestinal aminopeptidase were additionally examined for their Ki values with the above substrates in comparison to the classic protease substrate leucine p-nitroanilide. Especially the intestinal mucosa hydrolases are shown to be efficient in cleaving epsilon-peptide bonds.

A granulopoiesis inhibitor partially purified from large-scale serum-free cultures of porcine leukocytes.

A method is described for the partial purification of a granulomonopoiesis inhibitor derived from 200 l-batches equivalent to 1 kg or 2 X 10(12) leukocytes of porcine blood. Serum-free conditioned media were concentrated and separated via ammonium sulfate precipitation, acetone partitioning, ultrafiltration and gel chromatography. The active substance revealed a molecular mass of 500-700 Da and was not thiol-dependent, thus contrasting it with previously-reported granulopoiesis inhibitors. Inhibition of colony formation in an in vitro myeloid stem cell assay using various colony-stimulating factors showed that mature granulocytes and monocytes were equally affected. Apparent lack of cytotoxicity was suggested using a new reversibility test.

Identification of two granulocyte/macrophage colony-stimulating factors from porcine leukocyte cultures.

Granulocyte/macrophage colony-stimulating factors (CSF) were found in large-scale serum-free cultures of porcine blood leukocytes. The factors were concentrated from the conditioned culture medium of Concanavalin A-stimulated leukocytes using ammonium sulfate salting-out fractionation. The protein precipitate at 45-90% salt saturation contained two CSF types upon gel filtration on Sephadex G-100: One type was found to be active on mouse, but not on human bone marrow stem cells and had an apparent molecular mass of 85 000-100 000 Da; the other of about 40 000-45 000 Da stimulated both mouse and human bone marrow stem cells.