Local Factor Production Research Articles

Human endothelial cells prolong eosinophil survival. Regulation by cytokines and glucocorticoids.

Many recent studies have established the eosinophil as an active proinflammatory participant in a variety of disease states, most notably in allergic and helminthic disorders. In order to understand the effector role of eosinophils, factors which promote a selective eosinophilic infiltrate must be delineated. Eosinophil adherence to vascular endothelium is the first step in the formation of such an infiltrate. However, studies thus far have failed to identify factors which selectively activate the adherence of eosinophils. We have therefore speculated that the selective enrichment of eosinophils may result from nonselective recruitment of several leukocyte types combined with the production of local factors that promote the survival of eosinophils and not of other cells. We report that endothelial cell-conditioned medium selectively prolongs eosinophil survival up to 6 days in culture in a dose- and time-dependent manner. Stimulation of human vascular endothelial cells with IL-1 caused an increase in the generation of eosinophil survival-promoting activity, whereas stimulation with platelet-activating factor did not. Supernatants from human vascular endothelial cells cultured for 48 h in the presence of the glucocorticoid, dexamethasone, were less active in promoting eosinophil survival than control supernatants. These results suggest that factors produced locally in the vascular microenvironment may selectively promote eosinophil survival and may be under the regulation of cytokines and glucocorticoids.

Further studies on mechanisms regulating the voiding cycle of the rat urinary bladder

1. The effect of transection of pudendal or hypogastric nerves and of various pharmacological pretreatments on the bladder voiding cycle elicited by saline filling was investigated in urethane-anesthetized rats. 2. Sectioning of pudendal nerves reduced efficiency of the expulsive phase of the voiding cycle while sectioning of hypogastric nerves enhanced voiding efficiency and reduced residual volume. 3. An increased voiding efficiency was also observed in 6-hydroxydopamine-treated rats. 4. Atropine and physostigmine decreased and increased voiding efficiency, respectively. Indomethacin pretreatment produced a marked increase in residual volume. 5. These findings indicate that the autonomic innervation of the bladder and urethra as well as production of local factors such as prostanoids regulates not only the collecting phase of the cystometragram but also influence markedly voiding efficiency.

Mepacrine impairs neutrophil response after acute lung injury in rats. Effects on neutrophil migration.

Intraalveolar leukocytosis is integral in initiating and perpetuating airspace inflammatory reactions. We used intratracheal instillation of silica suspensions in adult male rats to cause neutrophil flux (32% increase over saline controls) without creating a protein leak, so simulating an early inflammatory response. We examined the in vivo effects of a known phospholipase A2 inhibitor (mepacrine) and the two mast cell active agents (cyproheptadine and reserpine) on lung lavage fluid chemotactic capability, alveolar macrophage (AM) production of chemotactic factor(s), and neutrophil diapedesis. Only mepacrine significantly depressed the leukocytosis (from 32% to 8% of total cells), with a similar diminution in AM chemotaxin production. Separate in vitro experiments using mepacrine-pretreated neutrophils and macrophages gave evidence that mepacrine: (1) diminishes neutrophil response to chemotaxin(s), (2) inhibits spontaneous, random neutrophil movement, and (3) diminishes macrophage-derived chemotactic factor production. These observations suggest that the earliest events in alveolar inflammatory reactions probably involve local production of chemotactic factors by AM, and that mepacrine's anti-inflammatory action results from inhibitory influences on both macrophage and neutrophil populations.

Elevation of PMN cytosolic free calcium and locomotion stimulated by novel peptides from IL-1-treated human synovial cell cultures

Treatment of human synovial cell cultures with human recombinant interleukin 1 (IL-1) results in the appearance of activity in the supernatant which stimulates human polymorphonuclear neutrophils (PMNs), as assessed by increased chemokinesis and elevation of intracellular free calcium concentration. IL-1 (or related cytokines) were unable to stimulate these responses. This activity chromatographed as a single peak on C8 reversed-phase HPLC and subsequent size exclusion HPLC revealed two peaks of chemokinetic activity with apparent molecular masses of approximately equal to 13kDa and 6kDa. Fractions containing the higher molecular mass material also elevated PMN cytosolic free calcium. The local production of such factors may mediate IL-1-induced PMN accumulation in vivo.

Interactions between the immune system and connective tissue in arthritis. Possible significance of an affinity between IgG and native type II collagen.

The synovial inflammation in rheumatoid arthritis (RA) resembles inflammatory reactions in other tissues concerning features such as increased expression of MHC class II antigens and infiltration of large amounts of activated T lymphocytes. The present communication is concerned with how to explain features such as local production of rheumatoid factors that distinguish the synovial inflammation in seropositive RA from other chronic inflammatory reactions; We show here that monomeric IgG and, to an even higher extent, aggregated IgG show a high binding capacity for native collagen type II. This finding is discussed in the light of previous findings that native collagen II structures are readily exposed to the environment in the cartilage of inflamed joints, and the evidence that T-cell reactivity to cartilage-derived molecules among them collagen II appears to be a common feature in seropositive RA. We suggest that an enhanced formation of IgG-collagen II complexes in RA joints, together with activation of T-cells to collagen II or collagen II-associated structures may constitute the basis for local rheumatoid factor production and to disease perpetuation in seropositive RA.

Neuroendocrine control processes: Pubertal onset and progression

This discussion has outlined current concepts in neuroendocrinologic control of pubertal onset and progression. Central nervous system regulation of the arcuate nucleus (ventromedial hypothalamus) pulse generator that subsequently controls pituitary gonadotropin synthesis and secretion has been highlighted. Significant investigative issues that deserve assessment in the next several years include the following: 1. Systematic neuropharmacologic, electrophysiologic, and anatomic assessment of the hypothalamic arcuate nucleus. These assessments would include the use of recombinant DNA technology to probe cellular regulation of GnRH production. 2. Physiologically oriented examination of hypothalamic GnRH synthesis and secretion, along with function in the remaining reproductive endocrine system, during situations of nutritional impairment and excessive energy utilization and psychologic stress. 3. Further assessment of the neurophysiologic inhibition of GnRH production during childhood and the late prepubertal reactivation of the arcuate nucleus pulse generator. Roles of opioids, dopamine, other neurotransmitters, and metabolic signals remain to be clarified. 4. Exploration of regulators of hypothalamic, pituitary, and gonadal function when pulsatile GnRH administration has replaced the usual hypothalamic mechanisms. Pituitary-gonadal interactions may be independently assessed. 5. Assessment of pubertal growth, endocrine function, and neuropharmacologic control mechanisms in circumstances of chemical removal of pituitary gonadotrope function by GnRH agonists or antagonists. 6. Concordance and discordance of potency estimates of gonadotropins made by bioassay and immunoassay. The biologic basis for qualitative changes in bioassayable levels of LH and FSH, often related to carbohydrate content of the glycoprotein, may help to explain changes of gonadal function during the pubertal process. The potential for significant molecular heterogeneity of the gonadotropins is recognized and suggests substantial posttranslational changes of LH and FSH. 7. A cogent delineation of the hormonal, nutritional, and energy regulators of the pubertal growth spurt, though not discussed in this manuscript, remains to be accomplished. The relationship between pituitary gonadotropins and growth hormone, sex steroids, and the various peptide growth factors, especially the relationship between the growth factors and intragonadal steroidogenesis and germ-cell production, remain to be resolved. The importance of local production and action of peptide-growth factors in diverse tissues, skeletal and other, is being increasingly recognized.(ABSTRACT TRUNCATED AT 400 WORDS)

Products of activated lymphocytes and macrophages inhibit mouse embryo development in vitro.

The effects of activated leukocyte products on embryonic development were assessed by adding mouse and human leukocyte culture supernatants and purified murine and human lymphokines and monokines to mouse embryos in tissue culture. Supernatants from mitogen-stimulated and mixed lymphocyte cultures arrested embryonic development at the two-cell to morula stage. Of a panel of six individual lymphokines and monokines tested for effects in this system, both murine and human forms of the lymphokines colony-stimulating factor, interferon-gamma, and human B cell growth factor significantly arrested embryonic development over a wide concentration range. The monokines, interleukin 1 and tumor necrosis factor, also had significant effects but only at high doses. These results indicate that products of activated lymphocytes and macrophages can have detrimental effects on preimplantation embryos. Early abortion could result from local (intrauterine) production of such embryotoxic factors by activated lymphocytes and macrophages in response to stimulation by microorganisms or reproductive tissue antigens.

Biologic Properties of LTB4 and Paf-acether In Vivo in Human Skin

Using an improved skin chamber technique, the consequences of prolonged contact of leukotriene B4 (LTB4) and platelet-activating factor (paf-acether) with human dermis were evaluated quantitatively and kinetically in vivo. Leukocyte chemotaxis, histoenzymologic alterations, and modifications in vascular permeability were studied in two sets of experiments. In a first set of experiments, the dose-effect relationship of LTB4 and paf-acether on leukocyte migration was studied. LTB4 (3 X 10(-8) M to 9 X 10(-7) M) in Hanks' balanced salt solution (HBSS) elicited an intense dose-dependent and time-dependent neutrophil migration. Paf-acether, at the same concentration range, induced a significant increase in cell migration only at 9 X 10(-7) M and when diluted in HBSS containing 0.25% serum albumin (HBSS-BSA). Histoenzymologic analysis demonstrated that LTB4 in vivo induced degranulation of most of the neutrophils migrating through the dermis. Paf-acether caused mild degranulation of neutrophils and induced the appearance of degranulated basophils in dermal vessels. A second set of experiments was designed to study simultaneously the modifications in vascular permeability and cell migration induced by LTB4 and paf-acether, with or without prostaglandin E2 (both at a concentration of 3 X 10(-7) M in HBSS). Since spontaneous protein diffusion in HBSS progressively declined up to a plateau reached after 20 h (1.2 +/- 0.15 mg of proteins/cm2/2 h), these experiments were carried out after a 20-h equilibration period. Leukotriene B4 induced a late and slight increase in vascular permeability. Paf-acether did so intensely and transiently. Prostaglandin E2 significantly enhanced protein diffusion and neutrophil migration induced by LTB4 and, to a lesser extent, by paf-acether. Interestingly, despite the reintroduction into the skin chambers of freshly prepared solutions containing the mediators, leukocyte migration and protein diffusion progressively decreased during the experiments. This suggests the local production of anti-inflammatory factors that inhibit local mediators and thus regulate the inflammatory response.

Human monocyte adherence: a primary effect of chemotactic factors on the monocyte to stimulate adherence to human endothelium.

Monocyte emigration into areas of inflammation is initiated by monocyte adherence to the microvascular endothelium which may be induced by the local production of chemotactic factors at the inflammatory site. However, it is not clear whether such stimuli act on the monocyte and/or the endothelial cell to promote this effect. Accordingly, the effect of the chemotactic peptides C5a des arg and formyl-methionyl-leucyl-phenylalanine (FMLP) on human monocyte adherence to human microvascular endothelial cell monolayers was investigated in vitro. Monocytes (92 to 98% pure) were isolated by discontinuous plasma-Percoll density gradients and cell elutriation, methods designed to minimize monocyte exposure to endotoxin. Mean spontaneous (unstimulated) adherence of 111Indium-tropolonate-radiolabeled monocytes to microvascular endothelial cell monolayers was 19.7% +/- 1.3. Monocyte adherence to microvascular endothelial cell monolayers was stimulated in a dose-response fashion in the presence of C5a des arg or FMLP to a maximum mean adherence of 47.2% +/- 2.9 or 43.8% +/- 2.2, respectively. C5a des arg or FMLP stimulated monocytes to adhere to monolayers of human vascular smooth muscle cells, human dermal fibroblasts, or serum-coated plastic wells in a comparable fashion as to endothelial cells. The simultaneous presence of both chemotactic peptides C5a des arg and FMLP in the assay system stimulated monocyte adherence to the same degree as either stimulus alone. This finding suggested that those monocytes stimulated to adhere by C5a des arg were the same subpopulation responding to FMLP. Spontaneous monocyte adherence (in the absence of chemotactic peptides) to both endothelial cell monolayers and serum-coated plastic wells was reduced in the presence of plasma, but chemotactic peptides induced a significant, albeit reduced, adhesion of monocytes in this circumstance. The pretreatment of monocytes with either C5a des arg or FMLP prior to the adherence assay induced stimulus-specific desensitization of monocyte adherence. Neither a desensitization nor stimulated monocyte adherence occurred when endothelial cell monolayers or serum-coated plastic wells were pretreated with either of the chemotactic peptides. The fixation of endothelial cell monolayers prior to the adherence assay did not alter the degree of spontaneous, C5a des arg-stimulated, or FMLP-stimulated monocyte adherence. These data suggest that the stimulated adhesion of monocytes to endothelial cells by C5a des arg or FMLP represents primarily an effect of these chemotactic peptides on the monocyte.

Effects of unilateral arterial infusion of GH and IGF-I on tibial longitudinal bone growth in hypophysectomized rats.

We have studied the effect of local arterial infusion of bacterially produced human growth hormone (hGH), insulinlike growth factor I (IGF-I), or pituitary-derived ovine prolactin (oPRL) on longitudinal bone growth of hypophysectomized rats. The substances were infused during a 14-day period by osmotic mini-pumps through a catheter which was implanted into the femoral artery of one hindlimb. Longitudinal bone growth was measured by the intravital marker tetracycline. Infusion of 1 microgram hGH per day stimulated bone growth only of the treated limb and not of the uninfused contralateral limb. Infusion of 10 micrograms hGH per day also stimulated unilateral longitudinal bone growth, but the uninfused contralateral limb also showed a significant growth response, probably because local administration of GH at this dose caused a significant elevation of GH in the systemic circulation. As a result, the differential growth response between the GH-treated and untreated limbs decreased compared to rats that were infused with 1 microgram hGH per day. Unilateral arterial infusion of 5 micrograms human IGF-I or 10 micrograms oPRL per day did not produce a significant growth response. The results of the present study confirm the observation by Schlechter and co-workers, who demonstrated that unilateral arterial infusion of GH maintained tibial cartilage width following hypophysectomy in the rat. The results of Schlechter and coworkers and the results of the present study show that GH in vivo stimulates epiphyseal cartilage growth directly. However, an increased local production of insulinlike growth factors is probably of importance for the expression of the direct effect of GH on longitudinal bone growth.(ABSTRACT TRUNCATED AT 250 WORDS)

EVIDENCE THAT RABBIT CULTURED GROWTH PLATE (GP) CHONDROCYTES SYNTHESIZE 40-60K and 7K PROTEINS WITH IMMUNOREACTIVE IGFI ACTIVITY (IR-IGFI A)

The local production of IGFI like factors by cultured GP Chondrocytes was studied. IR-IGFI A in the incubation medium of prepuhertal rabbit GP Chondrocytes grown in serum-free defined medium during 20 days was evaluated by RIA. Chondrocyte phenotype was followed by specific proteoglycan and Type II collagen biosynthesis. The mean IR-IGFI A measured every 48 h from day 6 to day 16 of the primary culture was constant at 40±18 ImU/106 cells. This activity was further characterized after extraction of the culture medium at neutral and acid pH successively. At neutral pH IR-IGFI A of the eluates of G100 Sephadex column appeared as two distinct peaks:a major one migrating in the 40-60K protein region plus an additive one in the 7K region with a 40-60K:7K ratio =2:l. The 40-60K protein peak material extracted from culture medium corresponding to 10 cells, was able to specifically and reversihly bind 42±10 fmoles 1251-IGFI. At acid pH,IR-IGFI A was similarly separate? as 40-60K and 7K peaks, but their respective amount was reversed resulting in a 40-60K:7K ratio =1:2. When Chondrocytes were incubated in the presence of 35S-Meth during 20 h, only the 40-60K IR-IGFI protein peak was radiolabeled after chronatography at neutral pH. After acid extraction of the radiolabeled 40-60K peak, 60 to 80% of the radioactivity was recovered in the 7K protein peak. This radiolabeled 7K protein peak analyzed by electrophoresis on SDS 10% polyacrylamide gel pH 8.3 was cornigrating with pure 125I-IGFI. These results suggest that chondrocyte synthesize and secrete IGFI-like peptide and a pool of higher MW proteins with IGFI binding capacity.

Ectopic Hematopoiesis in Peritoneal Tumor Nodules Induced by the Myeloproliferative Sarcoma Virus in DBA/2 Mice<xref ref-type="fn" rid="FN2">2</xref>

Tumor nodules composed of fibroblasts, large undifferentiated cells, granulocytes, and small lymphocytes develop in the spleens of adult DBA/2 mice infected with the myeloproliferative sarcoma virus (MPSV). They spread thereafter in the organism, and at the terminal stage of the disease they are especially numerous on the peritoneal membrane. The present study, performed on those tumor nodules to avoid contamination by exogenous hematopoietic cells, demonstrated that they were sites of granulopoiesis, which may have occurred via the local differentiation of granulomacrophage precursor cells (GM-CFC) and perhaps also from pluripotent hematopoietic stem cells, since these two populations were present in the tumor nodules (25 +/- 11 and 13 +/- 10, respectively, per 5-10(5) cells). Almost all (88%) those GM-CFC were able to clone in vitro without added colony-stimulating factor. A comparative study with the Moloney murine sarcoma virus-induced tumor indicated that the local production of hematopoiesis-stimulating factors was not sufficient to allow such ectopic granulopoiesis. These results imply the presence of a specific hematopoietic microenvironment in the MPSV-induced tumor nodules.

Mode of action of pituitary growth hormone on target cells.

Normal postnatal somatic growth becomes progressively dependent on GH with time. In contrast to other hormones, GH is the only hormone known to produce a dose-dependent stimulation of postnatal growth. Most of the effects attributed to GH action appear to be the result of a direct effect of GH on cells in different peripheral tissues, including cartilage. In addition to the growth-stimulating effect, GH has the intrinsic properties of being able to exert both insulin-like and insulin-antagonistic effects in adipose tissue and skeletal muscle. These two apparently antagonistic effects seem to be explained by the stage of responsiveness of the target cells to GH, which is determined by the previous influence of endogenous GH. An inhibition of adenylate cyclase with a concomitant decrease in intracellular cAMP might be an important early cellular event in the course of GH action, but it is not known whether or how this change in nucleotide metabolism relates to the various expressed effects of the hormone. The recognition that GH directly interacts with chondrocytes in cartilage suggests that alterations in the concentration of circulating somatomedins cannot be the only factor regulating skeletal growth. The recent discovery by Green and coworkers (42) demonstrating that GH specifically stimulates the differentiation of cloned preadipose cells and myoblasts in tissue culture may be a major breakthrough in the understanding of the mechanism of action of the growth-promoting effect of GH. Green (42) has proposed that GH directly stimulates terminal differentiation of cells in many different tissues including epiphyseal plate cartilage. The finding that GH binds specifically to cells in the resting cell zone but not to differentiated chondrocytes in the growth plate suggests that prechondrocytes in the growth plate are the target cells for GH action. If it is correct that GH directly stimulates the differentiation of prechondrocytes, we suggest that, during the process of chondrocyte differentiation in the growth plate, the genes that code for growth factors of the somatomedin class, such as IGF-I, are expressed. As a consequence, the clonal expansion of the chondrocytes in the proliferative zone of the growth plate that occurs in vivo during the process of normal growth is the result of this local production of growth factors.

Major histocompatibility complex-restricted, polyclonal B cell responses resulting from helper T cell recognition of antiimmunoglobulin presented by small B lymphocytes.

Anti-Ig has been widely used as a model for antigen receptor-mediated B cell activation. B cells activated with mitogenic concentrations of anti-Ig (approximately 10 micrograms/ml) become responsive to a set of T cell-derived, antigen-nonspecific helper factors that enable the B cells to proliferate, and, in some cases, mature to Ig secretion. In the present experiments, we show that anti-Ig can also be used as a model for major histocompatibility complex (MHC)-restricted, antigen-specific T-B cell collaboration. We used murine helper T cell lines and T cell hybridomas specific for a protein antigen, the F(ab')2 fragment of normal rabbit IgG. Small B cells are very efficient at presenting rabbit anti-IgM or rabbit anti-IgD to these rabbit Ig-specific T cell lines and hybridomas, and the responding (initially) small B cells, appear to be the only antigen-presenting cells required. Efficient presentation depends upon binding of rabbit antibody to mIg on the B cell surface. MHC-restricted recognition of rabbit Ig determinants on the B cell surface results in a polyclonal B cell response. This response is qualitatively different from the well-studied response to blastogenic concentrations of anti-Ig plus stable, T cell-derived helper factors, since it (a) requires 1,000-fold lower concentrations of anti-Ig, (b) involves helper T cell functions other than, or in addition to, the local production of the same stable helper factors, and (c) is largely MHC-restricted at the T-B cell level.

Production of mononuclear cell chemotactic factors during Sindbis virus infection of mice.

Draining lymph node cells of mice infected subcutaneously with Sindbis virus (SV) produced two mononuclear chemotactic factors in vitro. One factor did not require the addition of SV in vitro and was only detectable during the first week after infection. A second factor, resembling lymphocyte-derived chemotactic factor, required the addition of SV in vitro, was first detectable at 3 days, reached a peak at 15 to 18 days, and was gone by 29 days after infection. The production of this factor was virus specific. Diluent-inoculated mice produced no detectable mononuclear chemotactic factors in response to SV. In vitro production of the virus-specific chemotactic factor was dependent on both adherent cells and sensitized Lyt1+ T cells. In vitro production of the spontaneous factor was associated only with adherent cells but also appeared to be T cell dependent, since the lymph node cells from SV-infected athymic nude mice failed to produce either factor. Infectious center assays showed that adherent cells contained infectious SV without replicating it, suggesting the engulfment of virus by macrophages in the lymph node draining the area of virus replication. These cells probably process virus as antigen for presentation to T cells, resulting in local production of chemotactic factors as well as production in more distant sites of viral replication after leaving the lymph node. These virus-stimulated, mononuclear cell-produced chemotactic factors are likely to be of importance in generating the mononuclear inflammatory response.