Granulocyte/macrophage colony-stimulating factor and interleukin 3 release from human peripheral blood eosinophils and neutrophils.

Abstract

SummaryHuman peripheral blood eosinophils released eosinophil survival-enhancing activity when stimulated with the calcium ionophore, ionomycin . The release of activity was detected as early as 3 h after stimulation and was inhibited by an immunomodulating agent, cyclosporin A . The survivalenhancing activity was completely abolished by treatment with anti-interleukin 3 (IL3) and anti-granulocyte/macrophage colony-stimulating factor (GM-CSF) monoclonal antibodies . Moreover, IL-3 and GM-CSF were measurable in ionomycin-stimulated eosinophil supernatants by immunoassay. Eosinophils produced approximately one-half as much IL3 and one-fifth as much GM-CSF as ionomycin-stimulated mononuclear cells. Neutrophils also produced 11,3 and GM-CSF, but the amounts were less than those produced by eosinophils. These observations suggest a novel role for eosinophils in pathophysiology of allergic inflammation and host defense mechanisms. E osinophils are blood leukocytes associated with helminth infections and allergic diseases, especially bronchial asthma, and may mediate immunity and tissue damage (1) . Several rytokines, including IL3, 116, and granulocyte/macrophage colony-stimulating factor (GM-CSF), induce eosinophilopoiesis and activate mature peripheral blood eosinophils in vitro (reviewed in reference 2). Recently, mRNA for these rytokines was detected in allergen-induced late-phase cutaneous reactions in atopic patients, suggesting that these rytokines play an important role in allergic inflammation (3) .Because human peripheral blood eosinophils, despite their high degree of specialization, retain the ability to synthesize rytokines such as TGF-ot and IL1 (4, 5), we investigated whether they can produce IL-3, IL-5, and GM-CSF . We found that ionomycin-stimulated eosinophils produce GM-CSF and IL-3 as shown by enhanced eosinophil survival and by immunochemical measurement .Cells and Culture Conditions. Eosinophils, neutrophils, and mononuclear cells were isolated from peripheral blood of healthy volunteers or patients with mild hay fever by discontinuous Percoll density gradient as previously described (6) . Purities of eosinophils and neutrophils were 391% and 399%, respectively. Contaminating cells in the eosinophil preparations were only neutrophils and those in the neutrophil preparations were only eosinophils . Cell viabilities were >98% . Cells were incubated (2 x 105 cells per 0 .2 ml per well) in Hybri-Care medium (American Type Culture Collection, Rockville, MD) supplemented with 50 lAg/ml gentamicin and 10% defined calf serum in 96-well flat-bottomed microtiter plates (Falcon Labware, Lincoln Park, NJ) with ionomycin (Calbiochem Corp., San Diego, CA) in the presence or absence of PMA (500 pg/ml; Calbiochem Corp.) . After 24 h at 37°C, cellfree supernatants were harvested and frozen until assayed .Eosinophil Survival Assay. To measure eosinophil survival enhancing activity (7) in supernatants, eosinophils were freshly isolated from different donors and were cultured (2 .5 x 10" cells per 0 .2 ml per well) ...

MCP-1 is selectively expressed in the late phase by cytokine-stimulated human neutrophils: TNF-α plays a role in maximal MCP-1 mRNA expression

“…Among other PMN-stimulating cytokines, GM-CSF prolongs PMN survival by inhibiting programmed cell death [42,43] and also stimulates PMN to express several cytokine mRNAs [5,11,16,20,21]. Although rGM-CSF was able to induce a low-level MCP-1 mRNA accumulation in PMN, addition of neutralizing anti-GM-CSF IgG had no effect on the expression of MCP-1 mRNA.…”

“…It is now well known that many cytokines can be produced by stimulated human PMN [5]. These include IL-1␣ and ␤ [6], tumor necrosis factor ␣ (TNF-␣) [7], IL-1 receptor antagonist (IL-1Ra) [8,9], transforming growth factor ␤ (TGF-␤) [10], IL-6 [11,12], IL-8 [13][14][15][16], interferon-␥ (IFN-␥) [17], macrophage inflammatory protein-1␣ (MIP-1␣) and MIP-1␤ [18], IFN-␥-inducible protein-10 (IP-10) [19], granulocyte-macrophage colony-stimulating factor (GM-CSF) [20], granulocyte-CSF (G-CSF) [21], and macrophage-CSF (M-CSF) [21].…”

The role of the bone marrow in allergy and asthma

“…Lineagespecific cytokine effects on differentiated cells, which retain hemopoietic cytokine receptors on their surface, result in "activation" or "phenotype switching": IL-3, IL-5, and GM-CSF can activate and prevent apoptosis of basophils and eosinophils (42,59); G-CSF and GM-CSF do the same for neutrophils (60). Autocrine stimulation by eosinophils, basophils, or mast cells of their own differentiation and/or activation is thus possible, since they produce a wide range of hemopoietic and proinflammatory cytokines, including IL-3, IL-4, IL-5, IL-6, and GM-CSF (33,(61)(62)(63)(64)(65)(66)(67)(68)(69).…”

Eokines: synthesis, storage and release from human eosinophils

“…Ionomycin-induced release of GM-CSF resulted in the release of 45 ± 20 pg/10 6 cells in supernatants of cultured eosinophils (Kita et al 1991), while LPS induced the release of 23 ± 8 pg/mL of GM-CSF from 5 x 10 5 freshly isolated peripheral blood eosinophils (Takanaski et al 1994). LPS failed to stimulate significant GM-CSF release from a maximal number of potentially contaminating mononuclear cells or neutrophils, demonstrating that the LPS-induced release of GM-CSF is exclusively from eosinophils in these preparations.…”

“…GM-CSF can be released from peripheral blood eosinophils during in vitro incubation of cells with ionomycin or LPS (Kita et al 1991, Takanaski et al 1994. Ionomycin-induced release of GM-CSF resulted in the release of 45 ± 20 pg/10 6 cells in supernatants of cultured eosinophils (Kita et al 1991), while LPS induced the release of 23 ± 8 pg/mL of GM-CSF from 5 x 10 5 freshly isolated peripheral blood eosinophils (Takanaski et al 1994).…”