Post-transcriptional effects of interleukin-3, interferon-gamma, erythropoietin and butyrate on in vitro hemoglobin chain synthesis in congenital hemolytic anemia.
OBJECTIVE
Various agents modulate hemoglobin synthesis. In vitro modulation of translation in hemoglobin chain synthesis was analysed in patients with congenital hemolytic anemia (n=32) and healthy controls (n=17).


METHODS
Enriched reticulocytes were co-incubated with (3)H-leucine and cytokines or butyrate. Reversed-phase chromatography enabled separation of alpha-, beta- and gamma-globin chains. Globin chain synthesis was calculated from measured (3)H-leucine incorporation. Transferrin, erythropoietin, interleukin-3 and interferon-gamma receptors were detected by flow cytometry. Reverse-transcription polymerase chain reaction (RT PCR) was used to demonstrate changes of RNA stability.


CONCLUSIONS
Interleukin-3, interferon-gamma and butyrate caused a significant 2-fold increase (range 1.8-2.4; p<0.01) of the alpha- and beta-chain synthesis in congenital hemolytic anaemias. Analysis of gamma-globin chain synthesis revealed a lower, i.e. 1.4 fold increase (range 1.32 to 1.41; p<0.03). The absolute amount of globin synthesis was calculated to be 2.9 x 10(-12) g/reticulocyte/24h. After incubation with interleukin-3 the absolute additional synthesis of the alpha-globin chain reached 1.31 x 10(-12) g/reticulocyte/24h, of the beta-globin chain, 1.15 x 10(-12) g/reticulocyte/24h and of the gamma-globin chain, 0.26 x 10(-12) g/reticulocyte/24h. Butyrate and interferon-gamma had no or even an inhibiting effect on reticulocytes from normal controls, while interleukin-3 stimulated alpha- and gamma-chain synthesis (1.4 and 2.4 fold, respectively; p<0.03) suggesting an increase of fetal hemoglobin (HbF). Erythropoietin showed no stimulating influence. Membrane associated interleukin-3 receptors were detected in 0.78+/-0.14%, and interferon-gamma receptors in 0.1+/-0.015% of the red cells. Erythropoietin receptors were extremely rare (0.05+/-0.015%). The expression of transferrin receptors (CD71) correlated with the extent of globin chain stimulation. The alpha-, and beta-globin mRNA content of the reticulocytes after interleukin-3 incubation, as measured by RT-PCR, increased.


CONCLUSIONS
Hemoglobin chain synthesis could be modulated post-transcriptionally by interleukin-3, interferon-gamma and butyrate. Transferrin receptor and globin RNA stability might be involved in this phenomenon.
Reinhardt
D
D
Children's Hospital University Goettingen, Germany. dreinh@uni-muenster.de
Ridder
R
R
Kugler
W
W
Pekrun
A
A
D000293:Adolescent; D000328:Adult; D000745:Anemia, Hemolytic, Congenital / Q000097:blood*; D002087:Butyrates / Q000494:pharmacology; D016022:Case-Control Studies; D002478:Cells, Cultured; D002648:Child; D002675:Child, Preschool; D016207:Cytokines / Q000494:pharmacology*; D004921:Erythropoietin / Q000494:pharmacology; D005914:Globins / Q000096:biosynthesis / Q000187:drug effects / Q000235:genetics; D006454:Hemoglobins / Q000096:biosynthesis* / Q000187:drug effects; D006801:Humans; D007371:Interferon-gamma / Q000494:pharmacology; D007377:Interleukin-3 / Q000494:pharmacology; D007930:Leucine / Q000493:pharmacokinetics; D008875:Middle Aged; D020871:RNA Stability; D012333:RNA, Messenger / Q000032:analysis; D012156:Reticulocytes / Q000187:drug effects / Q000378:metabolism; D014316:Tritium
D002087:Butyrates; D016207:Cytokines; D006454:Hemoglobins; D007377:Interleukin-3; D012333:RNA, Messenger; D014316:Tritium; D004921:Erythropoietin; D007371:Interferon-gamma; D005914:Globins; D007930:Leucine