Hemoglobin induction in mouse macrophages

Abstract

The common perception that hemoglobin is involved solely in the transport of oxygen and carbon dioxide has been challenged by recent studies with nitric oxide (NO). These studies have shown that the primordial bacterial flavohemoglobin functions to consume NO enzymatically (to protect from nitrosative stress), whereas mammalian hemoglobin functions to deliver NO (thus maximizing oxygen delivery in the respiratory cycle). Here we report that murine macrophages stimulated to produce NO with lipopolysaccharide and interferon-gamma express the betaminor hemoglobin subunit. Consumption of NO, however, was not increased by cytokines or by hemoglobin expression. These data suggest alternative functions for globins in mammalian cells, and they challenge the prevailing view that the expression of alpha- and beta-globin genes is always balanced and coordinated.

Figure 1

Detection of hemoglobin by Western blotting. Antiserum against hemoglobin (Research Plus) was used in lanes 1–4 and antibody against myoglobin in lanes 5–8. Lanes 1 and 5, purified hemoglobin (100 ng, Sigma); lanes 2 and 6, mouse muscle extract (15 μg); lanes 3 and 7, lysate (50 μg) of resting RAW264.7 cells; lanes 4 and 8, lysate (40 μg) of RAW264.7 cells stimulated with IFN-γ/LPS for 16 hr. (An unidentified protein of ≈40 kDa is recognized by the anti-myoglobin antibody in lane 6.)

Figure 2

Expression of hemoglobin β^minor mRNA in IFN-γ/LPS-treated RAW264.7 cells. (A) RT-PCR was carried out with hemoglobin β-specific primers (β291se and β3′) at an annealing temperature of 55°C for 40 cycles. (B) RT-PCR was performed with GAPDH primers at an annealing temperature of 60°C for 40 cycles. N, no DNA templates; M (molecular weight markers), 100-bp DNA ladder; C (control), RAW264.7 cells without stimulation; I/L, RAW264.7 cells stimulated with IFN-γ/LPS for 12 hr.

Figure 3

Nucleotide and deduced amino acid sequence of macrophage hemoglobin.

Figure 4

Time course of globin mRNA and protein induction. RAW264.7 cells were incubated with IFN-γ/LPS for the times indicated (1.5–24 hr), after which equal amounts of RNA were used in RT-PCR with the primers β291se and β3′ at an annealing temperature of 60°C for 40 cycles (A), and cellular proteins were probed with hemoglobin antiserum as in Fig. 1 (B). N, no DNA templates; M (molecular weight markers), 100-bp DNA ladder; time 0, RAW264.7 cells without stimulation.

Figure 5

Induction of β^minor-globin in peritoneal macrophages. Macrophages treated (lanes 2 and 5) or not treated (lanes 1 and 4) with IFN-γ/LPS were probed by RT-PCR for β^minor transcripts (lanes 1 and 2) with primers β150se and β550as, and for GAPDH (lanes 4 and 5), at the PCR annealing temperature of 60°C. The 100-bp DNA ladder is shown in lane 3.

Figure 6

NO consumption by IFN-γ/LPS-activated macrophages. (A) NO (≈250 nM) was added to buffer (solid line), to a suspension of resting RAW264.7 cells (dotted line), or to RAW264.7 cells that had been previously treated with IFN-γ/LPS for 16 hr (dashed line). Decay of NO was monitored with an NO electrode (6). (B and C) NO was added at 100 nM (B) or 500 nM (C) to buffer (solid line) or to lysates of cells that had (dashed line) or had not (dotted line) been previously stimulated with IFN-γ/LPS, in the presence of 200 μM NADH and NADPH. Similar results were obtained in the added presence of flavins and/or biopterin.