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. 2004 Nov;52(11):1483-93.
doi: 10.1369/jhc.4A6306.2004.

Presence and localization of three lactic acid transporters (MCT1, -2, and -4) in separated human granulocytes, lymphocytes, and monocytes

Natalya Merezhinskaya  1 , Sunday A Ogunwuyi, Florabel G Mullick, William N Fishbein
Affiliations

Presence and localization of three lactic acid transporters (MCT1, -2, and -4) in separated human granulocytes, lymphocytes, and monocytes

Natalya Merezhinskaya et al. J Histochem Cytochem. 2004 Nov.

Abstract

We fractionated leukocytes from three donors into >90% pure samples of granulocytes, lymphocytes, and monocytes and tested them for transcriptional and translational expression of three physiologically-proven lactate transporters, monocarboxylate transporter 1(MCT1), MCT2, and MCT4, using RT-PCR and affinity-purified rabbit antibody (Ab) to the C-terminal segment of each human MCT. Transcripts of all three MCTs were identified in each leukocyte fraction by RT-PCR and proven by sequencing of fragments extracted after isolation on agarose gels. Transporter protein of the appropriate size was demonstrated for each of the monocarboxylate transporters MCTs in lymphocytes and monocytes by Western blot, while lower-molecular-weight bands were found in granulocytes and are presumed to be degraded forms, because they were blocked by antibody-antigen (Ab-Ag) preincubation. IHC demonstrated all three MCTs in methanol-fixed droplets of all three leukocyte fractions; stain was abolished on omission of the primary Ab. Plasmalemmal staining occurred with all MCTs in all leukocyte fractions. Because the K(m) for lactate increases approximately fivefold at each step, with MCT2<1<4, leukocytes must use the full range of lactate binding to survive in acidic and hypoxic environments. Except for MCT4 in lymphocytes, all the MCTs also stained leukocyte cytoplasm, often with distinct granularity. Nuclear membrane staining was also seen with MCT1 and MCT2, while platelet plasmalemma stained only with MCT2.

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Figures

Figure 1
Figure 1
Three amplimers identifying mRNA of MCT1 in granulocytes (G), lymphocytes (L), and monocytes (M). The oligonucleotide standards in lanes 1, 5, and 9 are 100-bp increments from 100 to 1000 bp. The blurry bands near the bottom are excess primers, while the strong bands just above that level in lanes 2 and 4 are primer-dimers of 90 bp. The expected amplimer sizes were, respectively, 799, 331, and 94, and presented strong bands for L and M. The corresponding bands for G are quite weak, but upon extraction and sequencing, gave exact matches to the database sequence, as did the strong bands. See Table 3 for further amplimer data.
Figure 2
Figure 2
Examples of amplimers of MCT1, −2, and −4 in the same acrylamide gel before excision for extraction and sequencing. Extraneous lanes have been removed and the oligonucleotide 100-bp stepladder is the same as in Figure 1. The amplimers here were obtained from a monocyte fraction, and the total base pairs sequenced (see Table 3) were, respectively, 331 for MCT1, 636 for MCT2, and 419 for MCT4.
Figure 3
Figure 3
Western blots of the three MCT transporter proteins in granulocytes (G), lymphocytes (L), monocytes (M), and heart (H) as the positive control. Each of the blots is from a single gel, with extraneous lanes removed. The numbers at left show the location of globular protein standards relevant to the bands of interest. For the MCT1 blot, G1 indicates that the granulocyte preparation is from a different blood sample than G, and the same distinction follows for L1 and M1 vs L and M in the MCT2 blot. Arrows point to the very weak granulocyte bands.
Figure 4
Figure 4
IHC of MCT1 in serial 6-μm sections of frozen human muscle after immersion in various fixatives for 30 min at 4C. (A) Unfixed section; (C) methanol, same field; (D) acetone, same field; (B) unfixed section, primary antibody omitted, different field, light decreased to increase refractive effects. Asterisks identify the same fibers in A, C, D. (E-H) A different field; again, the asterisks identify the same fibers in all four sections. (E) unfixed section; (F) formalin; (G) methanol; (H) ethanol. The essential feature for comparison is the plasmalemmal staining, not the cytoplasmic staining.
Figure 5
Figure 5
Wright's stain and ICC stain of the three transporters in the three human leukocyte fractions. All panels are ×415 save for M, which is ×700. The smears in F and K give better morphological distinction for the mononuclear cells than do the droplets in G and L, but the cells are much more widely spaced and difficult to quantify for specific features. ICC was therefore confined to droplet preparations. Arrows in C, H, I, M, and N show regions of suspected nuclear membrane staining. Arrowheads in D show unstained erythrocytes as a negative control, while those in N show convincing staining of platelet plasmalemma by MCT2.
Figure 6
Figure 6
Evidence for nuclear envelope staining by MCT1 and MCT2 antibodies in each of the three leukocyte fractions at high magnification (×1615). In each panel, the arrows touch a curvy line at nuclear folds or perimeter, which is darker than the nucleoplasm or cytoplasm on either side; this is our requirement for a convincing indicator. In F, the arrowheads point to two platelets with plasmalemmal staining.
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