Differences in the constitutive and SIV infection induced expression of Siglecs by hematopoietic cells from non-human primates

Abstract

The expression of the Siglec family of molecules by hematopoietic cells from uninfected and SIV infected disease susceptible rhesus macaques (RM) and SIV infected disease resistant sooty mangabeys (SM) and for comparison humans was carried out. The predominant cell lineage in all three species expressing Siglec's was monocytes. The major finding by both a cross sectional and a prospective SIV infection study showed that, whereas monocytes from RM show marked increase in each Siglec constitutively expressed, monocytes from SM showed marked decreases in Siglec-1 expression. While monocytes from all three species constitutively expressed Siglec-3, human monocytes in addition expressed Siglec-5 and -9 and to a lower density 7, monocytes from RM expressed Siglec-7 and those from SM expressed Siglec-1. Monocytes from all three species, however, expressed mRNA for Siglec-1, -5, -7 and -9. The reasons for the failure to detect these molecules at the protein level and the mechanisms for such distinct effects of SIV infection on Siglec expression are discussed.

Fig. 1

Aliquots of Ficoll hypaque purified PBMCs from healthy adult humans (n = 10), normal uninfected adult rhesus macaques (n = 13) and uninfected adult sooty mangabeys (n = 12) were stained with antibodies with specificity for human Siglec-1, 3, 5 (2 clones), 6, 7 (2 clones), 8, 9, 10 and11 and analyzed by flow cytometry by gating on monocytes based on scatter profiles. Only those showing significant staining with one of 3 species is depicted. Data are representative profiles from each of the 3 species.

Fig. 2

RT-PCR assisted detection of mRNA coding for Siglec-1 (128 bp), Siglec-5 (134 bp), Siglec-7 (192 bp) and Siglec-9 (293 bp) in RNA isolated from enriched populations of monocytes from humans, rhesus macaques and sooty mangabeys is shown. (M = molecular weight standards; S1, 5, 7, and 9 refer to Siglecs). Data are representative of 3 similar assays.

Fig. 3

The protein sequence of Siglec-5 from rhesus macaques and sooty mangabeys and their alignment with the published protein sequence of human Siglec-5 is shown. Note there is 87% homology between the human and non-human primate sequences and there are 8 amino acid residues that are similar between human and mangabeys but not rhesus macaques.

Fig. 4

Flow cytometric analysis of Siglec expression by gated populations of myeloid (mDC) and plasmacytoid dendritic (pDC) cells from rhesus macaques and sooty mangabeys. The gating strategy utilized for human samples is shown in Fig. 6 A and for rhesus macaques and sooty mangabeys on left portions of Fig. 6C and 6D, respectively. PBMC samples were first gated on forward and side scatter profiles and then on cells that were lineage negative, HLA-DR⁺ and were either CD11c⁺ (mDC) or CD123⁺ (pDC). Representative data from one of the 3 similar experiments is depicted. Fig. 6B, C and D show the profiles of human, rhesus macaques and sooty mangabeys, respectively. Note the 2 distinct populations of mDC in samples from sooty mangabeys based on differences in the expression of HLA-DR.

Fig. 4

Flow cytometric analysis of Siglec expression by gated populations of myeloid (mDC) and plasmacytoid dendritic (pDC) cells from rhesus macaques and sooty mangabeys. The gating strategy utilized for human samples is shown in Fig. 6 A and for rhesus macaques and sooty mangabeys on left portions of Fig. 6C and 6D, respectively. PBMC samples were first gated on forward and side scatter profiles and then on cells that were lineage negative, HLA-DR⁺ and were either CD11c⁺ (mDC) or CD123⁺ (pDC). Representative data from one of the 3 similar experiments is depicted. Fig. 6B, C and D show the profiles of human, rhesus macaques and sooty mangabeys, respectively. Note the 2 distinct populations of mDC in samples from sooty mangabeys based on differences in the expression of HLA-DR.

Fig. 4

Flow cytometric analysis of Siglec expression by gated populations of myeloid (mDC) and plasmacytoid dendritic (pDC) cells from rhesus macaques and sooty mangabeys. The gating strategy utilized for human samples is shown in Fig. 6 A and for rhesus macaques and sooty mangabeys on left portions of Fig. 6C and 6D, respectively. PBMC samples were first gated on forward and side scatter profiles and then on cells that were lineage negative, HLA-DR⁺ and were either CD11c⁺ (mDC) or CD123⁺ (pDC). Representative data from one of the 3 similar experiments is depicted. Fig. 6B, C and D show the profiles of human, rhesus macaques and sooty mangabeys, respectively. Note the 2 distinct populations of mDC in samples from sooty mangabeys based on differences in the expression of HLA-DR.

Fig. 4

Flow cytometric analysis of Siglec expression by gated populations of myeloid (mDC) and plasmacytoid dendritic (pDC) cells from rhesus macaques and sooty mangabeys. The gating strategy utilized for human samples is shown in Fig. 6 A and for rhesus macaques and sooty mangabeys on left portions of Fig. 6C and 6D, respectively. PBMC samples were first gated on forward and side scatter profiles and then on cells that were lineage negative, HLA-DR⁺ and were either CD11c⁺ (mDC) or CD123⁺ (pDC). Representative data from one of the 3 similar experiments is depicted. Fig. 6B, C and D show the profiles of human, rhesus macaques and sooty mangabeys, respectively. Note the 2 distinct populations of mDC in samples from sooty mangabeys based on differences in the expression of HLA-DR.

Fig. 5

Effect of chronic SIV infection and viral load on the expression of Siglec’s by monocytes from rhesus macaques (n = 13) and sooty mangabeys (n = 13) as determined by flow cytometry. Aliquots of gated population of monocytes in the PBMCs from 13 uninfected and 13 SIVmac239 infected rhesus macaques and 12 uninfected and 13 naturally SIV infected sooty mangabeys were analyzed for the expression of Siglec-1, 3, 5, 6, 7, 8, 9, 10 and 11. Data presented illustrate the Mean +/− S.D. of the frequency of monocytes from Rhesus macaques (Fig. 5A) and sooty mangabeys (Fig. 5B) that express the appropriate Siglecs. Fig. 5C shows the effect viral load has on the expression of Siglec-1 by monocytes from 12 chronically infected rhesus macaques with low (< 10,000 viral copies/ml of plasma), 11 samples with high (> 100,000 viral copies/ml of plasma) viral loads and for comparison uninfected monkeys of each of the 2 species.

Fig. 5

Effect of chronic SIV infection and viral load on the expression of Siglec’s by monocytes from rhesus macaques (n = 13) and sooty mangabeys (n = 13) as determined by flow cytometry. Aliquots of gated population of monocytes in the PBMCs from 13 uninfected and 13 SIVmac239 infected rhesus macaques and 12 uninfected and 13 naturally SIV infected sooty mangabeys were analyzed for the expression of Siglec-1, 3, 5, 6, 7, 8, 9, 10 and 11. Data presented illustrate the Mean +/− S.D. of the frequency of monocytes from Rhesus macaques (Fig. 5A) and sooty mangabeys (Fig. 5B) that express the appropriate Siglecs. Fig. 5C shows the effect viral load has on the expression of Siglec-1 by monocytes from 12 chronically infected rhesus macaques with low (< 10,000 viral copies/ml of plasma), 11 samples with high (> 100,000 viral copies/ml of plasma) viral loads and for comparison uninfected monkeys of each of the 2 species.

Fig. 5

Effect of chronic SIV infection and viral load on the expression of Siglec’s by monocytes from rhesus macaques (n = 13) and sooty mangabeys (n = 13) as determined by flow cytometry. Aliquots of gated population of monocytes in the PBMCs from 13 uninfected and 13 SIVmac239 infected rhesus macaques and 12 uninfected and 13 naturally SIV infected sooty mangabeys were analyzed for the expression of Siglec-1, 3, 5, 6, 7, 8, 9, 10 and 11. Data presented illustrate the Mean +/− S.D. of the frequency of monocytes from Rhesus macaques (Fig. 5A) and sooty mangabeys (Fig. 5B) that express the appropriate Siglecs. Fig. 5C shows the effect viral load has on the expression of Siglec-1 by monocytes from 12 chronically infected rhesus macaques with low (< 10,000 viral copies/ml of plasma), 11 samples with high (> 100,000 viral copies/ml of plasma) viral loads and for comparison uninfected monkeys of each of the 2 species.

Fig. 6

Prospective analysis of the effect of SIV infection on Siglec-1 expression by monocytes from a minimum of 3 rhesus macaques and 3 sooty mangabeys as determined by flow cytometry. A) Aliquots of PBMC samples from each of the 6 monkeys prior to and at 4, 8, 16 and 36 weeks post experimental SIV infection were stained with anti-Siglec-1 antibody and the frequency of the gated population of monocytes evaluated. B) The absolute number of CD4⁺ T cells and plasma SIV viral load were determined in aliquots of the same blood sample as used for Siglec studies. Data shown are Mean +/− S.D. of the 3 samples from each of the 2 species.

Fig. 6

Prospective analysis of the effect of SIV infection on Siglec-1 expression by monocytes from a minimum of 3 rhesus macaques and 3 sooty mangabeys as determined by flow cytometry. A) Aliquots of PBMC samples from each of the 6 monkeys prior to and at 4, 8, 16 and 36 weeks post experimental SIV infection were stained with anti-Siglec-1 antibody and the frequency of the gated population of monocytes evaluated. B) The absolute number of CD4⁺ T cells and plasma SIV viral load were determined in aliquots of the same blood sample as used for Siglec studies. Data shown are Mean +/− S.D. of the 3 samples from each of the 2 species.