Abstract
Considering that human milk (HM) supresses multiple Ca++ -regulated human polymorphonuclear leukocyte (PMN) functions, we sought to examine the effects of HM on PMN Ca++ metabolism. Fura-2 loaded PMN were exposed to HM, infant formula (FOR) or Hanks Balanced Salt Solution + Ca++ (HBSS) for 10 sec - 30 min and re-suspended in HBSS ± Ca++. Cytosolic Ca++ concentration [CaF] was monitored ± 10-6M f MLP, ± 100nM thapsigargin, ± 1mM extracellular CaCl2. HM exposure did not alter peak [CaF] following f MLP exposure but lowered the subsequent [CaF] fMLP response based on area under the curve (nmol*min) [HBSS: 828±36(Mean±SE) nmol*min; FOR: 638±42 nmol*min; HM: 405±42 nmol*min, p<0.02 HM vs HBSS or FOR]. This effect was observed with HM exposures as brief as 10 sec, persisted for 4 hr after removal from HM, was concentration-dependent and was associated with secondary Ca++-stores depletion. HM-exposure did not affect [CaF] after fMLP when cells were re-suspended in a zero Ca++-buffer, but extracellular addition of 1mM CaCl2 significantly increased [CaF] in HBSS and FOR -exposed PMN, but not HM-treated PMN [HBSS: 437±35 nmol*min; FOR: 718±57 nmol*min; HM: 302±15 nmol*min, p<0.02 HM vs HBSS or FOR], suggesting that HM exposure resulted in blockade of Ca++ influx. HM also blocked spontaneous Mn++ uptake by PMN over 20 min, based on loss of fura-2 fluorescence (%) [HBSS: 39±4%; FOR: 31±4%; HM: 23±4%, p<0.05 HM vs HBSS or FOR]. These data show that HM exposure blocks Ca++ influx in PMN. We speculate that this effect potentially explains the anti-inflammatory properties of HM on PMN.
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