Quinine-dependent antibodies to neutrophils react with a 60-Kd glycoprotein on which neutrophil-specific antigen NB1 is located and an 85-Kd glycosyl-phosphatidylinositol-linked N-glycosylated plasma membrane glycoprotein

We have previously described a 24-year-old woman with quinine-dependent antibodies that reacted with neutrophils, red blood cells (RBCs), platelets, and T lymphocytes. The drug-dependent neutrophil antibody was found to react with 85- and 60-Kd neutrophil membrane molecules. In these studies, we further characterized these molecules and found that both were glycosyl-phosphatidylinositol (GPI)-linked and contained sialic acid residues and N-linked carbohydrate side chains, but neither contained O-linked carbohydrates. The protein backbone of the 60-Kd molecule was 45 Kd, and the 85 Kd glycoprotein (GP) was made up of 33- and 31-Kd proteins. While some GPI-anchored neutrophil GPs are released by stimulated neutrophils, neither the 85- nor the 60-Kd GP was released by neutrophil stimulated with C5a, f-met-leu-phe (FMLP), or phorbol myristate acetate (PMA). Neutrophil-specific antigen NB1 is located on a 58- to 64-Kd GP. To determine if the quinine-dependent antibody and anti-NB1 recognize the same GP, immunoprecipitation studies were performed with the quinine-dependent antibody using neutrophils with varying NB1 phenotypes. The 60-Kd GP was detected on NB1-positive neutrophils from 11 of 12 donors tested, but not on NB1- negative neutrophils from two donors tested. After solubilized 125I- labeled neutrophils were absorbed with anti-NB1, the quinine-dependent antibody immunoprecipitated the 85-Kd GP, but not the 60-Kd GP. These results indicate that anti-NB1 and the quinine-dependent antibody identified the same GP. The 85-Kd GP was detected on neutrophils from all 14 donors tested. The electrophoretic mobility of the 85-Kd GP was similar to the electrophoretic mobility of the major 125I-labeled neutrophil protein.     

Quinine-induced immune thrombocytopenia associated with hemolytic uremic syndrome: a new clinical entity

Three patients are described who developed severe thrombocytopenia, microangiopathic hemolytic anemia, and acute renal failure after ingestion of quinine. In one patient, the same clinical findings recurred several months later after another exposure to quinine. Serum from one patient contained quinine-dependent IgG antibodies reactive with the platelet glycoprotein (GP) Ib/IX complex. In the second and third cases, serum contained IgG and IgM antibodies reactive with both the GP Ib/IX and IIb/IIIa complexes in the presence of quinine. Quinine appears to have induced both immune thrombocytopenia and the hemolytic uremic syndrome (HUS) in these individuals. Findings made in these cases may have implications for the pathogenesis of some forms of HUS.     

Quinine-induced thrombocytopenia following intravenous use of heroin

Profound thrombocytopenia developed in a 22-year-old man after intravenous use of heroin. A high-titer, quinine-dependent, platelet-specific antibody was detected in his serum using lysis of normal platelets labeled with chromium 51 and an electroimmunoassay for measurement of platelet-associated IgG. The antibody was specific for quinine and failed to react with platelets in the presence of quinidine hydrochloride or two structural analogues of heroin. Quinine, a common adulterant found in heroin, was detected in the patient's blood and urine. On the basis of these observations, the patient was judged to have quinine-induced immunologic thrombocytopenia. To our knowledge, this report is the first to confirm that quinine used as an adulterant can induce immunologic thrombocytopenia following an injection of heroin.     

Alloimmune neonatal neutropenia due to an antibody to the neutrophil Fc-gamma receptor III with maternal deficiency of CD16 antigen.

Antibodies to the neutrophil-specific antigens NA1 and NA2 are associated with alloimmune neonatal neutropenia (ANN), autoimmune neutropenia of childhood, and acute pulmonary transfusion reactions. These antigens have been found to be located on the neutrophil Fc-gamma receptor III (FcRIII). The mother of a child with ANN was found to lack both NA antigens and to produce an antibody that reacted with all normal neutrophils tested. We used maternal antibody and a CD16 monoclonal antibody (MoAb) that has specificity for FcRIII to immunoblot and immunoprecipitate neutrophil membranes of various NA phenotypes. Both antibodies immunoblotted an approximately 40- to 70-Kd glycoprotein (GP) on NA1, NA2-positive membrane, an approximately 40- to 55-Kd GP on NA1-homozygous membranes, and an approximately 55- to 70-Kd GP on NA2-homozygous membranes. Both antibodies also immunoprecipitated a 50- to 80-Kd GP from NA1, NA2-positive cells, a 50- to 60-Kd GP from NA1-homozygous cells, and a 55- to 80-Kd GP from NA2-homozygous cells. To further examine the specificity of the maternal antibody, sequential immunoprecipitation studies were performed using maternal antisera and a CD16 MoAb. After extracts of 125I surface-labeled neutrophils were precleared with maternal serum, CD16 MoAbs no longer immunoprecipitated any GP. Neither the CD16 MoAb nor a rabbit polyclonal antibody specific for FcRIII detected any GP in maternal neutrophil membranes by immunoblotting. Neutrophil FcRIII is a glycosyl-phosphatidylinositol anchored membrane GP as is decay accelerating factor and both are absent from neutrophils of patients with paroxysmal nocturnal hemoglobinuria (PNH). Maternal neutrophil membranes were probed with antibody specific for DAF and an 80-Kd GP was detected. This woman also has had no clinical evidence of PNH. These studies provide further evidence that the NA1 and NA2 antigens are on FcRIII and identify a healthy person whose neutrophils lack not only the neutrophil specific antigens NA1 and NA2 but multiple other epitopes of FcRIII and, therefore, likely lack FcRIII entirely.     

K-cell lymphocytosis/neutropenia syndrome: the neutropenia is not caused by autoimmunity

The role of humoral immune mechanisms in the pathogenesis of the neutropenia in five patients with chronic killer (K)-cell lymphocytosis was studied. For the detection of neutrophil antibodies in the patient's blood, immunofluorescence, cytotoxicity and agglutination tests and a sensitive antibody-dependent cellular cytotoxicity (ADCC) assay were applied. An assay for bone-marrow colony growth (CFU-GM, BFU-E) inhibition was applied as well. A C1q-binding test was used to detect immune complexes. The lymphocytes of all five patients had the capacity to lyse alloantibody sensitized neutrophils. Neutrophil-bound IgG was found only in one patient. Two patients had neutrophil-reactive antibodies in their serum; in one patient these antibodies were only detectable in the ADCC using the patient's serum to sensitize target cells. However, these antibodies reacted also with lymphocytes, were absorbable with platelets and thus probably were HLA antibodies. The serum of one of these two patients showed complement-dependent CFU-GM and BFU-E inhibition, but the observed inhibition was probably also due to the anti-HLA antibodies present in his serum, as the inhibiting effect disappeared after absorption of the serum with platelets. The serum of only one patient contained low amounts of immune complexes, as measured in the C1q-binding test. Our data suggest that humoral autoimmune mechanisms, such as autoantibodies against neutrophils or neutrophil precursors or circulating immune complexes, do not seem to play an important role in the K-cell lymphocytosis/neutropenia syndrome. The possible role of the expanded K-cell population, and its humoral products (tumour necrosis factor, interferons), in the syndrome is discussed.     

Characterization of the binding domains on platelet glycoproteins Ib-IX and IIb/IIIa complexes for the quinine/quinidine-dependent antibodies.

Sera of 12 patients with quinine/quinidine-induced thrombocytopenia showed drug-dependent antibody binding to glycoprotein (GP) Ib-IX complex. The reaction with GPIb-IX complex of 11 of these 12 sera was strongly inhibited by the complex-specific monoclonal antibodies (MoAbs) AK1 and SZ1. The exception was a quinine-induced serum designated BU. The reaction of the six quinidine-induced sera was also partially blocked by an anti-GPIX MoAb, FMC25. Only 3 of the 12 patient sera showed drug-dependent antibody binding to GPIIb/IIIa, which was strongly inhibited by the anti-GPIIIa MoAb 22C4, and the anti-GPIIb alpha MoAb SZ22. With detergent-solubilized Serratia metalloprotease-treated platelets, quinine/quinidine-induced sera, except BU, immunoprecipitated a membrane-bound proteolytic fragment of GPIb-IX complex. In contrast, BU immunoprecipitated glycocalicin and a 40-Kd peptide tail fragment of GPIb alpha from the cell supernatant. Using purified GPIb-IX complex or its components as the target antigen, all the quinine-induced sera, except BU, immunoprecipitated GPIb-IX complex but failed to immunoprecipitate GPIb, GPIX, or the complex reformed from GPIb and GPIX. The quinidine-induced sera strongly immunoprecipitated purified GPIb-IX complex, weakly immunoprecipitated purified GPIX and the recombined complex, but did not immunoprecipitate purified GPIb. The combined data suggest that one quinine-dependent antibody (BU) recognizes an epitope in the peptide tail region of GPIb alpha and the other five quinine-dependent antibodies react with a complex-specific epitope on the membrane-associated region of GPIb-IX complex, whereas each of the six quinidine-induced sera contain two drug-dependent antibodies, one reactive with the GPIb-IX complex-specific epitope and the other reactive with GPIX. The binding domain(s) on GPIIb/IIIa for the quinine/quinidine-dependent antibodies appear to be sterically close to the epitopes for 22C4 and SZ22.     

Antibodies to actin in autoimmune neutropenia

In an effort to characterize the cellular antigens recognized by anti-neutrophil antibodies in autoimmune neutropenia, we studied sera, purified immunoglobulin G (IgG) and isolated F(ab')2 from 70 neutropenic patients suspected of this diagnosis. Anti-neutrophil antibodies were found in the sera of 36 of these patients by either 125I-staph A binding or immunofluorescence cytometric techniques that detected increased binding of patients' IgG to normal neutrophils. Anti-neutrophil antibody positive sera were then evaluated for specific binding to electrophoretically separated neutrophil membrane-associated proteins by immunoblotting. A 43-Kd protein was consistently identified by eight anti-neutrophil antibody positive sera. The specificity of binding to this protein was confirmed with affinity purified IgG and F(ab')2 fragments prepared from these sera. Sera from 20 healthy normal controls and from 22 non-neutropenic, anti-neutrophil antibody negative rheumatoid arthritis patients failed to bind this protein. Separate studies identified the 43-Kd protein as actin. Purified Acanthamoeba actin comigrated with the protein and was specifically bound by anti-neutrophil antibody positive IgG. Moreover, two actin-specific monoclonal antibodies bound to the 43-Kd membrane-associated protein in immunoblots. In addition, a rabbit anti-actin antiserum not only bound to this same 43-Kd protein but also expressed anti-neutrophil antibody activity against normal human neutrophils, as did purified human anti-actin IgG prepared by affinity chromatography from the serum of one of the index patients. These studies indicate that the anti-neutrophil antibodies of certain patients with autoimmune neutropenia include autoantibodies specific for actin. The molecules on the surface of neutrophils, which have actin-like antigenic epitopes and are recognized by these anti-actin antibodies, remain to be characterized.     

Hemolytic anemia and acute renal failure associated with temafloxacin-dependent antibodies

Quinine-ingestion has been associated with immune-mediated recurrent pancytopenia, hemolysis, and renal failure. The structure of fluoroquinolone antibiotics is similar to the structure of quinine. Over a 3 month period, three patients at our institution developed hemolysis and renal failure following ingestion of the fluoroquinolone antibiotic temafloxacin. Two of the three patients required hemodialysis. Following withdrawal from the drug, the hemolysis resolved and the renal function eventually returned to normal in all three patients. One patient also had a transient mild thrombocytopenia. Sera from all three patients were tested for drug-dependent antibodies to red blood cells, platelets, and neutrophils. Temafloxacin-dependent red cell antibodies were detected in one patient, and temafloxacin-dependent red cell and neutrophil antibodies were detected in a second patient. No temafloxacin-dependent antibodies were detected in the third patient. Sera from all three patients were also tested for quinine and quinidine-dependent antibodies to red cells, platelets, and neutrophils. Sera from the patient without temafloxacin-dependent red cell antibodies reacted with red cells in the presence of quinine. These results suggest that, at least in some patients, the toxicities associated with temafloxacin are immune mediated. © 1994 Wiley-Liss, Inc.     

Heterogeneity of drug-dependent platelet antigens and their antibodies in quinine- and quinidine-induced thrombocytopenia: involvement of glycoproteins Ib, IIb, IIIa, and IX

The molecular nature of platelet receptors for quinine- and quinidine- dependent antiplatelet antibodies (Q.Ab and Qd.Ab) was studied by immunoblotting. One Q.Ab caused quinine-dependent IgG binding to platelet proteins with molecular weights (mol wts) of 174 Kd and 93 Kd and another to only a 93-Kd protein. A third Q.Ab caused binding to 174- , 140-, 93-, and 57-Kd proteins, while a fourth Q.Ab and a Qd.Ab caused IgG binding to 174- and 18-Kd proteins. Using platelets from patients with Glanzmann's thrombasthenia or Bernard Soulier syndrome and purified GPIIIa, these proteins were shown to be GPIb, GPIIb, GPIIIa, GPIX, and an unidentified 57-Kd protein missing in Bernard Soulier syndrome. Binding to the 93-Kd protein was independent of the PIA1 antigen. Absorption of one Q.Ab with Glanzmann's thrombasthenia platelets revealed different populations of antibodies with different specificities within the one patient. Thus Q.Ab and Qd.Ab are heterogeneous and may be directed toward different epitopes on major platelet glycoproteins.     

Autoimmune neutropenia in systemic lupus erythematosus

The mechanism of granulocyte depletion in a patient with systemic lupus erythematosus and neutropenia was investigated. Neutrophil kinetic studies showed a shortened intravascular survival (t1/2 of 1.6 hours) in the face of an increased marrow neutrophil pool. IgG bound to the patient's neutrophils, measured by the Fab antiF(ab')2 assay, was nearly three times normal. The IgG neutrophil-binding activity of the patient's serum was elevated in serial samples obtained over two years. In addition, his serum was able to opsonize normal neutrophils for ingestion by other neutrophils as detected by 14C-1-glucose oxidation. Enhanced IgG PMN-binding activity was observed with sucrose density gradient fractions of the patient's serum containing either large complexes (19S or greater in size), intermediate complexes (between 7S and 19S), or monomeric IgG. Only the momomeric IgG fraction from the patient's serum, however, opsonized normal neutrophils for ingestion by other neutrophils. These results support the hypothesis that anti-cell antibodies were responsible for the neutropenia in this patient by opsonizing neutrophils for ingestion by other phagocytic cells.     

Drug-induced thrombocytopenia: localization of the binding site of GPIX-specific quinine-dependent antibodies

Immune thrombocytopenia is a common complication of therapy with a large number of drugs. The most widely studied drug-induced immune thrombocytopenia (DIT) is caused by quinine. In most cases of DIT, antibodies bind to the platelet membrane glycoprotein (GP) Ib-IX complex in a drug-dependent fashion and bring about increased platelet clearance by the reticuloendothelial system resulting in thrombocytopenia. Here, we report the characterization of the quinine-dependent antibody activity of sera from 13 patients with quinine-induced thrombocytopenia. In our series of patients, GPIX was the most prevalent target of quinine-dependent antibodies. To identify the structural determinants of GPIX recognized by quinine-dependent antibodies, 4 chimeric mouse/human GPIX constructs and stable Chinese hamster ovary (CHO) cell lines that expressed the chimeras in association with GPIbalpha and GPIbbeta were produced. The analysis of 6 patient sera with the chimeric cell lines provided evidence for localization of the anti-GPIX quinine-dependent antibody binding site to the C-ext region (amino acid [aa] 64-135) of human GPIX. Further characterization of the C-ext region of the GPIX indicated that replacement of the Arg110 and Gln115 of the human GPIX with the corresponding residues from mouse (Gln and Glu, respectively) resulted in a significant reduction in the binding of GPIX antibodies in our series of patients, with Arg110Gln, giving a more pronounced effect than Gln115Glu. Hence, these 2 residues, particularly Arg110, play an important role in the structure of the antigenic site on GPIX recognized by anti-GPIX antibodies.     

Activated platelets and endothelial cell interaction with neutrophils under flow conditions.

OBJECTIVE: The mechanism of neutrophil adhesion to the endothelium during the earliest stages of acute inflammation, especially before the induction of adhesion molecules on endothelial cells, remains unknown. We studied the possible involvement of platelets in this process. METHODS: Neutrophils were added to human umbilical vein-derived endothelial cells (HUVEC) with or without adherent platelets in the presence or absence of adhesion-blocking monoclonal antibodies (mAbs). Adhesion of neutrophils to HUVEC at dynamic flow conditions was assessed using a flow chamber. RESULTS: 1) Thrombin-activated platelets adhered to resting-HUVEC at dynamic flow conditions through platelet glycoprotein IIb/IIIa and RGD proteins. 2) Neutrophils tethered to P-selectin induced on thrombin-activated platelets, which were immobilized on HUVEC. 3) Activated neutrophils adhered, via LFA-1, to ICAM-1 on HUVEC. 4) Activated platelets induced interleukin (IL)-8 secretion by HUVEC. CONCLUSIONS: Immobilized platelets on the vessel wall with induced P-selectin on the surface biochemically and functionally promote the adhesion of neutrophils to endothelial cells.     

CD31 regulates direction and rate of neutrophil migration over and under endothelial cells

Mechanisms guiding migration of neutrophils through endothelium are poorly understood. We showed previously that CD31-CD31 binding acted as an 'accelerator' for neutrophils migrating on platelets, while neutrophil alpha(v)beta3-integrin acted as a sensor to align migration with the direction of imposed flow. Here, we perfused neutrophils over human umbilical vein endothelial cells (HUVEC) treated with tumour necrosis factor-alpha, and characterised the kinetics of migration over, through and underneath the HUVEC. Before penetrating the monolayer, activated neutrophils migrated relatively slowly over the surface (approximately 6 microm/min), preferentially in the direction of flow. Once transmigrated, neutrophils moved more rapidly (approximately 14 microm/min) without preferred direction. Treatment of HUVEC and/or neutrophils with function-blocking antibodies against CD31 reduced directionality but not velocity of migration on top of HUVEC, and reduced velocity of migration underneath the monolayer. If neutrophils were pre-activated with formyl peptide, they did not migrate through the HUVEC, but migrated with increased velocity and directionality on top. Under these circumstances, both velocity and directionality were reduced by blocking CD31. alpha(v)beta3-integrin did not regulate migration under any conditions. We conclude that CD31-CD31 bonds act as robust sensors which can guide neutrophil migration, and also modify its velocity. Thus mechanical and adhesive signals can regulate neutrophil migration driven by locally-acting chemotactic agents.     

Heparin-induced thrombocytopenia: confirmation of diagnosis with in vitro methods

Profound thrombocytopenia developed in a patient during treatment with heparin for venous thrombosis. The platelet count increased toward normal when heparin administration was stopped, but fell abruptly when the drug was again given. Platelet aggregation occurred when heparin was added to the patient's platelet-rich plasma, or to normal platelets plus the patient's serum. This serum also effected release of 3H- serotonin from normal platelets. This pattern of aggregation was clearly different from that occasionally caused by heparin in a control population. The data is consistent with an effect of heparin on platelets, possibly mediated by on immune mechanism.     

Biochemical characterization of the neutrophil-specific antigen NB1

Neutrophil-specific alloantibodies and the antigens they recognize are important in clinical medicine, but little is known about the structure of these antigens. Alloimmunization to the antigen NB1 is a clinically important cause of neonatal neutropenia and febrile transfusion reactions. To study the immunochemistry of the NB1 antigen, we prepared neutrophil plasma membranes and granules by nitrogen cavitation and differential centrifugation and then analyzed them by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting with alloantibodies to several neutrophil-specific antigens. Two different antisera to the neutrophil-specific antigen NB1 identified an approximately 55-Kd protein by immunoblotting on neutrophil membranes from four NB1-positive donors but not on neutrophil membranes from five NB1-negative donors. Four anti-NB1 antisera immunoprecipitated a 58- to 64-Kd protein from extracts of NB1-positive neutrophils surface-labeled with 125I using lactoperoxidase, but not from similarly treated NB1-negative neutrophils. Normal human serum did not immunoprecipitate or immunoblot any proteins from these same neutrophil preparations. The NB1 antigen was detected by immunoblotting in secondary granules but was not found in primary granules. The electrophoretic mobility of the antigen was decreased slightly by reduction, suggesting that intrachain disulfide bonds were present. After reduction, the antigen could no longer be recognized by anti-NB1 antisera, but treatment of the antigen with periodate had no effect on the ability of anti-NB1 antisera to recognize the antigen, suggesting that it is not a carbohydrate. The data suggest that the neutrophil-specific antigen NB1 is present on a 58- to 64-Kd surface glycoprotein that is also present in secondary granules, and that the NB1 epitope is not a carbohydrate but probably resides in the tertiary structure of the protein backbone.     

Platelet activation by Shiga toxin and circulatory factors as a pathogenetic mechanism in the hemolytic uremic syndrome

Thrombocytopenia caused by platelet consumption in thrombi is a major manifestation of hemolytic uremic syndrome (HUS) associated with Shiga toxin (Stx) producing Escherichia coli. Platelets have glycosphingolipid receptors capable of binding Stx, but a direct interaction between the toxin and platelets, leading to platelet activation, has not been reported. In this study, it is shown that Stx1 and its B (binding) subunit (Stx1B), at 10 pg/mL to 10 ng/mL, bound to platelets. Toxin was internalized in platelets within 2 hours. This led to increased platelet aggregation, as demonstrated by confocal microscopy. Preincubation of Stx1B with anti-Stx1 antibody inhibited this reaction. Stx1 induced morphologic changes in platelets seen on scanning electron microscopy. In the presence of platelets and tumor necrosis factor-pretreated human umbilical vein endothelial cells (HUVEC), Stx1 and Stx1B induced the binding of platelets to the endothelial cell membrane and were present at this binding site. Incubation of Stx1 and Stx1B with whole blood increased fibrinogen binding to platelets detected by flow cytometry. Fibrinogen binding was partially inhibited by preincubation with anti-Stx1. Stx1 increased platelet retention measured in a glass bead assay. In addition, plasma from 17 patients with HUS, taken during the acute phase of the disease, increased the retention of normal platelets and normalized after recovery. Taken together, the results of this investigation show that Stx1, Stx1B, and a factor or factors in the plasma of patients with HUS activate platelets. The presence of Stx1 at the binding site of platelets to HUVEC suggests that Stx may be directly involved in the prothrombotic state seen in HUS.     

Comparative evaluation of the role of the adhesion molecule CD177 in neutrophil interactions with platelets and endothelium

Neutrophil-specific glycoprotein CD177 is expressed on a subset of human neutrophils and has been shown to be a counter-receptor for platelet endothelial cell adhesion molecule-1 (PECAM-1, CD31). Previous studies have demonstrated that the interaction of CD177 with endothelial PECAM-1 supports neutrophil transendothelial migration resulting in preferential transmigration of the CD177-expressing neutrophil subset. As PECAM-1 is also abundantly expressed on platelets, we addressed a follow-up suggestion that CD177/PECAM-1 adhesive interaction may mediate platelet-neutrophil interactions and CD177-positive neutrophils may have a competitive advantage over CD177-negative neutrophils in binding platelets. Here, we report that CD177-positive and CD177-negative neutrophils do not differ significantly in their capacity to form platelet-neutrophil conjugates as assayed in whole blood and in mixed preparations of isolated platelets and neutrophils. Under flow conditions, neither platelet nor neutrophil activation resulted in preferential binding of platelets to CD177-expressing neutrophils. Furthermore, no significant difference was found in the ability of both neutrophil subsets to adhere to and migrate across surface-adherent activated platelets, whereas predominantly CD177-positive neutrophils migrated across HUVEC monolayers. In addition, we demonstrated that S(536) N dimorphism of PECAM-1, which affects CD177/PECAM-1 interaction, did not influence the equal capacity of the two neutrophil subsets to interact with platelets but influenced significantly the transendothelial migration of CD177-expressing neutrophils. Thus, CD177/PECAM-1 adhesive interaction, while contributing to neutrophil-endothelial cell interaction in neutrophil transendothelial migration, does not contribute to or is redundant in platelet-neutrophil interactions.     

Direct evidence for the existence of a neutrophil-derived platelet activator (neutrophilin).

Human neutrophils and platelets were loaded with the intracellular calcium indicator fura-2. The chemotactic peptide N-formyl-Met-Leu-Phe (fMet-Leu-Phe) induced a rapid elevation of cytosolic free calcium in cytochalasin B-treated neutrophils but failed to increase the cytosolic calcium in platelets. On the other hand, when unloaded neutrophils were incubated together with autologous fura-2-loaded platelets, fMet-Leu-Phe stimulated a 6-fold increase in platelet cytosolic calcium subsequent to a brief lag. Parallel experiments demonstrated that the addition of fMet-Leu-Phe to neutrophil/platelet incubates also elicited platelet aggregation and serotonin release. Platelet activation showed a positive correlation with the concentration of fMet-Leu-Phe added to the mixed cell population. Cell-free supernatants prepared from fMet-Leu-Phe-stimulated neutrophils were capable of inducing platelet calcium mobilization, aggregation, and secretion. The amount of platelet-activating material present in the supernatant was proportional to the number of activated neutrophils. Preincubation of platelets with BN 52021, acetylsalicylic acid, SQ-29,548, or hirudin did not modify the aggregation response induced by the supernatant collected from fMet-Leu-Phe-activated neutrophils, suggesting that the material was not 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine (paf-acether), arachidonic acid, thromboxane A2, or thrombin. Pretreatment of the neutrophil supernatant with an ADP (creatine phosphate/creatine phosphokinase) or a superoxide/peroxide (superoxide dismutase/catalase) scavenging system also had no effect on aggregation or secretion, indicating that these substances did not participate in platelet activation. The biological activity present in the neutrophil supernatant was destroyed by heat and inactivated by treatment with phenylmethylsulfonyl fluoride, indicating that it is a protein and most probably an enzyme with serine protease activity. These data provide the direct observation of secondary signal transmission to platelets following primary activation of neutrophils. We propose the name neutrophilin for the neutrophil-derived mediator.     

Synergistic effects of thrombopoietin and granulocyte colony- stimulating factor on neutrophil recovery in myelosuppressed mice

Severe suppression of the hematopoietic system is a major factor in limiting chemotherapy dose escalation. To determine whether a combination of human recombinant granulocyte colony-stimulating factor (G-CSF) and thrombopoietin (TPO) would alter recovery of platelets, red blood cells (RBCs), or neutrophils after myeloablative therapy, myelosuppressed mice were treated with sc injections of TPO (90 micrograms/kg), G-CSF (250 micrograms/kg). TPO plus G-CSF or vehicle and complete blood counts were measured. Marrow and spleen cells were obtained at various times and assayed for erythroid, myeloid, and megakaryocytic progenitors. The prolonged neutropenia in vehicle controls (14 days) was significantly shortened in mice treated with G- CSF or TPO for 14 days. The combination of TPO plus G-CSF further reduced the duration of neutropenia. TPO and TPO plus G-CSF treatments also significantly shortened thrombocytopenia compared to vehicle. Recovery of RBCs was also enhanced in mice treated with either G-CSF or TPO, or the combination. Furthermore, treatment with G-CSF and/or TPO hastened myeloid, erythroid, and megakaryocyte progenitor recovery compared to vehicle controls. These results show that the combination of TPO plus G-CSF acts synergistically to accelerate neutrophil recovery in myelosuppressed mice and does not compromise the platelet or RBC response to TPO therapy.     

Non Hodgkin’s lymphoma presenting as neutropenia related to an IgM monoclonal anti-i antibody

A sixty-year old female was referred to the Internal Medicine Department for the treatment of a diffuse high-grade non Hodgkin’s lymphoma. She presented episodes of fever in a context of neutropenia (neutrophils 0.35 × 10⁹/l from 1.6 × 10⁹/l white blood cells). Hemoglobin level was 8.2 g/dl and platelets 132 × 10¹²/l. A monoclonal IgM-(kappa) protein (48 g/l) was detected in her serum. A direct antiglobulin test on the red cells proved positive with anti-C3d but not with anti-IgG antiglobulin, due to the presence of an IgM cold antibody with a serological anti-i specificity. The IgM antibody was found on the patient’s neutrophils as well as in her serum. This antibody recognized all neutrophils tested in conventional serological tests whatever the neutrophil phenotypes in systems NA, NB, and 5. It was demonstrated that it recognized the i antigen expressed on the neutrophils. These results suggest that a cold agglutinin anti-i might be responsible for neutropenia in some patients.