异常形态红细胞百分比法和芽孢状等特殊异常形态红细胞百分比法在小儿肾性血尿诊断中临床价值的对比观察

目的对小儿肾性血尿进行红细胞形态学分析,评价异常形态红细胞百分比法和芽孢状等特殊形态红细胞百分比法在小儿肾性血尿鉴别诊断中的临床价值。方法回顾性对来自首都医科大学附属北京友谊医院的117例非重复性小儿肾性血尿标本红细胞形态检查结果进行分析,比较尿红细胞形态检查两种判断标准的敏感性。结果在肾小球疾病组和对照组,异常形态红细胞占红细胞总数80%的标本百分比分别为62. 5%(73/117)和12. 0%(15/125),差异具有明显统计学意义(P <0. 01);芽孢状红细胞等特殊异常形态红细胞百分比≥30%标本百分比分别为83. 8%(98/117)和8. 0%(10/125),差异具有非常显著性统计学意义(P <0. 01)。明确诊断为肾小球疾病患儿,尿液异常形态红细胞占红细胞总数≥80%百分比显著低于芽孢状红细胞等特殊异常形态红细胞百分比≥30%者(62. 4%,73/117 vs.83. 8%,98/117),差异具有非常显著性统计学意义(P <0. 01)。结论以芽孢状红细胞等特殊异常形态红细胞百分比法作为判断标准对小儿肾性血尿的诊断具有较好的敏感性。     

UF-100尿液自动分析仪和显微镜检测RBC形态在鉴别肾性血尿中的比较

目的探讨尿液RBC形态检测的临床意义。方法采用UF-100和显微镜法对118例尿液标本进行正形(均一性)和异形(非均一性)RBC的检测,将两者结果进行比较分析。结果62例肾性血尿患者中显微镜检测有56例异形RBC增高,临床诊断符合率为90.3%;UF-100检测有58例为非均一性RBC,临床诊断符合率为93.5%。56例非肾性血尿中显微镜检测有54例正形RBC增高,临床诊断符合率为96.4%;UF-100检测有55例为均一性RBC,临床诊断符合率为98.2%。两法比较,经配对x2检验,P>0.05,结果无显著性差异。而肾性和非肾性血尿组相比,经四格表资料x2检验,P<0.05,结果显示两组有显著性差异。结论UF-100和显微镜对RBC形态的检测都可以用于鉴别诊断肾性和非肾性血尿。尤其是UF-100可为临床诊断疗效观察和判断预后起到非常重要的作用。     

Erythrocyte morphology in asymptomatic microhematuria: experimental studies and clinical significance

Renal and postrenal origin of hematuria can be differentiated by analysis of the morphology of the erythrocytes in the urinary sediment. In order to investigate the mechanisms which cause the membrane changes in dysmorphic erythrocytes, indicating renal origin of bleeding, normal red blood cells were exposed in vitro to an osmotic environment similar to that of the nephron. Upon exposure of osmotically challenged erythrocytes to a hemolytic environment, 50 to 90% of the cells became dysmorphic in a time- and dose-dependent manner and were indistinguishable on light and electron microscopy from those obtained in vivo from a patient with proven glomerular microhematuria. The reliability of erythrocyte morphology in differentiating between renal and postrenal microhematuria was evaluated by performing microscopic analysis as the initial step in the investigation of 316 consecutive patients. In 123 patients with eumorphic red cells in their urine complete urological investigation revealed a postrenal source of bleeding in 85%. Out of 193 patients with dysmorphic erythrocytes, 132 were followed up for at least 2 years after only minimal diagnostic evaluation. An additional postrenal source of bleeding developed in two patients, which was easily diagnosed by the change in erythrocyte morphology. Our studies, representing experience over 6 years with asymptomatic microhematuria, show that microscopic examination of erythrocyte morphology as initial diagnostic step is a safe, inexpensive and efficient method which renders invasive investigations superfluous in the majority of patients.     

Acanthocytes in the urine: useful tool to differentiate diabetic nephropathy from glomerulonephritis?

OBJECTIVE: The presence of hematuria has been suggested to indicate nondiabetic nephropathy in diabetic patients with proteinuria. However, hematuria is frequently found in patients with biopsy-proven diabetic glomerulosclerosis without nondiabetic nephropathy. Urine microscopy allows discrimination of glomerular hematuria, which is defined as acanthocyturia (urinary excretion of acanthocytes, which are dysmorphic erythrocytes with vesicle-like protrusions), from nonglomerular hematuria. We hypothesized that acanthocyturia is an uncommon finding in diabetic nephropathy, which suggests the presence of a nondiabetic nephropathy in diabetic patients with proteinuria. RESEARCH DESIGN AND METHODS: Urine samples of patients with the clinical diagnosis of diabetic nephropathy (n = 68), of patients with biopsy-proven glomerulonephritis (n = 43), and of age-matched healthy control subjects (n = 20) were examined by phase-contrast microscopy for the presence of hematuria (>/=8 erythrocytes/ micro l) and acanthocyturia. Acanthocyturia of >/=5% (5 acanthocytes among 100 excreted erythrocytes) was classified as glomerular hematuria; acanthocyturia of 2-4% was classified as suspected glomerular hematuria. RESULTS: Hematuria was found in 62% of patients with the clinical diagnosis of diabetic nephropathy, in 84% of patients with glomerulonephritis, and in 20% of the healthy control subjects upon a single urine examination. In contrast, glomerular hematuria occurred in 4% of patients with diabetic nephropathy and in 40% of patients with glomerulonephritis (P < 0.001). CONCLUSIONS: In contrast to hematuria, acanthocyturia is uncommon in patients with the clinical diagnosis of diabetic nephropathy. In diabetic patients with proteinuria, the finding of acanthocyturia points to nondiabetic glomerulopathies, and renal biopsy should be considered.     

Hematuria. When is it cause for alarm?

The urine of less than 3% of healthy persons shows more than three red cells per high-power field, which is about the limit of sensitivity of test strips for occult blood. Even minimal hematuria may herald serious problems and must be investigated. Common causes include infection, urethritis, and urethrotrigonitis (25%); stone (20%); and tumor (15%). Although hematuria may be caused by coagulopathy, patients with anticoagulant-induced hematuria must be examined for anatomic defects. Hematuria may originate from the kidney or the urinary tract; red cell casts and dysmorphic red cells suggest a renal source. Hematuria arising from the kidney is classified as glomerular (eg, various forms of glomerulonephritis) or nonglomerular (eg, polycystic kidney disease, cancer). If the urine is pigmented but test strips are negative, or test strips are negative but no red cells are seen, pseudohematuria must be considered.     

尿红细胞Tamm-Horsfall蛋白检测在儿童血尿诊断中的意义探讨

目的 :探讨尿红细胞Tamm Horsfall蛋白 (THP)检测在确定血尿来源上的诊断意义。方法 :对诊断明确的 86例肾小球性血尿和 2 6例非肾小球性血尿患儿尿样本同时进行尿红细胞THP间接免疫荧光法检测和尿红细胞的形态检测 ,比较这两种确定血尿来源方法的差异。结果 :在 86例肾小球性血尿中 ,红细胞THP检测法确定肾小球性血尿 79例 ,7例与诊断不符 ,在 2 6例非肾小球性血尿中 ,红细胞THP检测法确定为非肾小球性血尿 2 4例 ,2例与诊断不符 ,灵敏度为 91 9% ,特异性为 92 3 % ,准确性为 92 0 % ,漏诊率为 8 1% ,误诊率为 7 7% ,尿红细胞的形态检测法敏感性为 77 9% ,特异性为 69 2 % ,准确性为 76 7% ,漏诊率为 2 2 1% ,误诊率为 2 2 1% ,两者在灵敏性、准确性和漏诊率上的差异有极显著意义 (P <0 0 1) ,在特异性及误诊率上的差异有显著意义 (P <0 0 5 )。结论 :尿红细胞THP检测法确定血尿来源优于红细胞形态检查法 ,值得临床上推广应用。     

Urine flow cytometry and detection of glomerular hematuria.

BACKGROUND: The UF-100 is a flow cytometer designed for automated cellular urinalysis. In this study, the usefulness of the UF-100 in laboratory investigation into the origin of hematuria was evaluated. METHODS: Results from flow cytometric urinalysis were used to classify urinary red blood cells (RBCs) according to glomerular and non-glomerular origin and the classification was compared to the patient's clinical diagnosis as the gold standard. In parallel, microscopic sediment analysis was carried out. RESULTS: A total of 206 urine samples from 129 patients were analyzed (127 from patients with glomerular hematuria, 79 from patients with non-glomerular hematuria). Of these, 136 samples (92 patients) showed overt hematuria (>or=20 RBC/microL). Urine flow cytometry correctly classified 61% (sediment analysis 69%) of urine samples with overt hematuria. If inconclusive results are excluded, the UF-100 correctly diagnosed 85% (sediment analysis 98%) of urine samples with overt hematuria. The UF-100 and microscopic sediment analysis both showed sensitivity of 99% for the detection of glomerular hematuria. The specificity of the UF-100 for the detection of glomerular bleeding was lower (42%) than the specificity of microscopic sediment analysis (93%). CONCLUSIONS: Owing to its low specificity, the UF-100 showed limited capacity to discriminate glomerular from non-glomerular causes of hematuria in a population with a high incidence of renal disease. Therefore, extensive microscopic urinalysis remains necessary to assess the origin of hematuria.     

Familial pseudohyperkalaemia: inhibition of erythrocyte K+ efflux at 4 degrees C by quinine

1. Mechanisms responsible for increased erythrocyte K+ efflux in vitro have been investigated in a patient with familial pseudohyperkalaemia. Mean net K+ efflux (4 degrees C) was 108 nmol h-1 10(-9) erythrocytes, seven times greater than the mean for controls (15.2 nmol h-1 10(-9) erythrocytes). Net K+ efflux was not increased at 22 degrees C or 37 degrees C and losses at 4 degrees C were reversed by subsequent incubation at 37 degrees C. 2. Erythrocyte glucose consumption (4 degrees C) was 14 nmol h-1 10(-9) erythrocytes, similar to the mean for controls of 16.8 nmol h-1 10(-9) erythrocytes. This suggests that the increased net K+ efflux (4 degrees C) was not associated with abnormal energy consumption and was therefore unlikely to be due to an abnormality of the Na+, K+-pump. 3. Incubation of erythrocyte suspensions with ouabain (0.1 mmol/1) or frusemide (1 mmol/1) at 4 degrees C or 37 degrees C resulted in no differences in K+ efflux between patient and controls. Incubation with quinine (2 mmol/1), an inhibitor of the erythrocyte Ca2+-dependent K+ channel, reduced net K+ efflux at 4 degrees C, but the effect persisted in Ca2+-depleted erythrocytes, implying that quinine was acting in a non-specific fashion. 4. Chemical pathologists and clinicians must be aware of this condition if inappropriate treatment of pseudohyperkalaemia is to be avoided.     

尿变形红细胞新分类法在肾小球轻微病变中的应用

目的研究尿变形(D)红细胞的一种新分类法在肾小球轻微病变血尿中的应用价值。方法对84例确诊为肾小球轻微病变患者和73例非肾小球疾病患者的血尿标本应用相差显微镜观察进行分类,同步检测尿蛋白及管型。结果在84例肾小球轻微病变血尿中,经242次检测红细胞,以D1+D2≥5%为诊断标准的敏感性、特异性、准确性分别为49.6%、100%、67.4%,以新分类法(D1+D2≥5%或D3≥20%)为诊断标准的敏感性、特异性、准确性分别为90.1%、98.5%、93.0%。D1+D2≥5%组与D1+D25%而D3≥20%组间尿蛋白、细胞管型检出率有统计学差异(0.01P0.05),而透明管型、脂肪管型检出率无统计学差异(P0.05)。结论D1+D2≥5%或D3≥20%是诊断肾小球轻微病变血尿的敏感方法。     

Urinary macrophage counts and ratio to T lymphocytes: Possible use in differential diagnosis and management of glomerular disease

The noninvasive method that can differentiate hematuria-positive patients has not yet been developed. We evaluated the clinical value of the analysis of mononuclear cells in urine in combination with urinary erythrocytes as a noninvasive differential diagnostic tool of glomerular disease. The number of macrophages (CD14⁺ cells/ml urine) and T-lymphocytes (CD3⁺ cells/ml urine) were measured by flow cytometry using samples of freshly voided urine from 203 patients with hematuria. They had various types of proliferative glomerular disease, including rapidly progressive glomerulonephritis (RPGN), IgA nephropathy (IgAN), and membranoproliferative glomerulonephritis (MPGN), or nonproliferative glomerulopathy including idiopathic renal hematuria and hereditary nephropathy. Urinary macrophage counts increased significantly with the severity of glomerulonephritis; their number consistently exceeded that of T-lymphocyte counts in patients with active proliferative glomerulonephritis. Urinary macrophage counts in patients with proliferative GN were consistently higher than those of hematuria-matched nonproliferative GN. Moreover, urinary macrophage counts in patients with RPGN were significantly higher than those of MPGN and IgAN. Most of the patients with inactive proliferative glomerulonephritis or with nonproliferative glomerulopathy showed no marked increase in urinary macrophages. Although some patients with nonproliferative glomerulopathy who exhibited gross hematuria showed a slight increase in urinary macrophage counts, such counts were consistently lower than those of T lymphocytes. These observations suggests that urinary macrophage count and its ratio to T-lymphocyte count may provide useful information for clinicians in managing patients with proliferative glomerular disease as well as deciding whether to conduct renal biopsy in patients with hematuria. © 1996 Wiley Liss, Inc.     

Erythrocyte Adenosine Deaminase Deficiency without Immunodeficiency: EVIDENCE FOR AN UNSTABLE MUTANT ENZYME

Inherited deficiency of the purine salvage enzyme adenosine deaminase (ADA) gives rise to a syndrome of severe combined immunodeficiency (SCID). We have studied a 2.5-yr-old immunologically normal child who had been found to lack ADA in his erythrocytes during New York State screening of normal newborns. His erythrocytes were not detectably less deficient in ADA than erythrocytes of ADA(-)-SCID patients. In contrast, his lymphocytes and cultured long-term lymphoid cells contained appreciably greater ADA activity than those from patients with ADA(-)-SCID. This residual ADA activity had a normal molecular weight and K(m) but was markedly unstable at 56 degrees C. His residual erythrocytes-ADA activity also appeared to have diminished stability in vivo. ADA activity in lymphoid line cells of a previously reported erythrocyte-ADA-deficient!Kung tribesman was found to contain 50% of normal activity and to exhibit diminished stability at 56 degrees C. ATP content of erythrocytes from both partially ADA-deficient individuals was detectably greater than normal (12.3 and 6.1 vs. normal of 2.6 nmol/ml packed erythrocytes). However, the dATP content was insignificant compared to that found in erythrocytes of ADA(-)-SCID patients (400-1,000 nmol/ml packed erythrocytes). The New York patient, in contrast to normals, excreted detectable amounts of deoxyadenosine, but this was <2% of deoxyadenosine excreted by ADA(-)-SCID patients. Thus, the residual enzyme in cells other than erythrocytes appears to be sufficient to almost totally prevent accumulation of toxic metabolites.     

Measurement of acetate in human blood by gas chromatography: effects of sample preparation, feeding, and various diseases.

We measured acetate concentrations in whole blood, serum, and plasma by a modification of a previously described method involving vacuum distillation and gas chromatography. The mean acetate concentration of fresh venous plasma from 27 normal subjects was 51 +/- 5 mumol/L (95% confidence limits ranged from 0 to 103 mumol/L). The acetate concentrations of serum and plasma incubated for 2 h at either 4 degrees C or 27 degrees C were the same. The acetate concentration of whole blood incubated at 27 degrees C was significantly greater than that of blood incubated at 4 degrees C. This change may have resulted from the production of acetate by erythrocytes or from the hydrolysis of acetate esters. Storage of plasma at -20 degrees C for 24 h significantly increased acetate concentrations from 26 +/- 6 mumol/L to 63 +/- 4 mumol/L. After the subjects consumed a standard breakfast, venous plasma acetate concentrations increased from 58 to 97 mumol/L at 30 min. Acetate concentrations in arterial plasma exceeded those in venous plasma. Plasma acetate concentrations were not significantly altered in patients with malignancy or diabetes mellitus, but severe liver disease and severe acidosis were both associated with increased acetate concentrations. These preliminary observations suggest that plasma acetate concentrations may be altered in several disease states.     

Receptors for recombinant erythropoietin in human bone marrow cells

Human recombinant erythropoietin, labelled with 125I, was saturably bound to high affinity receptors, ka = 1.5 X 10(9) M-1, of bone marrow cells from 10 patients with various haematological disorders. Binding of labelled erythropoietin was specific, with less than 0.01% cross-reactivities of TSH, hCG and renin substrate (angiotensinogen). Binding capacity averaged 3 X 10(-14) moles/10(6) cells. Binding was maximal within 30 min at 37 degrees C. Low binding, less than 15% of that to bone marrow cells, was found to peripheral blood buffy coat, while erythrocytes and BALL cells did not bind labelled erythropoietin. Autoradiography showed binding of label confined to 4-5% of the bone marrow cells.     

Effects of myeloid leukaemic blast cell extract on sodium transport in human erythrocytes

1. Previous studies have shown that leukaemic plasma inhibits sodium efflux from erythrocytes. In this study the effect of an extract of myeloid leukaemic blast cells on sodium efflux was investigated. 2. Blast cell extract decreased the sodium efflux rate constant in erythrocytes from 0.395 +/- sem 0.028 to 0.310 +/- 0.018 (P less than 0.02), whereas a leucocyte extract had no significant effect. 3. The extract had an inhibitory effect after heating at 80 degrees C for 30 min, and in the presence of ouabain and frusemide alone or in combination. 4. These studies suggest that myeloid leukaemic blast cells contain a heat stable factor which inhibits the efflux of sodium at least in part through a pathway which is not inhibited by ouabain or frusemide.     

Paroxysmal nocturnal hemoglobinuria: deficiency in factor H-like functions of the abnormal erythrocytes

Erythrocytes from patients with paroxysmal nocturnal hemoglobinuria (PNH) contained a subpopulation that lacked membrane-associated Factor H-like activity present on normal human erythrocytes. Initial deposition of C3b on the erythrocytes was effected using a fluid phase C3 convertase. The cells were then treated with fluorescein-labeled C3 and the cell-bound C3 convertase. Analysis utilizing the fluorescence-activated cell sorter revealed two distinct cell populations, one of which was highly fluorescent, indicating a large number of C3b molecules per cell. Only this population (43%) was susceptible to lysis (44%) when exposed to acidified serum before C3b deposition. The less fluorescent population resembled normal human erythrocytes and was not affected by prior treatment with acidified serum. Since C3b deposition occurred almost exclusively on the complement-sensitive cells in the PNH erythrocyte population, these cells could be examined for the Factor H-like regulatory activities without prior isolation. These functions include enhancement of inactivation of erythrocyte-bound C3b by Factor I and acceleration of the decay of erythrocyte-bound C3 convertase, C3b,Bb. It was found that C3b on PNH erythrocytes was 100-fold less susceptible to inactivation by Factor I than C3b on normal human erythrocytes. The half-life at 22 degrees C of C3b,Bb on PNH erythrocytes was threefold greater than on normal human erythrocytes and similar to that of the enzyme bound to particles that do not possess Factor H-like activity. These observations suggest that the abnormal susceptibility of PNH erythrocytes to lysis by complement is due to a functional deficiency in one or more of the Factor H-like proteins present on normal human erythrocytes.     

Effects of in vitro treatment with 4-hydroperoxycyclophosphamide and hyperthermia on leukemic progenitor cells

A key point of autologous bone marrow transplantation for leukemic patients is how to remove leukemic cells from their own bone marrow grafts. In this study a leukemic progenitor cell assay was used to evaluate the antileukemic efficacy of marrow-purging protocols that employed hyperthermia or 4-hydroperoxycyclophosphamide (4HC) against leukemic blasts obtained from patients. After the treatment of 2 x 10(7) nucleated bone marrow cells/ml with 100 micrograms/ml of 4HC in the presence of 7% suspension of packed autologous erythrocytes, leukemic colonies were eradicated in 10 of 13 cases and reduced to less than 0.3% as compared with the colony count in untreated cultures in two cases. More than 10% of leukemic progenitor cells survived after hyperthermia treatment (42 degrees C 60 min) in 7 of 9 cases. It is suggested that treatment of leukemic cells and 7% autologous erythrocytes with 100 micrograms/ml of 4HC is effective to eliminate leukemic progenitor cells. Treatment with hyperthermia may not be effective enough to eliminate leukemic progenitor cells from autologous bone marrow.     

Role of Adherence in Cytopathogenic Mechanisms of Entamoeba Histolytica: STUDY WITH MAMMALIAN TISSUE CULTURE CELLS AND HUMAN ERYTHROCYTES

The enteric pathogen, Entamoeba histolytica, appears to cause disease by adhering to and then destroying mucosal barriers. Using an in vitro method of studying the interaction of E. histolytica with target cells (Chinese hamster ovary [CHO] and human erythrocytes [RBC]), we examined the mechanism of amebic adherence and its role in lysis of target cells. Killing and phagocytosis of target cells by amebas ceases at 4 degrees C, allowing observation of adherence. Amebas adhere to CHO cells at 4 degrees C, 78.9% formed rosettes (amebas with >/=3 adherent CHO cells each) at 2 h. At 37 degrees C, cytochalasins B and D inhibit adherence of amebas to CHO cells (P < 0.0005). Amebas adhere to and kill CHO cells in media with <0.1 muM calcium and magnesium plus 10 mM EDTA, indicating that divalent cations are not required in the medium. Adherence of amebas to human RBC was not ABO blood group specific and showed greater adherence to human than bovine or sheep RBC (P < 0.005). Neither Fc nor complement receptors were found on amebas by standard rosette studies. The amebic adherence receptor is not trypsin (0.125%) sensitive nor inhibited by trypan blue (1 mM). N-acetyl-d-galactosamine (GALNAc) inhibited the adherence of amebas to CHO cells and human RBC (0.1 g/100 ml or 4.5 mM GALNAc, P < 0.005) by binding to a receptor on the amebic surface. GALNAc abolishes amebic cytolysis of target CHO cells (determined by (111)Indium oxine release from CHO cells, P < 0.001) but not amebic phagocytosis of CHO cells. By suspending ameba-CHO cells rosettes in dextran, we found that GALNAc (1%) reversibly inhibits amebic adherence (P < 0.0005) and that cytochalasins decrease amebic killing of adherent CHO cells (P < 0.025).These findings indicate that the adherence of E. histolytica to target cells requires microfilament function, is via a specific amebic receptor that has affinity for GALNAc, and is required to lyse cells. Inhibition of the adherence of E. histolytica may alter the pathogenicity of this organism.     

Microcalorimetric measurements of heat production in human erythrocytes. III. Influence of pH, temperature, glucose concentration, and storage conditions.

Heat production in human erythrocytes has been measured under different conditions of pH, glucose concentration, and temperature. Storage conditions have also been varied. The erythrocytes, which were from healthy subjects, were suspended either in autologous plasma or in phosphate buffer. The heat effect, P, was shown to increase linearly in the physiological pH range: 1.2% per 0.01 pH unit. Variation of the glucose concentration within a wide range, 3-32 mmol/1, did not affect the P value. The temperature coefficient for P was determined to be Q10 = 2.8 for the temperature range 32-42 degrees C. A constant energy of activation was found, 82.6 kJ/mol, for the temperature range 25-42 degrees C. When the erythrocytes were stored at 4 degrees C, P values (measured at 37 degrees C) increased initially by 6%/h. After 24 h of storage P was about 50% higher than the initial value. Determinations of glucose consumption were made in parallel with most of the calorimetric experiments.     

Biological and environmental factors affecting ultrasound-induced hemolysis in vitro: 5. Temperature

This research project tested the hypothesis that cold-equilibrated (approximately 0 degrees C) human erythrocytes in vitro in the presence of an ultrasound contrast agent (Albunex) will undergo greater ultrasound-induced hemolysis than physiologically equilibrated (37 degrees C) human erythrocytes in vitro because of a temperature-related transition in membrane fluidity leading to increased fragility. First, it was shown that cold-equilibrated erythrocytes are more susceptible to mechanically induced hemolysis than physiologically equilibrated erythrocytes. Second, when adjustments were made for (1) temperature-dependent efficiencies of a 1-MHz transducer (200 micros pulse length, 20 ms interpulse interval, 30 s exposure duration) such that when cold or physiological temperatures were employed, there were equivalent acoustic outputs in terms of peak negative pressure (MPa P-) and (2) comparable viscosities of the 0 and 37 degrees C blood plasmas, the cold (approximately 0 degrees C) erythrocytes displayed substantially greater amounts of ultrasound-induced hemolysis than the physiological (37 degrees C) erythrocytes. The data supported the hypothesis.     

Binding characteristics of radioiodinated crystal-induced chemotactic factor to human neutrophils

The binding of radiolabeled crystal-induced chemotactic factor (CCF) to human neutrophils is characterized. Binding of 125I-CCF to the cells was higher at 4 degrees C than at 24 degrees or 37 degrees C and was found to be independent of CA2+ and Mg2+ ion concentration. The binding showed a pH optimum of 6.0. Tosylamido phenylethyl chloromethyl ketone at 100 mumol/L concentration inhibited 20% of 125I-CCF binding, but phenylmethylsulfonyl fluoride at 200 mumol/L had no effect. Approximately 50% of cell-associated 125I-CCF was released after treatment with proteases. The nonspecific uptake by the cells, as measured by the uptake of 3H-sucrose and 14C-inulin in the presence of CCF, was negligible. After the steady-state binding of 125I-CCF to the cells, approximately 15% of the cell-associated radioactivity at 4 degrees C and 40% to 50% at 24 degrees and 37 degrees C was released into the medium after 60 minutes of incubation in medium alone. Dissociation of the radioactive material was not affected by the presence of tosylamido phenylethyl chloromethyl ketone or phenanthroline in the media. The dissociated material was determined to be degraded 125I-CCF, suggesting that degradation of 125I-CCF occurs after binding to its specific receptor on the human neutrophil.